阅读说明：本技术 靶向抗癌核激素受体的化合物 (Compounds targeting anti-cancer nuclear hormone receptors ) 是由 范明山 S·查克拉瓦蒂 陈霁昀 J·坎卡纳拉 A·巴德 A·K·纳亚克 D·黄 于 2019-05-14 设计创作，主要内容包括：本公开涉及衍生自核类固醇受体结合剂的抗癌化合物、涉及含有其的产品以及涉及其使用和制备方法。(The present disclosure relates to anti-cancer compounds derived from nuclear steroid receptor binding agents, to products containing the same, and to methods of use and preparation thereof.)

1. A compound comprising at least one nuclear payload and at least one nuclear receptor-targeting epitope; with the proviso that when said compound comprises a nuclear payload and an epitope targeting a nuclear receptor:

The nuclear receptor-targeting epitope is not a peptide, protein, nanoparticle, or antibody; and is

When the nuclear receptor-targeting epitope is an androgen receptor-targeting epitope or an estrogen receptor-targeting epitope, the nuclear payload is not doxorubicin (doxorubicin) or an analog thereof, and is not a hydroxamic acid that binds Histone Deacetylase (HDAC).

2. A compound comprising at least one nuclear payload that binds to a catalytic domain of poly (ADP-ribose) polymerase (PARP) and at least one epitope that targets a nuclear receptor.

3. The compound of claim 1 or 2, wherein at least one nuclear receptor-targeting epitope is a nuclear steroid receptor-targeting epitope.

4. The compound of any preceding claim, wherein the nuclear payload is bonded to one nuclear receptor-targeting epitope via a non-biocleavable linking moiety and to one or more nuclear receptor-targeting epitopes via a biocleavable linking moiety.

5. The compound of any preceding claim, wherein the compound comprises at least one nuclear payload that binds to a protein involved in DNA damage repair processes.

6. The compound of any preceding claim, wherein the compound comprises at least one nuclear payload that binds poly (ADP-ribose) polymerase (PARP), DNA-dependent protein kinase (DNA-PK), Histone Deacetylase (HDAC), enhancer of zeste homolog 2 (EZH2), Histone Acetyltransferase (HAT), methyltransferase, bromodomain, myelin transcription factor 1(MYT1), p53, hormone stimulating Melanocyte (MSH), mutL homolog (MLH), ERCC1, depurination/depyrimidine endonuclease 1(APE1), topoisomerase i (topo i), topoisomerase ii (topo ii), Wee1, checkpoint kinase 1(Chk1), checkpoint kinase 2(Chk2), Ataxia Telangiectasia (ATR), or Ataxia Telangiectasia Mutation (ATM).

7. The compound of any preceding claim, wherein the nuclear payload comprises olaparib (AZD-2281), olaparib TOPARP- ｃA, rucapanib (AG014699, PF-01367338), nilapanib (niraparib), talaroparib (talazoparib) (BMN-673), veliparib (ABT-888), CEP 9722, E7016, BGB-290, 3-aminobenzamide, methoxyamine, CC-115, MSC2490484A, AZD6738, VX-970, AZD0156, GDC-0575, MK-8776, 2606368, AZD1775, belobitan (belotecan), CRLX101, irinotecan (irinotecan), LMP 400, LMP 776, NKTR-102, topotecan (topotecan), topotecan (epirubicin), epirubicin (epirubicin), etoposide (etoposide), tamiprodicin (BMN), BMN (BMN-673), trexaparib (BMN), BMN (BMN-673), vilazoparib-970), AZD-0156, GDC-0575, MK-76, 2606368, celadoxofenac (irinotecan), and topotecan (E), topotecan) in, Vorinostat (vorinostat), romidepsin (isotatax), cidentamine (chidamide), panobinostat (panobinostat) (Farydak), belinostat (belinostat) (PXD101), panobinostat (LBH589), valproic acid (in the form of magnesium valproate), moxetastat (MGCD0103), abexinostat (abexinostat) (PCI-7812458), entinostat (entinostat) (MS-275), SB939, renolostat (reminiostat) (4SC-201), givinostat (ITF) (ITF 7), quininostat (quisinostat) (JNJ-26481585), HBI-8000, trivirin (zetricin), dc-101, 2842, cumidin (r) 20046, cmantarcs (cmaxatrix), cmaxatrisentat (cgr) 20046, cmaxatrifostam-202, cmaxaster-202, cmaxtam (cgd), santopram), santopril (cgd-202), santopril-202, cmaxanthotac (cgd), santopril, cmaxanthotac-202, cmaxtam-202, tam, S-adenosylmethionine, JQ1, I-BET 151(GSK1210151A), I-BET 762(GSK525762), OTX-015, TEN-010, CPI-203, CPI-0610, olyrinone (olinone), LY294002, or an analog thereof.

8. The compound of claim 2, wherein the compound comprises at least one nuclear payload bound to a catalytic domain of poly (ADP-ribose) polymerase (PARP).

9. The compound of claim 2, wherein said catalytic domain comprises a conserved HYE motif.

10. The compound of any preceding claim, wherein the compound comprises at least one nuclear payload with an IC₅₀Less than about 500nM of poly (ADP-ribose) polymerase (PARP) binding.

11. The compound of claim 8, wherein said PARP comprises PARP-1 and/or PARP-2 or variants thereof.

12. The compound of any preceding claim, wherein the compound is of formula I or II:

A-(L-B)_m I

A-L-(B)_m II

wherein:

a is the core payload;

m is 1, 2 or 3;

each B is independently an epitope that targets a nuclear receptor; and is

Each L is independently a covalent bond or a linking moiety.

13. A compound of formula III, IV, V or VI, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

each R¹、R²、R³And R⁴Independently is-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C _2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-C(＝O)NR⁵R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more R¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_{1- 2}R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo or optionally with oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino _1-12Alkyl substitution;

each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

R⁹Is hydrogen or R²；

With the proviso that at least one R¹、R²、R³And R⁴is-L- (B)_m。

14. A compound of formula IIIB, IVA, VB, or VIA, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

15. The compound of any one of claims 12 to 14, wherein at least one L is a non-biocleavable linking moiety.

16. The compound of any one of claims 12 to 14, wherein at least one L is an acid-labile linking moiety.

17. The compound of any one of claims 12 to 16, wherein the linking moiety is alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene; wherein each alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene can optionally comprise an arylene, heteroarylene, cycloalkylene, or heterocycloalkylene group; and further wherein each alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, heteroalkynylene Aryl, cycloalkylene, or heterocycloalkylene are independently optionally substituted with one to five substituents independently selected from: oxo, halo, C_1-4Alkyl radical, C_1-4Alkoxy and C_1-4A haloalkyl group.

18. The compound of any one of claims 12 to 17, wherein the linking moiety has the formula:

-Y¹-(CH₂)_n'-Y²-(CH₂)_m'-Y³-

wherein:

Y¹、Y²and Y³Each of which is independently a bond, -NR¹¹-、-O-、-S(O)_0-2-、-NR¹¹C(O)-、-C(O)NR¹¹-、-NR¹¹S(O)₂-、-S(O)₂NR¹¹-、-CR¹²＝N-NR¹¹-、-NR¹¹-N＝CR¹²-, -C (O) -, arylene, heteroarylene, cycloalkylene or heterocycloalkylene; wherein each alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from: oxo, halo, C_1-4Alkyl radical, C_1-4Alkoxy and C_1-4A haloalkyl group;

each R¹¹Independently is C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl;

each R¹²Independently is C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; and is

n 'and m' are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

19. The compound of any one of claims 12 to 18, wherein at least one linking moiety comprises a hydrazone linkage.

20. The compound of any preceding claim, wherein the at least one nuclear receptor-targeting epitope is a nuclear steroid receptor-targeting epitope.

21. The compound of any preceding claim, wherein the at least one nuclear receptor-targeting epitope is independently selected from an estrogen receptor-targeting epitope, a glucocorticoid receptor-targeting epitope, a progesterone receptor-targeting epitope, or an androgen receptor-targeting epitope.

22. The compound of any preceding claim, wherein the at least one nuclear receptor-targeting epitope independently comprises an epitope derived from: an androgen receptor agonist, an androgen receptor antagonist, a Selective Androgen Receptor Modulator (SARM), an estrogen receptor agonist, an estrogen receptor antagonist, a Selective Estrogen Receptor Modulator (SERM), a glucocorticoid receptor antagonist, a glucocorticoid receptor agonist, a Selective Glucocorticoid Receptor Modulator (SGRM), a progesterone receptor antagonist, a progesterone receptor agonist, a Selective Progesterone Receptor Modulator (SPRM), or a combination thereof.

23. The compound of any preceding claim, wherein the at least one nuclear receptor-targeting epitope independently comprises an epitope derived from: estrogen, estetrol (estetrol), estriol (estriol), estrone, progesterone, enoboxate (enobosarm), bicalutamide (bicalutamide), apalutamide (apalutamide), testosterone (testosterone), dihydrotestosterone, estradiol (estradiol), flutamide (flutamide), nilutamide (nilutamide), enzalutamide (enzalutamide), tamoxifen (tamoxifen), toremifene (toremifene), raloxifene (raloxifene), bazedoxifene (bazedoxifene), speemifene (ospemifene), megestrol acetate (megestrol acetate), estramustine (estramustine), abiraterone (abiraterone), d-2941, BMS-564929, staphylin (tropine), or an analog thereof.

24. A compound according to any preceding claim, wherein at least one epitope targeting a nuclear receptor is capable of binding to the ligand binding domain of a nuclear steroid receptor.

25. The compound of claim 24, wherein the nuclear steroid receptor is an estrogen receptor, a glucocorticoid receptor, a progesterone receptor, or an androgen receptor.

26. The compound of any preceding claim, wherein the at least one nuclear receptor-targeting epitope is conjugated to IC₅₀Less than about 500nM or EC₅₀Less than about 1 μ M of nuclear steroid receptor binding.

27. The compound of any preceding claim, wherein the at least one nuclear receptor-targeting epitope is not a peptide, protein, nanoparticle, or antibody.

28. A compound as provided in table 1.

29. A pharmaceutical composition comprising a compound of any preceding claim and a pharmaceutically acceptable excipient.

30. A method for treating or preventing a condition capable of being ameliorated by the inhibition of PARP in an individual in need thereof, which comprises administering to the individual an effective amount of a compound or pharmaceutical composition according to any preceding claim, or a pharmaceutically acceptable salt or solvate thereof.

31. A method of treating or preventing cancer, the method comprising administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition of any preceding claim, or a pharmaceutically acceptable salt or solvate thereof.

32. The method of claim 30 or 31, wherein the administering comprises oral administration.

33. The method of any one of claims 30 to 32, further comprising administering an additional chemotherapeutic agent.

34. The method of claim 33, wherein the additional chemotherapeutic agent is cisplatin (cistatin) or etoposide, irinotecan, camptotheca (camptostar), topotecan, paclitaxel (paclitaxel), docetaxel (docetaxel), epothilones (epothilone), cloxacine (taxotere), tamoxifen, 5-fluorouracil, methotrexate (methoxtrexate), temozolomide (temozolomide), cyclophosphamide (cyclophosphamide), SCH 66336, R115777, L778123, BMS 214662, gefitinib (gefitinib), erlotinib hydrochloride (erlotinib), anti-EGFR antibodies, malinib (matrinib), introns, cytarabine (cytarabine), adriamycin (cytosin), cyclophosphamide (gemcitabine), gemcitabine (meprobamide), chlorambucil (meperidine), chlorambucil (mechlorethamine), mechlorethamine (mechlorethamine), mustard (mechlorethamine), mechlorethamine (mechlorethamine), an (mechlorethamine), mechlorethamine (TM), an (TM), or (TM) or (TM-E, a, Triethylenemelamine (triethylenemelamine), triethylenethiophosphoramide (triethyleneethiophosphoramine), busulfan (busulfan), carmustine (carmustine), lomustine (lomustine), streptozocin (streptozocin), dacarbazine (dacarbazine), floxuridine (floxuridine), cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate (fludarabine phosphate), pentostatin (pentostatin), vinblastine (vinblastine), vincristine (vincristine), vindesine (vindesine), bleomycin (bleomycin), doxorubicin, actinomycin D (dactinomycin), daunomycin (daunorubicin), epirubicin, idamycin, mithramycin (mithramycin), mycins (gentamycin), mitomycin (fumagiline), mitomycin (dihydrofenacin, mitomycin C), mitomycin (dihydrofenaminocycline, dihydrofenacin, dihydrofenaminocycline, dihydrofenacin, beta-C (dihydrofenacin), seco-17-D, dihydrofenaminocycline, dihydrofenacin, dihydrofenaminocycline, dihydrofenacin, dihydrofenaminocycline, dihydrofenapanomycin, dihydrofenaminocycline, dihydrofenapan, Fludroxymethyltestosterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone, leuprolide, flutamide, toremifene, sertraline, carboplatin, amsacrine, procarbazine, mitotane, levofloxacin, ketoprofen, levofloxacin, levo, Velcade (velcade), zerewirin (zevalin), arsenic trioxide (trisenox), hiloda (xeloda), vinorelbine (vinorelbine), porphine (porfimer), cetuximab (cetuximab), liposomes, Thiotepa (Thiotepa), Altretamine (Altretamine), melphalan, Trastuzumab (Trastuzumab), letrozole (lerozole), fulvestrant (fulvestrant), exemestane (exemestane), iformide (iformide), rituximab (itumab), C225, camphos (Campath), carboplatin, procarbazine, methyldiethylamine (mechlororethamine), cyclophosphamide, camptothecin (cetthecin), ifosfamide, melphalan, chlorambucil, dactinomycin (zeocin), netorubicin, bleomycin, netorubicin (VP 16), mitomycin (gentamycin), mitomycin (VP, mitomycin), mitomycin (gentamycin), mitomycin (16), mitomycin (gentamycin), mitomycin (gentamycin, gent, Gemcitabine, navelbine (navelbine), farnesyl-protein transferase (farnesyl-transferase) inhibitors, carboplatin (transplatinum), 5-fluorouracil, vincristine, vinblastine and methotrexate or analogues or derivatives thereof.

35. The method of any one of claims 31 to 34, further comprising administering radiation therapy to the patient.

36. The method of any one of claims 31-35, wherein the cancer is a BRCA-positive cancer.

37. The method of any one of claims 31-35, wherein the cancer is a solid tumor.

38. The method of any one of claims 31-35, wherein the cancer is a B cell affecting cancer.

39. The method of any one of claims 31 to 38, wherein the cancer is a blood cancer, a lung cancer, a breast cancer, a fallopian tube cancer, a brain cancer, a head and neck cancer, an esophageal cancer, an ovarian cancer, a pancreatic cancer, a peritoneal cancer, a prostate cancer, or a skin cancer.

40. The method of claim 39, wherein the cancer is liver cancer, melanoma, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, neuroblastoma, breast carcinoma, ovarian carcinoma, lung cancer, Wilms ' tumor, cervical cancer, testicular cancer, soft tissue sarcoma, chronic lymphocytic leukemia, primary macroglobulinemia, bladder cancer, chronic myelogenous leukemia, primary brain carcinoma, malignant melanoma, small cell lung carcinoma, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid cancer, malignant melanoma, choriocarcinoma, mycosis fungoides, head and neck carcinoma, osteogenic sarcoma, pancreatic carcinoma, acute myelogenous leukemia, hairy cell leukemia, malignant melanoma, cervical cancer, testicular cancer, soft tissue sarcoma, chronic lymphocytic leukemia, primary macroglobulinemia, bladder cancer, chronic myelogenous leukemia, primary brain carcinoma, malignant melanoma, small cell lung carcinoma, gastric cancer, colon cancer, malignant pancreatic insulinoma, choriocarcinoma, mycosis fungoid granuloma, head and neck carcinoma, osteogenic, Rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinoma, thyroid cancer, esophageal carcinoma, malignant hypercalcemia, cervical hyperplasia, renal cell carcinoma, endometrial cancer, polycythemia vera, essential thrombocythemia, adrenocortical carcinoma, skin cancer, or prostate cancer.

41. The method of claim 39, wherein the cancer is bladder cancer, breast cancer, fallopian tube cancer, ovarian cancer, prostate cancer, peritoneal cancer, testicular cancer, endometrial cancer, or uterine cancer.

42. A method of treating or preventing cancer that overexpresses androgen receptor, the method comprising administering to a subject in need thereof an effective amount of a compound or a pharmaceutically acceptable salt or solvate thereof comprising at least one nuclear payload and at least one epitope that targets androgen receptor.

43. The method of claim 42, wherein the cancer is prostate cancer, breast cancer, triple negative breast cancer, bladder cancer, or liver cancer.

44. The method of claim 42, wherein the androgen receptor targeted epitope comprises an androgen receptor agonist, a Selective Androgen Receptor Modulator (SARM), an androgen receptor antagonist, a Selective Estrogen Receptor Modulator (SERM), an estrogen receptor antagonist, a progestin, or an estrogen.

45. The method of claim 44, wherein the androgen receptor targeting epitope comprises enoboxate, bicalutamide, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, estramustine, ketoconazole (ketoconazole), abiraterone, daloluamide (daroluamide), or an analog thereof.

46. A method of treating or preventing cancer that overexpresses an estrogen and/or progesterone receptor, comprising administering to a subject in need thereof an effective amount of a compound or a pharmaceutically acceptable salt or solvate thereof comprising at least one nuclear payload and at least one epitope that targets an estrogen and/or progesterone receptor.

47. The method of claim 46, wherein the cancer is breast cancer, uterine cancer, or ovarian cancer.

48. A method of treating or preventing a cancer that overexpresses a glucocorticoid receptor, the method comprising administering to a subject in need thereof an effective amount of a compound or a pharmaceutically acceptable salt or solvate thereof comprising at least one nuclear payload and at least one epitope that targets the glucocorticoid receptor.

49. The method of claim 48, wherein the cancer is prostate cancer, breast cancer, uterine cancer, or ovarian cancer.

Background

The present disclosure relates to anti-cancer compounds derived from nuclear receptor binding agents, such as nuclear steroid receptor binding agents, to products containing the same, and to methods of use and preparation thereof.

PARP inhibitors are pharmacological agents that prevent DNA repair, cause cell death, and thus inhibit tumor growth. This mechanism of preventing cell growth causes significant antitumor activity in tumors with mutations in BRCA1, BRCA2 and PALB2, as these proteins are important for repair of double-stranded DNA breaks via the Homologous Recombination Repair (HRR) pathway. Normal cells divide less rapidly than tumors and do not carry the mutations BRCA1 or BRCA2, still leaving the HRR pathway intact, which allows it to survive in the face of PARP inhibition. In addition to catalyzing the inhibition of PARP, researchers at the National Cancer Institute in 2012 discovered another mechanism that drives PARP inhibitors to exert toxic effects in tumor cells. Their observation found that in addition to blocking the enzymatic activity of PARP, certain PARP inhibitors also have the ability to localize PARP proteins to sites of DNA damage, which is associated with the cytotoxicity of these inhibitors. This mode of action, termed "PARP trapping," is another mechanism by which such pharmacological agents prevent tumor growth and survival (Murai et al Cancer Research (2012)72 (21): 5588-99). Inhibitors of the PARP enzyme, such as olaparib, rucapanib, nilapanib, and talzopanib, have been approved for the treatment of breast cancer in patients with BRCA mutations and ovarian cancer. Several other inhibitors, such as verapamil (velaparib), are being clinically tested for breast, prostate and ovarian cancer. The use of PARP inhibitors is not without side effects and one of the major obstacles to the long-term use of PARP inhibitors is the rapid development and dose-dependent development of neutropenia. This requires dosing holidays and/or dose reductions in clinical practice, which compromises the ability to achieve maximum efficacy.

Disclosure of Invention

Provided herein are compounds comprising at least one nuclear payload and at least one epitope that targets a nuclear receptor. The compounds described herein are designed to bind nuclear receptors within cells and cause the compounds to accumulate in the nucleus of the cell along with their nuclear payloads.

Also provided herein are compounds comprising at least one nuclear payload and at least one epitope targeting a nuclear steroid receptor. The compounds described herein are designed to bind nuclear steroid receptors within cells (e.g., androgen receptors in the cytoplasmic matrix) and allow the compounds to accumulate in the nucleus of the cell along with their nuclear payloads. This approach allows for cell type selectivity of the compound, not only with enhanced potency, but also potentially with a higher therapeutic index.

In addition, the compounds described herein provide for targeted delivery of nuclear payloads. The compounds target and are located within tumor tissue. A compound comprising at least one epitope targeting a nuclear receptor, for example an epitope targeting a nuclear steroid receptor covalently attached to at least one nuclear payload, is transported into the nucleus of the cell such that the nuclear payload accumulates in the nucleus of the cell, thereby promoting tumor cell death. By doing so, the compounds described in the present disclosure may exhibit superior efficacy. In addition, the compounds described in the present disclosure will retain cells that do not express a specific nuclear steroid receptor by preferentially accumulating in nuclear receptor positive cells (e.g., steroid receptor positive cells), and thus reduce side effects.

In certain embodiments, compounds are provided that comprise at least one nuclear payload and at least one epitope that targets a nuclear receptor. In certain embodiments, when the compound comprises a nuclear payload and an epitope that targets a nuclear receptor, the epitope that targets a nuclear receptor is not a peptide, protein, nanoparticle, or antibody. In certain embodiments, when the compound comprises a nuclear payload and a nuclear receptor-targeting epitope, wherein the nuclear receptor-targeting epitope is an androgen receptor-targeting epitope or an estrogen receptor-targeting epitope, the nuclear payload is not doxorubicin (doxorubicin) or an analog thereof. In certain embodiments, when the compound comprises a nuclear payload and an epitope targeting a nuclear receptor, wherein the epitope targeting the nuclear receptor is an epitope targeting an androgen receptor or an epitope targeting an estrogen receptor, the nuclear payload is not a Histone Deacetylase (HDAC) -binding hydroxamic acid.

In certain embodiments, a compound is provided comprising at least one nuclear payload and at least one epitope that targets a nuclear receptor; with the proviso that when said compound comprises a nuclear payload and an epitope targeting a nuclear receptor:

An epitope that targets a nuclear receptor is not a peptide, protein, nanoparticle, or antibody; and is

When the epitope that targets the nuclear receptor is an epitope that targets the androgen receptor or an epitope that targets the estrogen receptor, the nuclear payload is not doxorubicin or an analog thereof, and is not a hydroxamic acid that binds Histone Deacetylase (HDAC).

In certain embodiments, a compound is provided comprising at least one nuclear payload that binds to a catalytic domain of poly (ADP-ribose) polymerase (PARP) and at least one epitope that targets a nuclear receptor. In certain embodiments, the at least one nuclear receptor-targeting epitope is a nuclear steroid receptor-targeting epitope.

In certain embodiments, a compound is provided comprising at least one nuclear payload that binds to poly (ADP-ribose) polymerase (PARP) (e.g., PARP-1 and/or PARP-2) and at least one epitope that targets a nuclear receptor. In certain embodiments, a compound is provided comprising at least one nuclear payload that binds to poly (ADP-ribose) polymerase (PARP) (e.g., PARP-1 and/or PARP-2) and at least one epitope that targets an estrogen receptor, an epitope that targets a glucocorticoid receptor, an epitope that targets a progesterone receptor, or an epitope that targets an androgen receptor.

In certain embodiments, a compound is provided comprising at least one nuclear payload that binds to poly (ADP-ribose) polymerase (PARP) (e.g., PARP-1 and/or PARP-2) and at least one epitope derived from a nuclear receptor targeted from: an androgen receptor agonist, an androgen receptor antagonist, a Selective Androgen Receptor Modulator (SARM), an estrogen receptor agonist, an estrogen receptor antagonist, a Selective Estrogen Receptor Modulator (SERM), a glucocorticoid receptor agonist, a glucocorticoid receptor antagonist, a Selective Glucocorticoid Receptor Modulator (SGRM), a progesterone receptor antagonist, a progesterone receptor agonist, a Selective Progesterone Receptor Modulator (SPRM), or a combination thereof.

In certain embodiments, a compound is provided comprising at least one nuclear payload that binds to poly (ADP-ribose) polymerase (PARP) (e.g., PARP-1 and/or PARP-2) and at least one epitope targeted to a nuclear steroid receptor derived from: an androgen receptor agonist, an androgen receptor antagonist, a Selective Androgen Receptor Modulator (SARM), an estrogen receptor agonist, an estrogen receptor antagonist, a progesterone receptor agonist, a Selective Progesterone Receptor Modulator (SPRM), or a combination thereof.

In certain embodiments, a compound is provided comprising at least one nuclear payload that binds to poly (ADP-ribose) polymerase (PARP) (e.g., PARP-1 and/or PARP-2) and at least one epitope derived from a nuclear receptor targeted from: estrogen, estetrol (estetrol), estriol (estriol), estrone, progesterone, enoboxate (enobosarm), bicalutamide (bicalutamide), apalutamide (apalutamide), testosterone (testosterone), dihydrotestosterone, estradiol (estradiol), flutamide (flutamide), nilutamide (nilutamide), enzalutamide (enzalutamide), tamoxifen (tamoxifen), toremifene (toremifene), raloxifene (raloxifene), bazedoxifene (bazedoxifene), speemifene (ospemifene), megestrol acetate (megestrol acetate), estramustine (estramustine), abiraterone (abiraterone), d-2941, BMS-564929, staphylin (tropine), or an analog thereof.

Also provided are compounds of formula I or II, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-(L-B)_m I

A-L-(B)_m II

wherein:

a is the core payload;

m is 1, 2 or 3;

each B is independently an epitope that targets a nuclear receptor; and is

Each L is independently a covalent bond or a linking moiety.

In certain embodiments of formula I or II, when m is 1, the epitope that targets the nuclear receptor is not a peptide, protein, nanoparticle, or antibody. In certain embodiments of formulas I or II, when m is 1 and B is an epitope that targets the androgen receptor or an epitope that targets the estrogen receptor, then a is not doxorubicin or an analog thereof. In certain embodiments of formulas I or II, when m is 1 and B is an epitope that targets the androgen receptor or an epitope that targets the estrogen receptor, then a is not a hydroxamic acid that binds Histone Deacetylase (HDAC).

Also provided are compounds of formula III, IV, V or VI, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

each R¹、R²、R³And R⁴Independently is-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more (e.g., 1 to 5 or 1 to 3) R ¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted by one or more (e.g. 1 to 5 or 1 to 3) halo groups or by oxo, halo, hydroxy or amino groups_1-12Alkyl substitution;

each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino _1-12Alkyl substitution; and is

R⁹Is hydrogen or R²；

With the proviso that at least one R¹、R²、R³And R⁴is-L- (B)_m。

Also provided are compounds of formula IIIA, IV, VA or VI, or stereoisomers, mixtures, hydrates, solvates, isotopically enriched analogs, or pharmaceutically acceptable salts thereof:

wherein:

R¹、R²、R³and R⁴Is independently-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more (e.g., 1 to 5 or 1 to 3) R¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted by one or more (e.g. 1 to 5 or 1 to 3) halo groups or by oxo, halo, hydroxy or amino groups _1-12Alkyl substitution;

each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

with the proviso that R¹、R²、R³And R⁴is-L- (B)_m。

Also provided are compounds of formula IIIB, IVA, VB or VIA, or stereoisomers, mixtures, hydrates, solvates, isotopically enriched analogs, or pharmaceutically acceptable salts thereof:

wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

Also provided are compounds of table 1, or stereoisomers, mixtures of stereoisomers, hydrates, solvates, isotopically enriched analogs, or pharmaceutically acceptable salts thereof.

Also provided is a composition comprising a compound as described herein, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

Also provided is a method of treating or preventing cancer comprising administering to an individual in need thereof an effective amount of a compound or composition as described herein. The cancer can be hematologic cancer, lung cancer, breast cancer, fallopian tube cancer, brain cancer, head and neck cancer, esophageal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, prostate cancer, or skin cancer, such as, but not limited to, liver cancer, melanoma, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, neuroblastoma, breast carcinoma, ovarian carcinoma, lung carcinoma, Wilms ' tumor, cervical cancer, testicular cancer, soft tissue sarcoma, chronic lymphocytic leukemia, primary macroglobulinemia, bladder cancer, chronic myelogenous leukemia, primary brain carcinoma, malignant melanoma, small cell lung carcinoma, gastric cancer, colon cancer, malignant pancreatic carcinoma, malignant carcinoid cancer, malignant melanoma, choriocarcinoma, mycosis fungoides granulomatosis, mycosis, ovarian cancer, melanoma, and skin cancer, Head and neck carcinomas, osteogenic sarcomas, pancreatic carcinomas, acute myelogenous leukemia, hairy cell leukemia, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid cancer, esophageal carcinoma, malignant hypercalcemia, cervical hyperplasia, renal cell carcinoma, endometrial cancer, polycythemia vera, essential thrombocythemia, adrenocortical carcinoma, skin cancer, or prostate carcinoma.

Also provided is a method of treating or preventing bladder cancer, breast cancer, fallopian tube cancer, ovarian cancer, prostate cancer, peritoneal cancer, testicular cancer, endometrial cancer, or uterine cancer, comprising administering to a subject in need thereof an effective amount of a compound or composition as described herein, or a pharmaceutically acceptable salt or solvate thereof.

Also provided is a method of treating or preventing cancer comprising administering an effective amount of a compound, or a pharmaceutically acceptable salt or solvate thereof, comprising at least one nuclear payload and at least one nuclear receptor targeting epitope comprising: testosterone, testosterone esters (e.g., testosterone enanthate, testosterone propionate, testosterone cypionate, etc. or analogs thereof), enoboxate, BMS-564929, PS178990, LGD-4033 (Ligandrol), LGD-2941, AC-262,356, JNJ-28330835, JNJ-37654032, JNJ-26146900, LGD-2226, LGD-3303, LGD-121071, LG-120907, S-40503, S-23, RAD-140, acetyl behenamide (acetylthiolutimide), adatansine (andrarine) (S-4), CHELG-121071, TFM-4AS-1, YK-11, MK-0773(PF-05314882), GSK2849466, GSK2881078, GSK8698, GSK4336, ACP-105, 701, CHETT 73, 1- (2-hydroxy-243-propionyl-2-phenoxy) -methyl propionyl-2-propionyloxy-indoline-20144 (M J-2014-4. multidrug-2014-4. multidrug-20144, 57(6),2462-71), norgestimate (anordrin), bazedoxifene (bazedoxifene), bropalestrrol (acnestrrol), clomiphene (clomifene) (cimid), cyclofenil (cycloofenil) (Sexovid), lasofoxifene (Fablyn), oxymethofene (ormeloxifene) (mentron), novice (Novex), novice-DS, Sevista (Sevista), ospemifene (Osphena), deaminohydroxy toremifene (deaminohydroxytoremifene)), valnoxifene (Evista), Nolvadex (Nolvadex), toremifene (toremifene) (fattone (falciprofen); 4-chloro-tamoxifen), acobifene (acolbifene), aficoxifene (afimoxifene) (4-hydroxy tamoxifen; metabolites of tamoxifen), elapsin (elacetrart), enclomifene (enclomifene) ((E) -clomiphene), tamoxifen (endoxifen) (4-hydroxy-N-demethyltamoxifen; metabolites of tamoxifen), dactylclomiphene (zuclomiphene) ((Z) -clomiphene), bazedoxifene (bazedoxifene), arzoxifene (arzoxifene), brisket (brilanestrant), clomiphene (clomiphene N-oxide); metabolites of clomiphene), droloxifene (droloxifene) (3-hydroxytamoxifene), etacetin (etastil), fispemifene (fispemifene), GW-7604 (4-hydroxyetacetin), idoxifene (idoxifene) (pyrrolidinyl-4-iodotamoxifen), levomethoxifene (levormeloxifene) ((L) -oxymetaxifene), miloxifene (miproxifene), naphthoxidine (nafoxidine), nifemifene (nitromifene) (CI-628), panomoxifene (panomoxifene), pipindoxifene (pipindoxifene) (ERA-indoxacarb), troxifene (trioxifene), raloxifene (raloxifene), 117018, onafenasone (octopiroxicam), ketoprofen (pyridoxine) (vera-indoxifene 768), valaciflufenacet (fentefuran-768), valacifenethazine (arvatine, valacil-r), valacifene (fentezine), valacil-768, valacifenethazine (arvatine), valacil, valacifene (naltrexone), valacine (naltrexone), naltrexone (e) (naltrexone), naltrexone (r (e) (r), naltrexone (e, naltrexone), naltrexone (r), naltrexone (r), naltrexone), sopranil (asprisnil) (J867), mifepristone (mifepristone), torasemide (telapristone) (CDB-4124, Proellex, progentia) or analogs thereof. In certain embodiments, the nuclear payload comprises a PARP inhibitor.

Also provided is a method of treating or preventing cancer that overexpresses androgen receptor, the method comprising administering to a human in need thereof an effective amount of a compound or a pharmaceutically acceptable salt or solvate thereof, comprising at least one nuclear payload and at least one epitope that targets androgen receptor. In certain embodiments, the cancer is prostate cancer, breast cancer, triple negative breast cancer, bladder cancer, or liver cancer. In certain embodiments, the epitope that targets an androgen receptor comprises an androgen receptor agonist, a Selective Androgen Receptor Modulator (SARM), an androgen receptor antagonist, a Selective Estrogen Receptor Modulator (SERM), an estrogen receptor antagonist, a progestin, or an estrogen. In certain embodiments, the epitope that targets the androgen receptor comprises enoboxate, bicalutamide, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, estramustine, ketoconazole (ketoconazole), abiraterone, daloluamide (daroluamide), or an analog thereof. In certain embodiments, the epitope that targets the androgen receptor comprises enoboxate, bicalutamide, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, estramustine, ketoconazole, abiraterone or an analog thereof. In certain embodiments, the nuclear payload comprises a PARP inhibitor.

Also provided is a method of treating or preventing cancer that overexpresses estrogen and/or progesterone receptors, comprising administering to a subject in need thereof an effective amount of a compound, or a pharmaceutically acceptable salt or solvate thereof, comprising at least one nuclear payload and at least one epitope that targets estrogen and/or progesterone receptors. In certain embodiments, the cancer is breast cancer, uterine cancer, or ovarian cancer. In certain embodiments, the nuclear payload comprises a PARP inhibitor.

Also provided is a method of treating or preventing a cancer that overexpresses a glucocorticoid receptor, the method comprising administering to a subject in need thereof an effective amount of a compound comprising at least one nuclear payload and at least one epitope that targets the glucocorticoid receptor, or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, the cancer is prostate cancer, breast cancer, uterine cancer, or ovarian cancer. In certain embodiments, the nuclear payload comprises a PARP inhibitor.

Also provided is a method of treating or preventing cancer, the method comprising administering to a subject in need thereof an effective amount of a compound or composition as described herein, or a pharmaceutically acceptable salt or solvate thereof, and an additional chemotherapeutic agent.

Also provided is a method of treating or preventing a condition ameliorated by the inhibition of PARP in a subject in need thereof, which comprises administering to the subject an effective amount of a compound or composition according to any preceding claim, or a pharmaceutically acceptable salt thereof.

Detailed Description

The following description sets forth illustrative embodiments of the present technology. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments.

1. Definition of

As used in this specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

The term "about" refers to a variation of ± 1%, ± 3%, ± 5% or ± 10% of the specified value. For example, "about 50" may include a range of 45 to 55 in some embodiments. For a range of integers, the term "about" can include one or two integers at each end of the range that are greater than and/or less than the recited integer. Unless otherwise indicated herein, the term "about" is intended to include values, e.g., weight percentages, close to the recited range that are equivalent in terms of functionality of the individual ingredient, composition or embodiment. In addition, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the compound" includes a plurality of such compounds, and reference to "the assay" includes reference to one or more compounds and equivalents thereof known to those skilled in the art.

"alkyl" refers to a non-branched or branched saturated hydrocarbon chain. As used herein, an alkyl group has from 1 to 10 carbon atoms (i.e., C)_1-10Alkyl), 1 to 8 carbon atoms (i.e., C)_1-8Alkyl), 1 to 6 carbon atoms (i.e., C)_1-6Alkyl) or 1 to 4 carbon atoms (i.e. C)_1-4Alkyl groups). Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methylpentyl. When an alkyl residue having a particular number of carbons is named by chemical name or identified by molecular formula, all positional isomers having the number of carbons can be encompassed; thus, for example, "butyl" includes n-butyl (i.e., - (CH)₂)₃CH₃) Sec-butyl (i.e., -CH (CH))₃)CH₂CH₃) Isobutyl (i.e., -CH)₂CH(CH₃)₂) And tert-butyl (i.e., -C (CH)₃)₃) (ii) a And "propyl" includes n-propyl (i.e., - (CH)₂)₂CH₃) And isopropyl (i.e., -CH (CH)₃)₂)。

"haloalkyl" refers to a non-branched or branched alkyl radical as defined above wherein one or more hydrogen atoms are replaced by a halogen. For example, where a residue is substituted with more than one halogen, it may be referred to by using a prefix corresponding to the number of halogen moieties attached. Dihaloalkyl and trihaloalkyl refer to alkyl substituted with two ("di") or three ("tri") halo groups, which may be, but are not necessarily, the same halo. Examples of haloalkyl groups include difluoromethyl (-CHF) ₂) And trifluoromethyl (-CF)₃)。

"heteroalkyl" refers to an alkyl group in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with the same or different heteroatom groups. The term "heteroalkyl" includes unbranched or branched saturated chains having carbon and heteroatoms. For example, 1,2, or 3 carbon atoms may be independently replaced by the same or different heteroatom groups. Heteroatom groups include, but are not limited to, -NH-, -O-, -S-, -S (O) -, -S (O)₂-and the like. As used herein, heteroalkyl groups include 1 to 8 carbon atoms, or 1 to 4 carbon atoms; and 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom.

"alkoxy" means a group "-O-alkyl".

"alkenyl" means containing at least one carbon-carbon double bond and having from 2 to 20 carbon atoms (i.e., C)_2-20Alkenyl), 2 to 8 carbon atoms (i.e., C)_2-8Alkenyl), 2 to 6 carbon atoms (i.e., C)_2-6Alkenyl) or 2 to 4 carbon atoms (i.e. C)_2-4Alkenyl) alkyl groups. Examples of alkenyl groups include, for example, ethenyl, propenyl, butadienyl (including 1, 2-butadienyl and 1, 3-butadienyl).

"alkynyl" means containing at least one carbon-carbon triple bond and having 2 to 20 carbon atoms (i.e., C)_2-20Alkynyl), 2 to 8 carbon atoms (i.e., C) _2-8Alkynyl), 2 to 6 carbon atoms (i.e., C)_2-6Alkynyl) or 2 to 4 carbon atoms (i.e., C)_2-4Alkynyl) alkyl. The term "alkynyl" also includes those groups having one triple bond and one double bond.

"alkoxy" refers to the group "alkyl-O-". Examples of the alkoxy group include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy and 1, 2-dimethylbutoxy.

"alkoxyalkyl" refers to the group "alkyl-O-alkyl".

"amino" means a radical-NR^yR^zWherein R is^yAnd R^zIndependently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroalkyl, or heteroaryl; each of which may be as described hereinDefinitions are optionally substituted.

"aryl" refers to an aromatic carbocyclic group having a single ring (e.g., monocyclic) or multiple rings (e.g., bicyclic or tricyclic), including fused systems. As used herein, an aryl group has from 6 to 20 ring carbon atoms (i.e., C)_6-20Aryl), 6 to 12 carbon ring atoms (i.e., C)_6-12Aryl) or 6 to 10 carbon ring atoms (i.e. C)_6-10Aryl). Examples of aryl groups include, for example, phenyl, naphthyl, fluorenyl, and anthracenyl. However, aryl does not encompass heteroaryl groups as defined below or overlap with heteroaryl groups in any way. If one or more aryl groups are fused to a heteroaryl group, the resulting ring system is a heteroaryl group. If one or more aryl groups are fused to a heterocyclyl group, the resulting ring system is a heterocyclyl group.

"cycloalkyl" refers to saturated or partially unsaturated cyclic alkyl groups having a single ring or multiple rings, including fused, bridged, and spiro ring systems. The term "cycloalkyl" includes cycloalkenyl (i.e., cyclic groups having at least one double bond) and cyclic groups having at least one sp³A carbocyclic fused ring system of carbon atoms (i.e., at least one non-aromatic ring). As used herein, cycloalkyl groups have from 3 to 20 ring carbon atoms (i.e., C)_3-20Cycloalkyl), 3 to 12 ring carbon atoms (i.e., C)_3-12Cycloalkyl), 3 to 10 ring carbon atoms (i.e., C)_3-10Cycloalkyl), 3 to 8 ring carbon atoms (i.e., C)_3-8Cycloalkyl) or 3 to 6 ring carbon atoms (i.e. C)_3-6Cycloalkyl groups). Monocyclic groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Furthermore, the term cycloalkyl is intended to encompass any non-aromatic ring that may be fused to an aryl ring, regardless of the attachment to the rest of the molecule. Still further, cycloalkyl groups, when two substitution positions are on the same carbon atom, also include "spirocycloalkyl".

"heteroaryl" refers to an aromatic group having a single ring, multiple rings, or multiple fused rings, wherein one or more ring heteroatoms are independently selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl includes 1 to 20 ring carbon atoms (i.e., C) _1-20Heteroaryl), 3 to 12 ring carbon atoms (i.e., C)_3-12Heteroaryl), or 3 to 8 carbon ring atoms (i.e., C)_3-8Heteroaryl radical) And 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen, and sulfur. In certain instances, heteroaryl includes a 5-10 membered ring system, a 5-7 membered ring system, or a 5-6 membered ring system, each independently having 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen, and sulfur. Any aromatic ring having single or multiple fused rings and containing at least one heteroatom is considered a heteroaryl group regardless of the attachment to the rest of the molecule (i.e., through either of the fused rings). Heteroaryl does not encompass aryl as defined above or overlap with aryl.

"Heterocyclyl" refers to a saturated or partially unsaturated cyclic alkyl group having one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. The term "heterocyclyl" includes heterocycloalkenyl (i.e., a heterocyclyl having at least one double bond), bridged heterocyclyls, fused heterocyclyls, and spiroheterocyclyls. A heterocyclyl group may be a single ring or multiple rings, where multiple rings may be fused, bridged or spiro, and may contain one or more (e.g. 1 to 3) oxo (═ O) or N-oxide (-O) ^-) And (4) partial. Any non-aromatic ring containing at least one heteroatom is considered to be heterocyclyl, regardless of attachment (i.e., may be bound through a carbon atom or heteroatom). Furthermore, the term heterocyclyl is intended to encompass any non-aromatic ring containing at least one heteroatom, which ring may be fused to an aryl or heteroaryl ring, regardless of the attachment to the rest of the molecule. As used herein, a heterocyclyl has 2 to 20 ring carbon atoms (i.e., C)_2-20Heterocyclyl), 2 to 12 ring carbon atoms (i.e. C)_2-12Heterocyclyl), 2 to 10 ring carbon atoms (i.e. C)_2-10Heterocyclyl), 2 to 8 ring carbon atoms (i.e. C)_2-8Heterocyclyl), 3 to 12 ring carbon atoms (i.e. C)_3-12Heterocyclyl), 3 to 8 ring carbon atoms (i.e. C)_3-8Heterocyclyl) or 3 to 6 ring carbon atoms (i.e. C)_3-6A heterocyclic group); having 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, sulfur, or oxygen. When in the same carbon atomThe term "heterocyclyl" when two substitution positions are indicated above also includes "spiroheterocyclyl".

"alkylene" refers to a divalent alkyl group as defined above. As used herein, an alkylene group has from 1 to 10 carbon atoms (i.e., C)_1-10Alkylene) of 1 to 8 carbon atoms (i.e. C) _1-8Alkylene) of 1 to 6 carbon atoms (i.e. C)_1-6Alkylene) or 1 to 4 carbon atoms (i.e. C)_1-4Alkylene).

"Heteroalkylene" refers to an alkylene group in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced by the same or different heteroatom groups. The term "heteroalkylene" includes unbranched or branched saturated chains having carbon and heteroatoms. For example, 1, 2, or 3 carbon atoms may be independently replaced by the same or different heteroatom groups. Heteroatom groups include, but are not limited to, -NH-, -O-, -S-, -S (O) -, -S (O)₂-and the like. As used herein, heteroalkylene groups include 1 to 8 carbon atoms or 1 to 4 carbon atoms; and 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom.

"alkenylene" means a compound containing at least one carbon-carbon double bond and having from 2 to 8 carbon atoms (i.e., C)_2-8Alkenylene), 2 to 6 carbon atoms (i.e., C)_2-6Alkenylene) or 2 to 4 carbon atoms (i.e. C)_2-4Alkenylene) group.

"heteroalkenylene" means a compound containing at least one carbon-carbon double bond and having 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms; and a heteroalkylene of 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom. The term "heteroalkynyl" also includes those groups having one triple bond and one double bond.

"Alkynylene" means a compound containing at least one carbon-carbon triple bond and having from 2 to 8 carbon atoms (i.e., C)_2-8Alkynylene), 2 to 6 carbon atoms (i.e., C)_2-6Alkynylene) or 2 to 4 carbon atoms (i.e., C)_2-4Alkynylene) is used. The term "alkynyl" also includes those groups having one triple bond and one double bond.

"heteroalkynylene" means a compound containing at least one carbon-carbon triple bond and having 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms; and a heteroalkylene of 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom. The term "heteroalkynyl" also includes those groups having one triple bond and one double bond.

"cycloalkylene" refers to divalent saturated or partially unsaturated cyclic alkyl groups having a single ring or multiple rings, including fused, bridged, and spiro ring systems. The term "cycloalkylene" includes cycloalkenylene (i.e., a cyclic group having at least one double bond). As used herein, cycloalkenylene has from 3 to 10 ring carbon atoms (i.e., C)_3-10Cycloalkyl), 3 to 8 ring carbon atoms (i.e., C)_3-8Cycloalkyl) or 3 to 6 ring carbon atoms (i.e. C)_3-6Cycloalkyl groups).

"heterocycloalkylene" refers to a cycloalkylene group in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with the same or different heteroatom group. For example, 1, 2, or 3 carbon atoms may be independently replaced by the same or different heteroatom groups. Heteroatom groups include, but are not limited to, -NH-, -O-, -S-, -S (O) -, -S (O) ₂-and the like. As used herein, heterocycloalkylene includes 1 to 9 carbon atoms or 1 to 4 carbon atoms; and 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom.

"oxo" refers to ═ O.

"halogen" or "halo" includes fluorine, chlorine, bromine and iodine.

The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur. The term "optionally substituted" means that any one or more hydrogen atoms on the indicated atom or group may or may not be replaced by a moiety other than hydrogen.

Stereoisomers, mixtures of stereoisomers, tautomers, hydrates, solvates, isotopically enriched analogs, and pharmaceutically acceptable salts of the compounds described herein are also provided.

The compounds disclosed herein, or pharmaceutically acceptable salts thereof, may include asymmetric centers and, thus, may give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined in absolute stereochemistry as (R) -or (S) -or for amino acids as (D) -or (L) -. The present disclosure is intended to include all such possible isomers, as well as racemic and optically pure forms thereof. The optically active (+) and (-), (R) -and (S) -, or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents, or decomposed using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing/separating the individual enantiomers include chiral synthesis from suitable optically pure precursors or decomposition of the racemate (or the racemate of a salt or derivative) using, for example, chiral High Pressure Liquid Chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers.

"stereoisomers" refers to compounds that are composed of the same atoms bonded by the same bond, but have different three-dimensional structures that are not interchangeable. The present disclosure encompasses various stereoisomers and mixtures thereof, and includes "enantiomers," which refers to two stereoisomers whose molecules are mirror images of each other that are not superimposable; and "diastereomers," which are stereoisomers having at least two asymmetric atoms, but which are not mirror images of each other. Accordingly, the present invention encompasses all stereoisomers (e.g., geometric isomers, optical isomers, etc.) of the inventive compounds (including compounds that are salts, solvates, and hydrates of the compounds), such as isomers that may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of an asymmetric carbon), rotameric forms, atropisomers, and diastereomeric forms.

Mixtures of diastereomers may be separated into their individual diastereomers on the basis of their physicochemical differences by methods well known to those skilled in the art, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture with an appropriate optically active compound (e.g., a chiral auxiliary, such as a chiral alcohol or a morser's acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. Furthermore, some of the compounds of formula (1) may be atropisomers and are considered part of the present disclosure. Stereoisomers may also be separated by using chiral HPLC.

Some compounds exist in tautomeric forms. Tautomers are in equilibrium with each other. For example, an amide-containing compound can exist in equilibrium with an imide tautomer. Regardless of which tautomers are shown, and regardless of the nature of the balance between tautomers, a person of ordinary skill in the art would understand that compounds encompass both amide and imide tautomers. Thus, an amide-containing compound is understood to include an imide tautomer thereof. Likewise, an imide acid-containing compound is understood to include amide tautomers thereof.

The term "hydrate" refers to a complex formed by combining a compound described herein with water.

"solvate" refers to the association or complexation of one or more solvent molecules with a compound of the present disclosure. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and ethanolamine.

Any compound or structure given herein is also intended to mean the compound in unlabeled form as well as in isotopically labeled form. These forms of the compound may also be referred to as "isotopically enriched analogs". Isotopically labeled compounds have the structure depicted herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as ²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、³⁶Cl、¹²³I and¹²⁵I. various isotopically-labeled compounds of the present disclosure, e.g., thereofIn which a radioactive isotope (e.g. is incorporated)³H、¹³C and¹⁴C) those of (a). Such isotopically labeled compounds are useful in metabolic studies, reaction kinetic studies, detection, or imaging techniques, such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), including drug or basal tissue distribution assays, or in the treatment of patients with radiation. Such compounds may exhibit increased metabolic resistance when administered to a mammal, particularly a human, and are therefore useful for extending the half-life of any compound. Such compounds are synthesized by means well known in the art, for example, by employing starting materials in which one or more hydrogens have been replaced with deuterium.

Certain compounds disclosed herein contain one or more ionizable groups (groups from which protons can be removed (e.g., -COOH) or added (e.g., amine) or which can be quaternized (e.g., amine)). All possible ionic forms of such molecules and salts thereof are intended to be included individually in the disclosure herein. With respect to the salts of the compounds described herein, one of ordinary skill in the art can select suitable ones from a variety of available counterions. In certain applications, the selection of a given anion or cation to make a salt may result in an increase or decrease in the solubility of the salt.

As used herein, the term "non-biocleavable linking moiety" refers to a linking moiety that does not readily hydrolyze under physiological conditions. As used herein, the term "biocleavable linking moiety" refers to a linking moiety that is readily hydrolyzed under physiological conditions. In certain embodiments, at least one linking moiety is hydrolyzed under intracellular conditions (e.g., low pH).

As used herein, the term "cancer" refers to a class of mammalian diseases characterized by uncontrolled cell growth. The term "cancer" may be used interchangeably with the terms "tumor", "solid tumor", "malignant disease", "hyperproliferative" and "neoplasm". Cancer includes all types of hyperproliferative growth, cell proliferative growth, neoplastic growth, cancerous growth or carcinogenic process, metastatic tissue or malignantly transformed cells, tissues or organs, regardless of histopathological type or invasive stage. Illustrative examples include lung, prostate, head and neck, breast and colorectal cancers, melanomas and gliomas (e.g., high grade gliomas, including glioblastoma multiforme (GBM), which is the most common and most fatal malignant primary brain tumor in adults).

The phrase "solid tumor" includes, for example, lung cancer, head and neck cancer, brain cancer, oral cancer, colorectal cancer, breast cancer, prostate cancer, pancreatic cancer, and liver cancer. Other types of solid tumors are named for the specific cells that form them, e.g., sarcomas formed from connective tissue cells (e.g., osteochondral, adipose), carcinomas formed from epithelial tissue cells (e.g., breast, colon, pancreas), and lymphomas formed from lymphoid tissue cells (e.g., lymph nodes, spleen, thymus). Regardless of the nomenclature, treatment of all types of solid tumors is within the scope of the present disclosure.

"chemotherapeutic agent" refers to any substance capable of reducing or preventing the growth, proliferation or spread of cancer cells, cancer cell populations, tumors or other malignant tissues. The term is also intended to encompass radiotherapy or any anti-tumour or anti-cancer agent.

As used herein, "treatment" is a method for obtaining beneficial or desired results, including clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, remission and/or reduction in the extent of symptoms and/or prevention of worsening of symptoms associated with the disease or condition. In one variation, beneficial or desired clinical results include, but are not limited to, symptom relief and/or reduction in the extent of symptoms and/or prevention of worsening of symptoms associated with cognitive, psychiatric, neurotransmitter-mediated conditions and/or neuronal disorders. Preferably, treatment of a disease or condition with a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, produces no, or fewer, side effects than those associated with currently available therapies for the disease or condition, and/or improves the quality of life of the individual.

The term "inhibit" refers to slowing, stopping or reversing the progression or worsening of a disease, infection, condition or cell population. For example, inhibition may be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99% as compared to development or worsening that occurs in the absence of treatment or contact.

As used herein, "combination therapy" means therapy that includes two or more different compounds. Thus, in one aspect, combination therapies comprising a compound detailed herein and another compound are provided. In some variations, the combination therapy optionally includes one or more pharmaceutically acceptable carriers or excipients, non-pharmaceutically active compounds, and/or inert substances. In various embodiments, treatment with a combination therapy can produce additive or even synergistic (e.g., greater than additive) results as compared to administration of a single compound of the disclosure alone. In some embodiments, lower amounts of each compound are used as part of a combination therapy as compared to the amounts typically used for individual therapies. Preferably, the same or greater therapeutic benefit is obtained using combination therapy as compared to the use of any individual compound alone. In some embodiments, fewer amounts (e.g., lower doses or less frequent dosing) than are typically used for individual compounds or therapies are used in combination therapy to achieve the same or greater therapeutic benefit. Preferably, the use of a small amount of a compound reduces the number, severity, frequency and/or duration of one or more side effects associated with the compound.

As used herein, the term "effective amount" means that the amount of a compound of the present disclosure in combination with its efficacy and toxicity parameters and should be effective in a given treatment modality based on the knowledge of the practitioner of practice. It is understood in the art that an effective amount may be divided into one or more doses, i.e., a single dose or multiple doses may be required to achieve a desired therapeutic endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and it is contemplated that a single agent may be given in an effective amount to achieve or obtain the desired or beneficial result if combined with one or more other agents. Suitable dosages of any of the co-administered compounds may optionally be reduced by a combined effect (e.g., additive or synergistic effect) of the compounds.

As used herein, the term "agonist" refers to a compound whose presence results in the biological activity of a protein that is the same as the biological activity resulting from the presence of a naturally occurring ligand of a protein, such as PARP.

As used herein, the term "partial agonist" refers to a compound that is present such that the biological activity of the protein is of the same type, but in a lower amount, as the biological activity of the protein caused by the presence of the naturally occurring ligand.

As used herein, the term "antagonist" or "inhibitor" refers to a compound whose presence causes a decrease in the amount of biological activity of a protein. In certain embodiments, the presence of the antagonist results in complete inhibition of the biological activity of the protein, e.g., poly (ADP-ribose) polymerase (PARP).

As used herein, IC₅₀Refers to the amount, concentration or dose of a particular test compound that achieves 50% inhibition of the maximal response, e.g., modulation of PARP in an assay that measures such response.

As used herein, EC₅₀Refers to a dose, concentration, or amount of a particular test compound that elicits a dose-dependent response at 50% of the maximum expression of a particular response that is elicited, caused, or enhanced by a particular test compound.

As used herein, the term "cancer" refers to an abnormal growth of cells that tends to proliferate in an uncontrolled manner and in some cases tends to metastasize (spread). Cancer types include, but are not limited to, solid tumors (e.g., bladder, intestinal, brain, breast, endometrial, heart, kidney, lung, lymphoid tissue (lymphoma), ovarian, pancreatic or other endocrine organ (thyroid) tumors), prostate, skin (melanoma), or hematological tumors (e.g., leukemia).

As used herein, the term "carrier" refers to a relatively non-toxic compound or agent that facilitates incorporation of the compound into a cell or tissue.

As used herein, "unit dosage form" refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The unit dosage form may contain monotherapy or combination therapy.

As used herein, the term "controlled release" refers to a drug-containing formulation or fraction thereof in which the release of the drug is not immediate, i.e., by means of a "controlled release" formulation, the dosage administered does not result in immediate release of the drug into the absorption cell. The term encompasses long acting formulations designed to gradually release a pharmaceutical compound over an extended period of time. Controlled release formulations may include a variety of drug delivery systems, typically involving mixing a drug compound with a carrier, polymer, or other compound having a desired release profile (e.g., pH-dependent or pH-independent solubility, varying degrees of water solubility, etc.), and formulating the mixture according to the desired delivery route (e.g., coated capsule, implantable reservoir, biodegradable capsule containing injectable solution, etc.).

As used herein, "pharmaceutically acceptable" or "pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effect or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The pharmaceutically acceptable carrier or excipient preferably meets the required standards for toxicological and manufacturing testing and/or is included in the Inactive Ingredient Guide (Inactive Ingredient Guide) as planned by the U.S. food and Drug administration.

"pharmaceutically acceptable salts" are those salts that retain at least some of the biological activity of the free (non-salt) compound and that can be administered to an individual as a medicament or drug product. Such salts include, for example: (1) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like); or an acid addition salt formed from an organic acid (e.g., acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid, etc.); (2) salts formed when an acidic proton present in the parent compound is replaced with a metal ion (e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion); or a salt complexed with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. Other examples of pharmaceutically acceptable Salts include Berge et al, in Pharmaceutical Salts (Pharmaceutical Salts), in the journal of the Pharmaceutical sciences (J.pharm.Sci.) in 1977, month 1; 66(1) salts listed in 1-19. Pharmaceutically acceptable salts can be prepared in situ by manufacturing processes or by separately reacting a purified compound of the disclosure in its free acid or base form with a suitable organic or inorganic base or acid, respectively, and isolating the salt thus formed during subsequent purification. It is to be understood that reference to a pharmaceutically acceptable salt includes solvent addition forms or crystal forms thereof, especially solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and are typically formed during crystallization. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Polymorphs include different crystal packing arrangements of compounds of the same elemental composition. Polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shape, optical and electrical properties, stability and solubility. Various factors may cause a single crystal form to dominate, such as recrystallization solvent, crystallization rate, and storage temperature.

The term "excipient", as used herein, means an inert or inactive substance that can be used in the manufacture of a medicament or medicine, e.g., a tablet containing a compound of the present disclosure as an active ingredient. The term excipient may encompass a variety of substances including, but not limited to, any substance that acts as a binding agent, disintegrant, coating, compression/encapsulation aid, cream or emulsion, lubricant, solution for parenteral administration, material for chewable tablets, sweetener or flavoring agent, suspending/gelling agent, or wet granulation agent. Binders include, for example, carbomer (carbomer), povidone (povidone), xanthan gum (xanthan gum), and the like; coatings include, for example, cellulose acetate phthalate (cellulose acetate phthalate), ethyl cellulose, gellan gum (gellan gum), maltodextrin, enteric coatings, and the like; compression/encapsulation aids include, for example, calcium carbonate, dextrose, direct compressible fructose (fructose direct compressible; fructose dc), honey dc, lactose (anhydrous or monohydrate; optionally in combination with aspartame, cellulose or microcrystalline cellulose), starch dc, sucrose, and the like; disintegrants include, for example, croscarmellose sodium, gellan gum, sodium starch glycolate, and the like; creams or lotions include, for example, maltodextrin (maltodextrin), carrageenan (carrageenan), and the like; lubricants include, for example, magnesium stearate, stearic acid, sodium stearyl fumarate, and the like; materials for chewable tablets include, for example, dextrose, fructose dc, lactose (monohydrate, optionally in combination with aspartame or cellulose), and the like; suspending/gelling agents include, for example, carrageenan, sodium starch glycolate, xanthan gum, and the like; sweeteners include, for example, aspartame, dextrose, fructose dc, sorbitol, sucrose dc, and the like; and wet granulating agents include, for example, calcium carbonate, maltodextrin, microcrystalline cellulose, and the like.

Compound (I)

Provided herein are compounds comprising at least one nuclear payload and at least one epitope that targets a nuclear receptor. The compounds described herein are capable of delivering nuclear payloads to the nucleus of the cell by recognizing that the nucleus is targeted and binding an epitope of the targeted nuclear receptor to the corresponding binding site. The nuclear payload is then capable of binding to one or more target sites within the nucleus and/or disrupting one or more cellular processes, causing cell death. In certain embodiments, the nuclear payload is bound to an epitope that targets the nuclear receptor via a linking moiety. In certain embodiments, the linking moiety provides a single or single linkage, meaning that the linker binds to only one atom of each of the payload and the epitope.

The compounds described herein may comprise more than one epitope that targets a nuclear receptor. The epitopes may be the same or different, such that the compound is directed against one or more cellular targets, in addition to the nucleus.

Accordingly, there is provided a compound of formula I, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-(L-B)_m I

wherein:

A is the core payload;

m is 1, 2 or 3;

each B is independently an epitope that targets a nuclear receptor; and is

Each L is independently a covalent bond or a linking moiety.

In certain embodiments, one or more epitopes that target a nuclear receptor are bound to the nuclear payload via a single linking moiety. Accordingly, there is also provided a compound of formula II, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-L-(B)_m II

wherein:

a is the core payload;

m is 1, 2 or 3;

each B is independently an epitope that targets a nuclear receptor; and is

L is a linking moiety.

In certain embodiments, the nuclear receptor-targeting epitope of formula I or II is a nuclear hormone receptor-targeting epitope. In certain embodiments, the nuclear receptor-targeting epitope of formula I or II is a nuclear steroid receptor-targeting epitope.

Also provided are compounds of formula I or II, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-(L-B)_m I

A-L-(B)_m II

wherein:

a is the core payload;

m is 1, 2 or 3;

each B is independently an epitope derived from a nuclear receptor targeting: estrogen, estetrol, estriol, estrone, progesterone, enoboxate, bicalutamide, apalutamide, testosterone, dihydrotestosterone, testosterone, 19-nortestosterone, progesterone, adatansine, cortisol, prednisone (prednisone), estradiol, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, bazedoxifene, ospemifene, megestrol acetate, estramustine, abiraterone, LGD-2941, BMS-564929, osputan, ulipristal acetate, sorrel (J867), mifepristone, torasemide (CDB-4124, Proellex, Progenta), or an analog thereof; and

Each L is independently a covalent bond or a linking moiety.

Also provided are compounds of formula I, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-(L-B)_m I

wherein:

a is a nuclear payload that binds to poly (ADP-ribose) polymerase-1 (PARP-1) and/or poly (ADP-ribose) polymerase (PARP-2);

m is 1, 2 or 3;

each B is independently an epitope derived from a nuclear receptor targeting: estrogen, estetrol, estriol, estrone, progesterone, enoboxate, bicalutamide, apalutamide, testosterone, dihydrotestosterone, estradiol, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, bazedoxifene, ospemifene, megestrol acetate, estramustine, abiraterone, LGD-2941, BMS-564929, osthole, or an analog thereof; and is

Each L is independently a covalent bond or a linking moiety.

Also provided are compounds of formula II, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-L-(B)_m II

wherein:

a is a nuclear payload that binds to poly (ADP-ribose) polymerase-1 (PARP-1) and/or poly (ADP-ribose) polymerase (PARP-2);

m is 1, 2 or 3;

each B is independently an epitope derived from a nuclear receptor targeting: estrogen, estetrol, estriol, estrone, progesterone, enoboxate, bicalutamide, apalutamide, testosterone, dihydrotestosterone, estradiol, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, bazedoxifene, ospemifene, megestrol acetate, estramustine, abiraterone, LGD-2941, BMS-564929, osthole, or an analog thereof; and is

Each L is independently a covalent bond or a linking moiety.

The "linking moiety" of any of the compounds described herein can be either biocleavable (e.g., acid labile) or non-biocleavable. The linking moiety may be linear, branched, saturated, unsaturated, all carbon or heteroatom. The linking moiety may also contain one or more fused, saturated, unsaturated, and all carbon or heteroatom rings. In certain embodiments, the linking moiety is a non-biocleavable linking moiety. In certain embodiments, the linking moiety is a biocleavable linking moiety. In certain embodiments, the nuclear payload is bound to one nuclear steroid receptor-targeting epitope via a non-biocleavable linking moiety, and to one or more nuclear steroid receptor-targeting epitopes via a biocleavable linking moiety. In certain embodiments, the biocleavable linking moiety is an acid labile linking moiety. In some embodiments, the linking moiety comprises a hydrazone linkage.

It is contemplated that any linking moiety may be used in the compounds described herein, provided that it does not significantly interfere with or disrupt the desired binding of the nuclear payload or epitope targeted to the nuclear receptor. In some embodiments, the linking moiety is alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene; wherein each alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene may optionally comprise an arylene, heteroarylene, cycloalkylene, or heterocycloalkylene group; and further wherein each alkylene, heteroalkylene, alkenylene, heteroalkyleneIndependently, the alkyl, alkynyl, heteroalkynyl, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene group is optionally substituted with one to five substituents independently selected from the group consisting of: oxo, halo, C_1-4Alkyl radical, C_1-4Alkoxy and C_1-4A haloalkyl group.

In certain embodiments, the linking moiety has the formula:

-Y¹-(CH₂)_n'-Y²-(CH₂)_m'-Y³-

wherein:

Y¹、Y²and Y³Each of which is independently-NR¹¹-、-O-、-S(O)_0-2-、-NR¹¹C(O)-、-C(O)NR¹¹-、-NR¹¹S(O)₂-、-S(O)₂NR¹¹-、-CR¹²＝N-NR¹¹-、-NR¹¹-N＝CR¹²-, -C (O) -, arylene, heteroarylene, cycloalkylene or heterocycloalkylene; wherein each alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from: oxo, halo, C _1-4Alkyl radical, C_1-4Alkoxy and C_1-4A haloalkyl group;

each R¹¹Independently is C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl;

each R¹²Independently is C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; and is

n 'and m' are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

Also provided are compounds of formula IA, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-(Y¹-(CH₂)_p-Y²-(CH₂)_q-Y³-B)_r IA

wherein:

a is a nuclear payload that binds to poly (ADP-ribose) polymerase-1 (PARP-1) and/or poly (ADP-ribose) polymerase (PARP-2);

r is 1, 2 or 3;

each B is independently an epitope derived from a nuclear receptor targeting: estrogen, estetrol, estriol, estrone, progesterone, enoboxate, bicalutamide, apalutamide, testosterone, dihydrotestosterone, estradiol, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, bazedoxifene, ospemifene, megestrol acetate, estramustine, abiraterone, LGD-2941, BMS-564929, osthole, or an analog thereof; and is

Y¹、Y²And Y³Each of which is independently a bond, -NR ¹¹-、-O-、-S(O)_0-2-、-NR¹¹C(O)-、-C(O)NR¹¹-、-NR¹¹S(O)₂-、-S(O)₂NR¹¹-、-CR¹²＝N-NR¹¹-、-NR¹¹-N＝CR¹²-, -C (O) -, arylene, heteroarylene, cycloalkylene or heterocycloalkylene; wherein each alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from: oxo, halo, C_1-4Alkyl radical, C_1-4Alkoxy and C_1-4A haloalkyl group;

each R¹¹Independently is C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl;

each R¹²Independently is C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; and is

p and q are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

Also provided are compounds of formula IIA, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-Y¹-(CH₂)_p-Y²-(CH₂)_q-Y³-(B)_r IIA

wherein:

a is a nuclear payload that binds to poly (ADP-ribose) polymerase-1 (PARP-1) and/or poly (ADP-ribose) polymerase (PARP-2);

r is 1, 2 or 3;

each B is independently an epitope derived from a nuclear receptor targeting: estrogen, estetrol, estriol, estrone, progesterone, enoboxate, bicalutamide, apalutamide, testosterone, dihydrotestosterone, estradiol, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, bazedoxifene, ospemifene, megestrol acetate, estramustine, abiraterone, LGD-2941, BMS-564929, osthole, or an analog thereof; and is

Y¹、Y²And Y³Each of which is independently a bond, -NR¹¹-、-O-、-S(O)_0-2-、-NR¹¹C(O)-、-C(O)NR¹¹-、-NR¹¹S(O)₂-、-S(O)₂NR¹¹-、-CR¹²＝N-NR¹¹-、-NR¹¹-N＝CR¹²-, -C (O) -, arylene, heteroarylene, cycloalkylene or heterocycloalkylene; wherein each alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from: oxo, halo, C_1-4Alkyl radical, C_1-4Alkoxy and C_1-4A haloalkyl group;

each R¹¹Independently is C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl;

each R¹²Independently is C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; and is

p and q are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

In some embodiments, the connecting portion is not a key. In certain embodiments, each R¹¹Independently of each other is hydrogen, C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R¹²Independently of each other is hydrogen, C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl.

In certain embodiments, the linking moiety has the formula:

-Y¹-(CH₂)_n'-Y²-(CH₂)_m'-Y³-

wherein:

Y¹、Y²and Y³Each of which is independently-NR¹¹-、-O-、-S(O)_0-2-、-NR¹¹C(O)-、-C(O)NR¹¹-、-NR¹¹S(O)₂-、-S(O)₂NR¹¹-、-CR¹²＝N-NR¹¹-、-NR¹¹-N＝CR¹²-, -C (O) -, arylene, heteroarylene, cycloalkylene or heterocycloalkylene; wherein each alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from: oxo, halo, C _1-4Alkyl radical, C_1-4Alkoxy and C_1-4A haloalkyl group;

each R¹¹Independently of each other is hydrogen, C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl;

each R¹²Independently of each other is hydrogen, C_1-4Alkyl radical, C_1-4Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; and is

n 'and m' are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

Also provided are compounds of formula I or II, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-(L-B)_m I

A-L-(B)_m II

wherein:

a is the core payload;

m is 1, 2 or 3;

each B is independently an epitope that targets a nuclear receptor; and is

Each L is independently a covalent bond or a linking moiety.

Also provided are compounds of formula I or II, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

A-(L-B)_m I

A-L-(B)_m II

wherein:

a is the core payload;

m is 1, 2 or 3;

each B is independently an epitope that targets a nuclear steroid receptor; and is

Each L is independently a covalent bond or a linking moiety.

In certain embodiments of any formula or subformula (e.g., formula I or II) disclosed herein, when m is 1, then the epitope targeted to the nuclear receptor is not a peptide, protein, nanoparticle, or antibody. In certain embodiments of any formula or subformula disclosed herein (e.g., formula I or II), when m is 1 and B is an epitope that targets the androgen receptor, then a is not doxorubicin or an analog thereof. In certain embodiments of any formula or subformula disclosed herein (e.g., formula I or II), when m is 1 and B is an epitope that targets the androgen receptor, then a is not a hydroxamic acid that binds Histone Deacetylase (HDAC). In certain embodiments of any formula or subformula disclosed herein (e.g., formula I or II), when m is 1 and B is an epitope that targets an estrogen receptor, then a is not doxorubicin or an analog thereof. In certain embodiments of any formula or subformula disclosed herein (e.g., formula I or II), when m is 1 and B is an epitope that targets the estrogen receptor, then a is not a hydroxamic acid that binds Histone Deacetylase (HDAC).

Also provided are compounds of formula III, IV, V or VI, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

each R¹、R²、R³And R⁴Independently is-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more (e.g., 1 to 5 or 1 to 3) R¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted by one or more (e.g. 1 to 5 or 1 to 3) halo groups or by oxo, halo, hydroxy or amino groups _1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

R⁹Is hydrogen or R²；

With the proviso that at least one R¹、R²、R³And R⁴is-L- (B)_m。

Also provided are compounds of formula IIIA, IV, VA or VI, or stereoisomers, mixtures, hydrates, solvates, isotopically enriched analogs, or pharmaceutically acceptable salts thereof:

wherein:

R¹、R²、R³and R⁴Is independently-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR ⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more (e.g., 1 to 5 or 1 to 3) R¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted by one or more (e.g. 1 to 5 or 1 to 3) halo groups or by oxo, halo, hydroxy or amino groups_1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

with the proviso that R¹、R²、R³And R⁴is-L- (B)_m。

Also provided are compounds of formula IIIB, IVA, VB or VIA, or stereoisomers, mixtures, hydrates, solvates, isotopically enriched analogs, or pharmaceutically acceptable salts thereof:

wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

Also provided are compounds of formula III ', IV', V 'or VI', or stereoisomers, mixtures, hydrates, solvates, isotopically enriched analogs, or pharmaceutically acceptable salts thereof:

wherein:

R¹、R²、R³and R⁴Is independently-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence ¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more (e.g., 1 to 5 or 1 to 3) R¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear steroid receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ C)O)R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted by one or more (e.g. 1 to 5 or 1 to 3) halo groups or by oxo, halo, hydroxy or amino groups_1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino _1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

with the proviso that R¹、R²、R³And R⁴is-L- (B)_m。

Also provided are compounds of formula IIIA ', IVA', VA ', or VIA', or stereoisomers, mixtures, hydrates, solvates, isotopically enriched analogs, or pharmaceutically acceptable salts thereof:

wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear steroid receptor.

Nuclear payload

A nuclear payload as used herein is generally capable of binding to any site involved in a cellular process important for the development of cancer or cell replication. In certain embodiments, the nuclear payload binds to a target site within the nucleus and disrupts one or more cellular processes, thereby causing cell death. Target sites within the nucleus include, but are not limited to, a sub-nuclear compartment (e.g., promyelocytic leukemia nucleus (PML NB), nucleolus), a protein-protein interaction within the nucleus (e.g., hypoxia inducible factor 1 α (HIF-1 α), FKBP25), or a modification of chromatin structure. In certain embodiments, the nuclear payload targets a protein involved in DNA damage repair processes, such as, but not limited to, poly (ADP-ribose) polymerase (PARP), DNA-dependent protein kinase (DNA-PK), myelin transcription factor 1(MYT1), p53, hormone stimulating Melanocytes (MSH), mutL homolog (MLH), ERCC1, apurinic/apyrimidinic endonuclease 1(APE1), topoisomerase i (topo i), topoisomerase ii (topo ii), Wee1, checkpoint kinase 1(Chk1), checkpoint kinase 2(Chk2), Ataxia Telangiectasia (ATR), or Ataxia Telangiectasia Mutation (ATM).

In certain embodiments, the nuclear payload comprises olaparib (AZD-2281), olaparib TOPARP-A, lucapanib (AG014699, PF-01367338), nilapanib, talaronib (talazoparib) (BMN-673), veliparib (veliparib) (ABT-888), CEP 9722, E7016, BGB-290, 3-aminobenzamide, methoxyamine, CC-115, MSC2490484A, AZD6738, VX-970, VX 0156, GDC-0575, MK-8776, LY2606368, AZD1775, belotecan (belotecan), CRLX101, irinotecan (irinotecan), LMP 400, LMP 776, NKTR-102, topotecan, doxorubicin, epirubicin (epirubicin), etoposide (etoposide), teniposide (timinoside), timinoside (amitriptonide), or an analog thereof. In certain embodiments, the core payload comprises AZD-1775(MK-1775, Adavostib), SCH900776(MK-8776), AZD0156, M6620(VX-970, VE-822, Besselt cloth (Berzosertib)), AZD6738, or CC-115, or an analog thereof. In certain embodiments, the core payload comprises a combination of CC-115 and an additional core payload. In certain embodiments, the nuclear payload comprises CC-115 and the compound comprises enzalutamide or an analog thereof.

The analogs are derived from known nuclear payloads, already named herein, and are modified to bind, optionally via a linking moiety, to at least one epitope that targets a nuclear receptor. Even after modification to achieve the compounds described herein, the analogs maintain biological activity, similar to that observed in the initial unmodified nuclear payload. In certain embodiments, the analog exhibits at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of the binding activity or inhibition observed in the initial unmodified core payload.

In certain embodiments, the terms "modified" and "derived from" as used with respect to a nuclear payload mean that at most one non-hydrogen atom of the initial unmodified nuclear payload (i.e., the known nuclear payload) is replaced by a covalent bond, optionally linked via a linking moiety to at least one epitope targeting a nuclear receptor. In certain embodiments, the terms "modified" and "derived from" as used with respect to a nuclear payload mean that only up to one hydrogen atom of the initial unmodified nuclear payload (i.e., the known nuclear payload) is replaced by a covalent bond, optionally linked via a linking moiety to at least one epitope targeting a nuclear receptor. In certain embodiments, one hydrogen atom of a heteroatom (e.g., N, O or S) bound to an initially unmodified nuclear payload (i.e., a known nuclear payload) is replaced with a covalent bond, optionally linked via a linking moiety to at least one epitope of a targeted nuclear receptor.

In certain embodiments, the nuclear payload is conjugated to an epigenetic target, such as a Histone Deacetylase (HDAC) (e.g., vorinostat (vorinostat), romidepsin (isotaxax), cidamide (chidamide), panobinostat (Farydak), belinostat (belinostat) (PXD101), panobinostat (LBH589), valproic acid (in the form of magnesium valproate), mocetinostat (mocetinostat) (MGCD0103), abexinostat (abexinostat) (PCI-24781), entinostat (MS-275), SB939, raninostat (resinostat) (4SC-201), virinostat (ITF) (ITF 7), quinisitostat (quisinostat) (HBJ-265), Reynant I-488000, JNK-3976, JNK-202, JNK-26CG, JNK-202, JR-202, JC-202, JR-C-3, JR-C, JR-202, JR-3, J, Enhancer of zeste homolog 2 (EZH2) (e.g., tasetastat, MAK638, CPI-1205, DS-3201b, etc. or analogs thereof), Histone Acetyltransferases (HAT) (e.g., anacardic acid, MG149, C646, etc. or analogs thereof), methyltransferases (e.g., S-adenosylmethionine, etc. or analogs thereof), bromodomains (e.g., JQ1, I-151 BET (GSK1210151A), I-BET 762(GSK525762), OTX-015, TEN-010, CPI-203, CPI-0610, orinon, LY294002, or analogs thereof), and the like.

Any known nuclear payload that targets one or more cellular processes of a protein can be used as the nuclear payload of the compounds described herein. Small molecule nuclear payloads (i.e., molecular weights less than about 1,000g/mol) are expected to be particularly useful for the compounds described herein (e.g., tipapamine (tripazamine), clarithromycin (chetomin), rapamycin (rapamycin), PARP inhibitors, etc.).

In certain embodiments, the compounds comprise at least one nuclear payload that binds to poly (ADP-ribose) polymerase (PARP) and is referred to herein as a "PARP inhibitor". PARP inhibitors are cytotoxic agents that prevent such DNA repair, thereby causing cell death and tumor growth inhibition. In certain embodiments, the PARP is human PARP and comprises PARP-1 and/or PARP-2 or variants thereof. In certain embodiments, the nuclear payload is capable of blocking the enzymatic activity of PARP and/or localizing PARP protein to the site of DNA damage (i.e., "PARP trapping"). Thus, in certain embodiments, the nuclear payload binds to PARP and induces an allosteric conformational change in the enzyme.

In certain embodiments, the nuclear payload is bound to a PARP-1 catalytic domain. In certain embodiments, the nuclear payload is bound to a PARP-2 catalytic domain. In certain embodiments, the nuclear payload binds to a conserved HYE motif. In certain embodiments, the nuclear payload binds to the nicotinamide binding pocket in the PARP protein.

In one embodiment, the nuclear payload is an analog of a known PARP inhibitor. Exemplary PARP inhibitors that may be used as nuclear payloads in the compounds described herein include, but are not limited to, Olaparib (AZD-2281), Olaparib TOPARP-A, Ruvacarib (AG014699, PF-01367338), Nilaparib, Tarlaparib (BMN-673), Veliparib (ABT-888), CEP 9722, E7016, BGB-290, and 3-aminobenzamide, or analogs thereof.

PARP inhibitor analogs are derived from PARP inhibitors and are modified to bind, optionally via a linking moiety, to at least one epitope that targets a nuclear steroid receptor. Even after modification to achieve the compounds described herein, PARP inhibitor analogs maintain biological activity, similar to that observed in the original unmodified PARP inhibitors. In certain embodiments, the PARP inhibitor analogs maintain the ability to inhibit PARP. In certain embodiments, the PARP inhibitor analogs exhibit at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of the binding activity observed in an initially unmodified PARP inhibitor. In certain embodiments, compounds and ICs as described herein ₅₀Less than about 500nM, or less than about 400nM, or less than about 350nM, or less than about 300nM, or less than about 200nM, or less than about 100nM, or less than about 50nM of poly (ADP-ribose) polymerase (PARP) (e.g., PARP-1 and/or PARP-2) binding.

In certain embodiments, a nuclear payload (e.g., PARP inhibitor analog) comprises one or more moieties capable of having a binding interaction with G863, Y907, S904, a898, K903, E988, Y896 and/or Y889 of PARP-1. In certain embodiments, a nuclear payload (e.g., PARP inhibitor analog) comprises one or more moieties capable of having a binding interaction with Y889, Y896, H862, G863, S904, Y907, K903, E988, and/or M890 of PARP-1. In certain embodiments, a nuclear payload (e.g., PARP inhibitor analog) comprises one or more moieties capable of having a binding interaction with Y896, Q763, G863, S904, Y907, K903 and/or E988 of PARP-1. In certain embodiments, the nuclear payload (e.g., PARP inhibitor analog) comprises one or more positively charged moieties (e.g., amino groups) that interact with the side chains of Q763, D766, and/or Y896 of PARP-1. In certain embodiments, a nuclear payload (e.g., PARP inhibitor analog) comprises one or more moieties capable of having a binding interaction with E322, D326, I425, S417, H415, E545, and/or Y449 of PARP-2.

In certain embodiments, the core payload comprises akapani (AG014699, PF-01367338) or an analog thereof (i.e., an analog containing akapani). Accordingly, there is provided a compound of formula III, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R¹、R²、R³and R⁴Is independently-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more R¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo or optionally with oxo, halo, hydroxy or amino _1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;and is

Each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

with the proviso that R¹、R²、R³And R⁴is-L- (B)_m。

In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, R¹、R²、R³And R⁴Only one of them is-L- (B)_m。

In certain embodiments, there is provided a compound of formula IIIA, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R¹、R²、R³and R⁴Is independently-L- (B) _mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted by one or moreR is¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo or optionally with oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino _1-12Alkyl substitution; and is

Each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸With the atom to which it is attachedTaken together to form a heterocyclic group, which is optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

with the proviso that R¹、R²、R³And R⁴is-L- (B)_m。

In certain embodiments, there is provided a compound of formula IIIB, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

In certain embodiments of formulas III, IIIA and IIIB, the epitope that targets the nuclear receptor is an epitope that targets the nuclear steroid receptor. In certain embodiments of formulas III, IIIA and IIIB, m is 1. In certain embodiments of formulas III, IIIA and IIIB, m is 2. In certain embodiments of formulas III and IIIA, R¹、R²、R³And R⁴Only one of them is-L- (B)_m。

In certain embodiments, the nuclear payload is derived from talaropab (BMN-673) or an analog thereof (i.e., an analog containing talaropab). Accordingly, there is provided a compound of formula IV, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

Wherein:

R¹、R²、R³and R⁴Is independently-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more R¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted by one or more halo groups or optionally by oxo,Halogen-, hydroxy-or amino-substituted C_1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino _1-12Alkyl substitution; and is

Each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

with the proviso that R¹、R²、R³And R⁴is-L- (B)_m。

In certain embodiments, there is provided a compound of formula IVA, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

In certain embodiments, there is provided a compound of formula IVB, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R²¹and R²³Each independently selected from hydrogen, halo, hydroxy, C_1-12Alkyl radical, C_3-10Cycloalkyl radical, C_1-12Alkoxy radical, C_1-12An alkoxyalkyl group; wherein each alkyl, cycloalkyl, alkoxy, alkoxyalkyl is independently optionally substituted with at least one substituent selected from the group consisting of: cyano, halo, hydroxy, nitro, C _1-12Alkyl and C_3-10Cycloalkyl, wherein R²³Is not a hydroxyl group;

R²²and R²⁴Each independently is hydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_1-12Alkoxy, -C (═ O) R⁵⁰、-C(＝O)OR⁵⁰、-C(＝O)N(R⁵⁰)₂、-S(＝O)_0-2R⁵⁰、-S(＝O)_1-2N(R⁵⁰)₂、-NR⁵⁰S(＝O)_1-2R⁵⁰、C_3-10Cycloalkyl, aryl, heterocyclyl or heteroaryl, wherein each C is_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_1-12Alkoxy radical, C_3-10The cycloalkyl, aryl, heterocyclyl or heteroaryl group may be independently optionally substituted with 1, 2 or 3R²⁹Substitution;

R²⁰and R²⁵Each independently selected from the group consisting of: hydrogen, C_1-12Alkyl radical, C_3-10Cycloalkyl radical, C_1-12Alkoxyalkyl group, C_1-12Haloalkyl, C_1-12alkyl-OH and C_1-12alkyl-NR⁵¹R⁵²；

R²⁶、R²⁷And R²⁸Each independently selected from the group consisting of: hydrogen, halo, cyano, nitro, amino, hydroxy, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_1-12Alkoxy radical, C_3-10A cycloalkyl group, a,-C (═ O) -alkyl, -C (═ O) -alkoxy, haloalkoxy, haloalkyl, heteroalkyl; wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo, hydroxy or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

each R²⁹Selected from hydroxy, halo, cyano, nitro, -OR⁵¹、-SR⁵¹、-SF₅、-NR⁵¹R⁵²、C_1-12Alkyl radical, C_2-12Alkenyl radical, C _2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵¹、-C(＝O)OR⁵¹、-OC(＝O)OR⁵¹、-OC(＝O)R⁵¹、-C(＝O)NR⁵¹R⁵²、-OC(＝O)NR⁵¹R⁵²、-NR⁵¹C(＝O)NR⁵¹R⁵²、-S(＝O)_1-2R⁵¹、-S(＝O)_{1- 2}NR⁵¹R⁵²、-NR⁵¹S(＝O)_1-2R⁵²、-NR⁵¹S(＝O)_1-2NR⁵¹R⁵²、-NR⁵¹C(＝O)R⁵²、-NR⁵¹C(＝O)OR⁵²or-C ═ NOR⁵¹Wherein R, when allowed by valence²⁹Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo or optionally with oxo, halo, hydroxy or amino_1-12Alkyl substitution;

each R⁵⁰Independently of each other is hydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, aryl, heterocyclyl or heteroaryl, wherein R, when valency permits⁵⁰Each alkyl, alkenyl, alkynyl, cycloalkylheterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo or optionally with oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R⁵¹And R⁵²Independently of hydrogen、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein R, when valency permits⁵¹And R⁵²Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is optionally substituted with oxo, halo, hydroxy or amino; or R⁵¹And R⁵²Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino _1-12Alkyl substitution;

with the proviso that at least one R²²Or R²⁴comprises-L- (B) bonded thereto_mA group, wherein each L is independently a covalent bond or a linking moiety, each B is independently an epitope that targets a nuclear receptor, and m is 1, 2, or 3.

In the compound of formula VIB, at least one R²²Or R²⁴Comprises a compound having-L- (B) bonded thereto_mA substituent of the group. Thus, it should be understood that-L- (B)_mThe group is not directly bound to the tricyclic core.

In certain embodiments of formulas IV, IVA and IVB, the epitope that targets the nuclear receptor is an epitope that targets a nuclear steroid receptor. In certain embodiments of formulas IV, IVA, and IVB, m is 1. In certain embodiments of formulas IV, IVA, and IVB, m is 2. In certain embodiments of formula IV, R¹、R²、R³And R⁴Only one of them is-L- (B)_m。

In certain embodiments, the nuclear payload is derived from olaparib (AZD-2281) or an analog thereof (i.e., an olaparib-containing analog). In certain embodiments, the compound is a compound of formula V, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

each R¹、R²、R³And R⁴Independently is-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C _2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more R¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted by one or more halo groups or by oxo, halo, hydroxyRadical or amino substituted C_1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino _1-12Alkyl substitution;

each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

R⁹is hydrogen or R²(ii) a And is

With the proviso that at least one R¹、R²、R³And R⁴is-L- (B)_m。

In certain embodiments, there is provided a compound of formula VA, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R¹、R²、R³and R⁴Is independently-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more R¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR ⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo or optionally with oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Together with the atom to which they are attached to form a heterocyclic group, optionally substituted by halo or optionally substituted by oxoC substituted by radicals, halogens, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

with the proviso that R¹、R²、R³And R⁴is-L- (B)_m。

In certain embodiments, there is provided a compound of formula VB, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

Wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

In certain embodiments, there is provided a compound of formula VC, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

a and B together represent an optionally substituted fused aromatic ring:

R³⁰and R³¹Independently is hydrogen or C_1-12Alkyl, or when X is-CR³³R³⁴When R is³⁰、R³¹、R³³And R³⁴Together with the carbon atoms to which they are attached may form an optionally substituted fused aromatic ring;

R³²is hydrogen or halogen;

x is-NR³³or-CR³³R³⁴(ii) a Wherein if X is-NR³³Then t is 1 or 2; and if X is-CR³³R³⁴Then t is 1;

R³³is hydrogen, optionally substituted C_1-12Alkyl, aryl, heterocyclyl, -C (═ O) R⁵⁰、-C(＝O)OR⁵⁰、-C(＝O)N(R⁵⁰)₂、-S(＝O)_0-2R⁵⁰、-S(＝O)_1-2N(R⁵⁰)₂、-NR⁵⁰S(＝O)_1-2R⁵⁰；

R³⁴Is hydrogen, hydroxy or amino;

R³³and R³⁴May be taken together to form C_3-10Cycloalkyl or heterocyclyl; and is

Each R⁵⁰Independently of each other is hydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, aryl, heterocyclyl or heteroaryl, wherein R, when valency permits⁵⁰Each alkyl, alkenyl, alkynyl, cycloalkylheterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo or optionally with oxo, halo, hydroxy or amino _1-12Alkyl substitution;

with the proviso that at least one R³⁰、R³¹、R³³Or R³⁴The group comprises-L- (B) bonded thereto_mA group, wherein each L is independently a covalent bond or a linking moiety, each B is independently an epitope that targets a nuclear receptor, and m is 1, 2, or 3.

In certain embodiments of formulas V, VA, VB and VC, the epitope that targets the nuclear receptor is an epitope that targets a nuclear steroid receptor. In certain embodiments of formulae V, VA, VB, and VC, m is 1. In certain embodiments of formulae V, VA, VB, and VC, m is 2. In the formulae V and VIn certain embodiments of A, R¹、R²、R³And R⁴Only one of them is-L- (B)_m。

In certain embodiments, the nuclear payload is derived from verapamil (ABT-888) or an analog thereof (i.e., a verapamil-containing analog). Also provided is a compound of formula VI, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R¹、R²、R³and R⁴Is independently-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²、R³And R⁴Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently optionally substituted with one or more R ¹⁰Substitution;

m is 1, 2 or 3;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ C)O)R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo or optionally with oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁷And R⁸Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino _1-12Alkyl substitution;

with the proviso that R¹、R²、R³And R⁴is-L- (B)_m。

In certain embodiments, there is provided a compound of formula VIA, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

In certain embodiments of formulas VI and VIA, the epitope that targets a nuclear receptor is an epitope that targets a nuclear steroid receptor. In certain embodiments of formulas VI and VIA, m is 1. In certain embodiments of formulas VI and VIA, m is 2. In certain embodiments of formula V, R¹、R²、R³And R⁴Only one of them is-L- (B)_m。

In certain embodiments, the nuclear payload comprises CC-115 or an analog thereof (an analog comprising CC-115). Also provided are compounds of formula VII, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

R¹、R²and R³Is independently-L- (B)_mHydrogen, C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R ⁵、-C(＝O)OR⁵、-OC(＝O)R⁵、-C(＝O)NR⁵R⁶、-NR⁵C(＝O)R⁶、-S(＝O)_1-2R⁵、-S(＝O)_1-2NR⁵R⁶、-NR⁵S(＝O)_1-2R⁶or-C ═ NOR⁵Wherein R, when allowed by valence¹、R²And R³Each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and hetero ofAryl is independently optionally substituted with one or more R¹⁰Substitution;

each L is independently a covalent bond or a linking moiety;

each B is independently an epitope that targets a nuclear receptor;

each R¹⁰Independently halo, cyano, nitro, -OR⁷、-SR⁷、-SF₅、-NR⁷R⁸、C_1-12Alkyl radical, C_2-12Alkenyl radical, C_2-12Alkynyl, C_3-10Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (═ O) R⁷、-C(＝O)OR⁷、-OC(＝O)OR⁷、-OC(＝O)R⁷、-C(＝O)NR⁷R⁸、-OC(＝O)NR⁷R⁸、-NR⁷C(＝O)NR⁷R⁸、-S(＝O)_1-2R⁷、-S(＝O)_1-2NR⁷R⁸、-NR⁷S(＝O)_1-2R⁸、-NR⁷S(＝O)_1-2NR⁷R⁸、-NR⁷C(＝O)R⁸、-NR⁷C(＝O)OR⁸or-C ═ NOR⁷Wherein R, when allowed by valence¹⁰Each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl of (a) is independently C optionally substituted with one or more halo or optionally with oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁵And R⁶Independently hydrogen, deuterium, C optionally substituted by oxo, halo, hydroxy or amino_1-12Alkyl or C_3-12A cycloalkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution; and is

Each R, when allowed by valence⁷And R⁸Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino _1-12An alkyl group; or R⁷And R⁸Together with the atoms to which they are attachedTo form a heterocyclic group, C optionally substituted with halo or optionally oxo, halo, hydroxy or amino_1-12Alkyl substitution;

with the proviso that R¹、R²And R³is-L- (B)_m。

In certain embodiments, there is provided a compound of formula VIIA, or a stereoisomer, mixture of stereoisomers, hydrate, solvate, isotopically enriched analog, or pharmaceutically acceptable salt thereof:

wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

In certain embodiments of formulas VII and VIIA, the epitope that targets the nuclear receptor is an epitope that targets a nuclear steroid receptor. In certain embodiments of formulas VII and VIIA, m is 1. In certain embodiments of formulas VII and VIIA, m is 2. In certain embodiments of formula VII, R¹、R²、R³And R⁴Only one of them is-L- (B)_m。

In certain embodiments of the formulae disclosed herein, each R, where permitted by valence⁵And R⁶Independently is hydrogen, deuterium or C optionally substituted by oxo, halo, hydroxy or amino_1-12An alkyl group; or R⁵And R⁶Taken together with the atoms to which they are attached to form a heterocyclyl, optionally substituted with halo or optionally oxo, halo, hydroxy or amino _1-12Alkyl substitution.

In certain embodiments, the nuclear payload binds to a DNA-dependent protein kinase (DNA-PK). In certain embodiments, the nuclear payload is an inhibitor of a DNA-dependent protein kinase (DNA-PK). In certain embodiments, the nuclear payload is derived from AZD-1775(MK-1775, Adawatt), SCH900776(MK-8776), AZD0156, M6620(VX-970, VE-822, Besselt), AZD6738, or CC-115, or an analog thereof. In certain embodiments, compounds of the formula:

wherein one hydrogen atom is substituted by-L- (B)_mReplacement; wherein

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

In certain embodiments, is-L- (B)_mThe replacing hydrogen atom is on the hetero atom. In certain embodiments, is-L- (B)_mThe replacing hydrogen atom is on the nitrogen. In certain embodiments, is-L- (B)_mThe replacing hydrogen atom is on oxygen. In certain embodiments, is-L- (B)_mThe replacing hydrogen atom is on carbon. In certain embodiments, the epitope that targets a nuclear receptor is an epitope that targets a nuclear steroid receptor. In certain embodiments, m is 1. In certain embodiments, m is 2.

In certain embodiments, compounds of the formula:

Wherein:

R²is-L- (B)_m；

m is 1, 2 or 3;

l is a covalent bond or a linking moiety; and is

Each B is independently an epitope that targets a nuclear receptor.

In certain embodiments of any of the compounds, formulae, or embodiments disclosed herein, the epitope that targets a nuclear receptor is an epitope that targets a nuclear steroid receptor. In certain embodiments, m is 1. In certain embodiments, m is 2.

Epitopes targeting nuclear receptors

As used herein, "epitope targeting a nuclear receptor" refers to a portion of a compound described herein (e.g., a "B" portion of formula I or formula II) that is derived from a nuclear targeting agent as disclosed herein and interacts with a ligand binding domain of the targeted nuclear receptor, i.e., the portion of the compound that drives the ligand binding interaction. Targeting epitopes of nuclear receptors serves to associate a compound with a target nuclear receptor, e.g., a nuclear steroid receptor, helps localize the compound to cells expressing the nuclear steroid receptor, and transports nuclear payloads from the cytosol into the nucleus, causing the compound to accumulate in the nucleus. The level of accumulation may be controlled by selecting an appropriate epitope for targeting the nuclear receptor. For example, the compounds described herein may accumulate in the nucleus to alter the degree via nuclear translocation of the nuclear steroid receptor that occurs upon binding of the epitope to the receptor, high in the case of full agonists (e.g., Dihydrotestosterone (DHT)), moderate in the case of partial agonists (e.g., bicalutamide), and low in the case of antagonists (e.g., enzalutamide).

The steroid receptor target can be any steroid receptor, including but not limited to receptors that are overexpressed on cancer cells. In certain embodiments, the at least one nuclear steroid receptor-targeting epitope is capable of binding to a ligand binding domain of a nuclear steroid receptor, such as a ligand binding domain on an estrogen receptor, a glucocorticoid receptor, a progesterone receptor, or an androgen receptor.

Exemplary epitopes targeting nuclear steroid receptors include epitopes derived from: an androgen receptor agonist, an androgen receptor antagonist, a Selective Androgen Receptor Modulator (SARM), an estrogen receptor agonist, an estrogen receptor antagonist, a Selective Estrogen Receptor Modulator (SERM), a glucocorticoid receptor antagonist, a glucocorticoid receptor agonist, a Selective Glucocorticoid Receptor Modulator (SGRM), a progesterone receptor antagonist, a progesterone receptor agonist, a Selective Progesterone Receptor Modulator (SPRM), or a combination thereof. Epitopes targeting nuclear steroid receptors are generally capable of associating with IC₅₀Less than about 500nM, smallAt about 400nM, less than about 300nM, less than about 200nM, less than about 100nM or EC₅₀Less than about 1 μ M, less than about 900nM, less than about 800nM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 3400nM, less than about 200nM, less than about 100nM of nuclear steroid receptor binding.

In certain embodiments, the epitope that targets the nuclear steroid receptor is an agonist at the androgen receptor. In certain embodiments, the epitope that targets the nuclear steroid receptor is an antagonist at the androgen receptor.

In certain embodiments, the epitope that targets the nuclear steroid receptor is steroidal (e.g., dihydrotestosterone). In certain embodiments, the epitope that targets the nuclear steroid receptor is nonsteroidal (e.g., enzalutamide, apalutamide, and bicalutamide).

The analogs are derived from epitopes described herein known to target nuclear steroid receptors, and are modified to bind to at least one nuclear steroid payload, optionally via a linking moiety. Even after modification to achieve the compounds described herein, the analogs maintain biological activity, similar to that observed in the original unmodified epitope targeting the nuclear steroid receptor. In certain embodiments, the analog exhibits at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of the binding activity or inhibition observed for the initially unmodified epitope that targets the nuclear steroid receptor.

In certain embodiments, the analogs are derived from epitopes known to target nuclear receptors, such as epitopes known to target nuclear steroid receptors. In certain embodiments, the term "derived from" as used in reference to an epitope that targets a nuclear receptor means that at most one non-hydrogen atom of an initially unmodified nuclear receptor-targeting compound (i.e., a compound known to target a nuclear steroid receptor) is replaced by a covalent bond, optionally linked to a nuclear payload via a linking moiety. Exemplary non-hydrogen atoms include, but are not limited to, -CH ₃-OH, ═ O and-NH₂. In certain embodiments, the term "derived from" as used in reference to an epitope that targets a nuclear receptor means that the originalAt most one non-hydrogen atom of an unmodified compound targeting a nuclear receptor (i.e., a compound known to target a nuclear steroid receptor) is replaced by a covalent bond, optionally linked to a nuclear payload via a linking moiety. In certain embodiments, one hydrogen atom of a heteroatom (e.g., N, O or S) bound to an initially unmodified nuclear receptor targeting compound (i.e., a compound known to target nuclear steroid receptors) is replaced with a covalent bond, optionally via a linking moiety, to the core payload.

In certain embodiments, the epitope that targets a nuclear steroid receptor is an epitope that targets an androgen receptor. As used herein, the term "epitope targeting an androgen receptor" is intended to refer to the portion of a compound that binds to an androgen receptor agonist or androgen receptor antagonist (including a partial androgen receptor agonist or partial androgen receptor antagonist) and is capable of shuttling the compound from the cytoplasm into the cell nucleus. "Androgen Receptor (AR)" also known as nuclear receptor subfamily 3, group C, member 4 (NR 3C4), is a type of nuclear receptor that is capable of translocating androgens into the nucleus when activated by binding androgen receptor binders (e.g., androgens such as testosterone or dihydrotestosterone) in the cytoplasm.

Exemplary androgen receptor targeting epitopes that can be used in the compounds described herein include, but are not limited to, androgen receptor agonists, Selective Androgen Receptor Modulators (SARMs) (e.g., inobocavirus), androgen receptor antagonists (e.g., bicalutamide, flutamide, nilutamide, or enzalutamide), Selective Estrogen Receptor Modulators (SERMs) (e.g., tamoxifen, toremifene, or raloxifene), estrogen receptor antagonists (e.g., fulvestrant), progestins (e.g., megestrol acetate), estrogens (e.g., estramustine), ketoconazole, abiraterone, dalulomide, or analogs thereof.

In certain embodiments, the epitope that targets a nuclear steroid receptor is a Selective Androgen Receptor Modulator (SARM). In certain embodiments, the compound comprises at least one epitope that targets a nuclear steroid receptor, which independently comprises an epitope derived from: testosterone, testosterone esters (e.g., testosterone enanthate, propionate, cypionic acid, etc. or analogs thereof), enoboxate, BMS-564929, PS178990, LGD-4033 (Liglycindello), LGD-2941, AC-262,356, JNJ-28330835, JNJ-37654032, JNJ-26146900, LGD-2226, LGD-3303, LGD-121071, LG-120907, S-40503, S-23, RAD-140, acetylbehenamide, adarine (S-4), LG-121071, TFM-4AS-1, YK-11, MK-0773(PF-05314882), GSK2849466, GSK2881078, GSK8698, GSK4336, ACP-105, TT701, LY 242424245873, 1- (2-hydroxy-2-methyl-3-phenoxypropionyl) -indoline-4-carbonitrile-derivatives (J.chem.2014, 57(6),2462-71) or the like.

In certain embodiments, a single atom on an epitope of a targeted nuclear receptor as disclosed herein is replaced to attach to the rest of the compound (e.g., moieties a-L of formula I and formula II). In certain embodiments, the halogen atom on the epitope of the targeted nuclear receptor disclosed herein is replaced to attach to the rest of the compound. In certain embodiments, a hydrogen atom on an epitope of a targeted nuclear receptor disclosed herein is replaced to attach to the rest of the compound. In certain embodiments, the hydrogen atom is on a heteroatom. In certain embodiments, the hydrogen atom is on nitrogen. In certain embodiments, the hydrogen atom is on oxygen. In certain embodiments, the hydrogen atom is on carbon.

In certain embodiments, moiety B of formula I or formula II comprises at least one nuclear receptor targeting epitope derived from:

or a stereoisomer thereof, or a mixture of stereoisomers, or an analog thereof.

These and other Selective Androgen Receptor Modulators (SARMs) that can be used as epitopes for targeted nuclear steroid receptors in the compounds described herein can be found in US 6,462,038, US 6,777,427, WO2001/027086, WO2004/013104, WO2004/000816, WO2004/0113309, US2006/0211756, US2006/0063819, US2005/245485, US2005/250741, US2005/277681, WO2006/060108, WO2004/041277, WO2003/034987, US2006/0148893, US2006/0142387, WO2005/000795, WO2005/085185, WO2006/133216, WO2006/044707, WO2006/124447, WO2007/002181, WO2005/108351, WO2005/115361 and US 2006/0160845.

In certain embodiments, the epitope that targets the nuclear steroid receptor is a Selective Estrogen Receptor Modulator (SERM). In certain embodiments, the compound comprises at least one epitope that targets a nuclear steroid receptor, which independently comprises an epitope derived from: diacetylnorcarb, bazedoxifene, bromphenpromestrene (Acnestrol), clomiphene (Clomid), cyclofennel (Sexovid), lasofoxifene (Fabryn), oxymetaxifene (Sentolone, Novex-DS, Sevista), ospemifene (ospemifene, deaminohydroxy toremifene), raloxifene (Eista), tamoxifen (Nolvadex), toremifene (Faleton; 4-chlorottamoxifen), acobiprofen, alfesifene (4-hydroxy tamoxifen; a metabolite of tamoxifen), elargualin, enclomiphene ((E) -clomiphene), tamoxifen (4-hydroxy-N-demethyltamoxifen; a metabolite of tamoxifen), clomiphene ((Z) -miphene), clomiphene, Arloxifen, Aristoxifen, N-clomiphene (a metabolite of clomiphene; metabolites of clomiphene; oxygen-N-oxide of clomip, Droloxifene (3-hydroxytamoxifene), etacetin, fispemifene, GW-7604 (4-hydroxyetacetin), idoxifene (pyrrolidinyl-4-iodotamoxifen), levomeloxifene ((L) -oxymifene), milofloxacin, naproxidine, nifemifene (CI-628), panomifene, pennoxifene (ERA-923), troloxifene, raloxifene, LY117018, onapristone, faletone (toremifene citrate), or indoxifene (D-16726), or an analog thereof.

In certain embodiments, the SERM is structurally classified as a triphenylethylene (tamoxifen, clomiphene, toremifene, droloxifene, idoxifene, ospemifene, fispemifene, alfifene, etc. or analogs thereof), a benzothiophene (raloxifene, arzoxifene, etc. or analogs thereof), an indole (bazedoxifene, indoxifene, pentoxifene, etc. or analogs thereof), a tetrahydronaphthalene (lasofoxifene, naproxidine, etc. or analogs thereof), or a benzopyran (acobiprofen, oxybenzone, levomeloxifene, etc. or analogs thereof).

In certain embodiments, the epitope that targets the nuclear steroid receptor is a selective estrogen receptor down-regulator (SERD). In certain embodiments, the compound comprises at least one epitope that targets a nuclear steroid receptor, which independently comprises an epitope derived from fulvestrant, ARN-810, GW5638, or GW 7604.

In certain embodiments, the epitope that targets the nuclear steroid receptor is a Selective Progesterone Receptor Modulator (SPRM). In certain embodiments, the compound comprises at least one epitope that targets a nuclear steroid receptor, which independently comprises an epitope derived from: ulipristal acetate, soprenil (J867), mifepristone, torasemide (CDB-4124, Proellex, Progenta) or analogs thereof.

In certain embodiments, the compound comprises at least one epitope that targets a nuclear steroid receptor, which independently comprises an epitope derived from: estrogen, estetrol, estriol, estrone, progesterone, enoboxate, bicalutamide, apalutamide, testosterone, dihydrotestosterone, estradiol, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, bazedoxifene, ospemifene, megestrol acetate, estramustine, abiraterone, LGD-2941, BMS-564929, osthole, or an analog thereof.

In certain embodiments, the at least one nuclear steroid receptor targeting epitope is an androgen receptor targeting epitope and comprises:

or a stereoisomer thereof, or a mixture of stereoisomers, or the like, wherein the wavy line indicates the point of attachment to the linking moiety or the nuclear payload.

In certain embodiments, the at least one nuclear steroid receptor targeting epitope is an estrogen receptor targeting epitope and comprises:

or a stereoisomer thereof, or a mixture of stereoisomers, or the like, wherein the wavy line indicates the point of attachment to the linking moiety or the nuclear payload.

In certain embodiments, the epitope that targets the nuclear steroid receptor is not or does not comprise a peptide, protein, nanoparticle, or antibody.

Exemplary compounds provided by the present disclosure include, but are not limited to, compounds as shown in table 1, or stereoisomers, mixtures of stereoisomers, hydrates, solvates, isotopically enriched analogs, or pharmaceutically acceptable salts thereof.

TABLE 1

Method of treatment

Provided herein are compounds useful for treating, preventing, and/or delaying the onset and/or development of cancer. Thus, in certain embodiments, there is provided a method for treating cancer, the method comprising administering to an individual in need of treatment a therapeutically effective amount of a compound or composition described herein. Certain embodiments provide a method of enhancing cytotoxic cancer therapy in a subject in recognized chronic need of such treatment comprising administering to the subject a therapeutically acceptable amount of a compound or composition described herein.

It is contemplated that patients with any cancer may benefit from treatment with the compounds and compositions described herein. Thus, in certain embodiments, the cancer is liver cancer, melanoma, hodgkin's disease, non-hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, neuroblastoma, breast carcinoma, ovarian carcinoma, lung carcinoma, wilms 'tumor, cervical carcinoma, testicular carcinoma, soft tissue sarcoma, chronic lymphocytic leukemia, primary macroglobulinemia, bladder carcinoma, chronic myelogenous leukemia, primary brain carcinoma, malignant melanoma, small cell lung carcinoma, gastric carcinoma, colon carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, malignant melanoma, choriocarcinoma, mycosis fungoides, head and neck carcinoma, osteogenic sarcoma, pancreatic carcinoma, acute myelogenous leukemia, hairy cell leukemia, rhabdomyosarcoma, kaposi's sarcoma, urogenital carcinoma, Thyroid cancer, esophageal carcinoma, malignant hypercalcemia, cervical hyperplasia, renal cell carcinoma, endometrial cancer, polycythemia vera, essential thrombocythemia, adrenocortical carcinoma, skin cancer, or prostate carcinoma. In certain embodiments, the cancer is bladder cancer, blood cancer (e.g., leukemia (e.g., chronic leukemia, Chronic Lymphocytic Leukemia (CLL), etc.)) or lymphoma (e.g., hodgkin's lymphoma, non-hodgkin's lymphoma, low grade lymphoma, high grade lymphoma), lung cancer (e.g., small cell lung cancer), breast cancer, fallopian tube cancer, glioblastoma multiforme, head and neck cancer, esophageal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, prostate cancer, testicular cancer, skin cancer (e.g., melanoma), or uterine cancer. In certain embodiments, the cancer is bladder cancer, breast cancer, fallopian tube cancer, ovarian cancer, prostate cancer, peritoneal cancer, testicular cancer, endometrial cancer, or uterine cancer.

In certain embodiments, the compounds and compositions as described herein are tailored to target cancers that overexpress a particular nuclear receptor, such as but not limited to, the androgen receptor, estrogen receptor, progesterone receptor, and/or glucocorticoid receptor, by including an epitope that targets the particular receptor. The epitope may be derived from a steroid hormone or any non-steroid drug which targets the particular receptor.

In certain embodiments, there is provided a method of treating or preventing a cancer that overexpresses an androgen receptor, the method comprising administering to a subject in need thereof an effective amount of a compound, or a pharmaceutically acceptable salt or solvate thereof, comprising at least one nuclear payload and at least one epitope that targets the androgen receptor. Particular cancers contemplated to be treated by such methods include, but are not limited to, prostate cancer, breast cancer, triple negative breast cancer, bladder cancer, or liver cancer. Also provided is a method of treating or preventing metastatic castration resistant prostate cancer (mCRPC), the method comprising administering to a subject in need thereof an effective amount of a compound or composition as described herein, or a pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, there is provided a method of treating or preventing a cancer that overexpresses an androgen receptor, the method comprising administering to a subject in need thereof an effective amount of a compound, or a pharmaceutically acceptable salt or solvate thereof, comprising at least one nuclear payload and at least one epitope that targets the androgen receptor. In certain embodiments, the cancer is prostate cancer, breast cancer, triple negative breast cancer, bladder cancer, or liver cancer. In certain embodiments, the epitope that targets an androgen receptor comprises an androgen receptor agonist, a Selective Androgen Receptor Modulator (SARM), an androgen receptor antagonist, a Selective Estrogen Receptor Modulator (SERM), an estrogen receptor antagonist, a progestin, or an estrogen. In certain embodiments, the epitope that targets the androgen receptor comprises enoboxate, bicalutamide, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, estramustine, ketoconazole, abiraterone, dalulomide, or an analog thereof. In certain embodiments, the epitope that targets the androgen receptor comprises enoboxate, bicalutamide, flutamide, nilutamide, enzalutamide, tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, estramustine, ketoconazole, abiraterone or an analog thereof. In certain embodiments, the nuclear payload comprises a PARP inhibitor.

In certain embodiments, there is provided a method of treating or preventing cancer that overexpresses estrogen and/or progesterone receptors, the method comprising administering to a subject in need thereof an effective amount of a compound, or a pharmaceutically acceptable salt or solvate thereof, comprising at least one nuclear payload and at least one epitope that targets estrogen and/or progesterone receptors. Particular cancers contemplated to be treated by such methods include, but are not limited to, breast, uterine or ovarian cancer.

In certain embodiments, there is provided a method of treating or preventing a cancer that overexpresses a glucocorticoid receptor, the method comprising administering to a subject in need thereof an effective amount of a compound comprising at least one nuclear payload and at least one epitope that targets the glucocorticoid receptor, or a pharmaceutically acceptable salt or solvate thereof. Particular cancers contemplated to be treated by such methods include, but are not limited to, breast, uterine or ovarian cancer. Particular cancers contemplated for treatment by such methods include, but are not limited to, prostate cancer, possibly breast cancer, uterine cancer, ovarian cancer.

Breast cancers include Ductal Carcinoma In Situ (DCIS) and invasive breast cancers. Breast cancer can occur in the milk ducts, milk-producing lobules and connective tissue. Breast cancer includes Estrogen Receptor (ER) negative and Hormone Receptor (HR) negative and can also be classified as group 3 (HER-2 positive) or group 4 (basal-like).

Prostate cancer is a cancer that develops in the glandular prostate in the male reproductive system. It occurs when prostate cells mutate and begin to multiply uncontrollably. These cells may be transferred from the prostate to almost any other part of the body (metastatic prostate cancer), especially the bones and lymph nodes, but also the kidneys, bladder and even the brain and other tissues. Prostate cancer can lead to painful urination, difficulty, problems with sexual intercourse, erectile dysfunction. Other symptoms may appear later in the disease. The detection rates for prostate cancer vary widely throughout the world, with south and east asia being detected less frequently than in europe, and particularly in the united states. Prostate cancer is most common in men over the age of fifty years and is one of the most common cancers in men. However, many men with prostate cancer are never symptomatic, have not been treated at all, and eventually die from other causes. This is because prostate cancer grows slowly in most cases and most are affected by over 60 years of age. Thus, they often die of causes unrelated to prostate cancer. The development of prostate cancer involves a number of factors, including genetics and diet. The presence of prostate cancer may be indicated by symptoms, physical examination, Prostate Specific Antigen (PSA), or biopsy. Concerns arise regarding the accuracy of PSA testing and its utility in screening. Suspected prostate cancer is typically confirmed by biopsy of the prostate and examination under a microscope. Further tests, such as CT scans and bone scans, can be performed to determine whether prostate cancer has spread. Also contemplated are combinations with primary surgery and radiotherapy or other treatments, such as hormone therapy, chemotherapy, proton therapy, cryosurgery, High Intensity Focused Ultrasound (HIFU).

Certain embodiments provide a method of inhibiting PARP in an individual in recognized need of such treatment comprising administering to the individual a therapeutically acceptable amount of a compound or composition described herein. In one embodiment, provided herein is a method of treating a disease ameliorated by the inhibition of PARP, comprising administering to an individual in need of treatment a therapeutically effective amount of a compound or composition described herein.

Certain embodiments provide a method of treating leukemia, colon cancer, glioblastoma, lymphoma, melanoma, breast carcinoma, or cervical cancer in a subject in recognized need of such treatment, the method comprising administering to the subject a therapeutically acceptable amount of a compound or composition described herein.

In some embodiments, provided herein is a method of treating a cancer that is deficient in a Homologous Recombination (HR) -dependent DNA Double Strand Break (DSB) repair pathway comprising administering to an individual in need of treatment a therapeutically effective amount of a compound or composition described herein. In certain embodiments, the cancer comprises one or more cancer cells having a reduced or eliminated ability to repair DNA DSBs by HR relative to normal cells. In some embodiments, the cancer cell has a BRCA1 or BRCA2 deficient phenotype. In some embodiments, the cancer cell lacks BRCA1 or BRCA 2. In some embodiments, the methods provided herein relate to the treatment of individuals who are heterozygous for a mutation in a gene encoding a component of the HR dependent DNA DSB repair pathway. In certain embodiments, the individual is heterozygous for a mutation in BRCA1 and/or BRCA 2. In some embodiments, the method of treating cancer comprises treatment of breast, ovarian, pancreatic and/or prostate cancer. In some embodiments, the method of treating cancer further comprises administering ionizing radiation or a chemotherapeutic agent.

The main function of the DNA mismatch repair (MMR) system is to eliminate single base mismatches and insertion-deletion loops that may occur during DNA replication. Insertion-deletion loops are caused by the acquisition or loss of short repeat units within the microsatellite sequence, also known as microsatellite instability (MSI). At least six different MMR proteins are required. For mismatch recognition, MSH2 protein forms a heterodimer with MSH6 or MSH3, depending on the type of lesion to be repaired (correction of single base mismatch requires MSH6, whereas both MSH3 and MSH6 may help correct insertion-deletion loops). The heterodimers of MLH1 and PMS2 coordinate mismatch recognition complexes with other proteins essential for MMR. These additional proteins may include at least exonuclease 1(EXO1), potential helicases, Proliferating Cell Nuclear Antigen (PCNA), single stranded DNA binding protein (RPA), and DNA polymerases δ and ∈. In addition to PMS2, MLH1 can heterodimerize with two additional proteins MLH3 and PMS 1. Recent observations indicate that PMS2 is necessary to correct single base mismatches, and that both PMS2 and MLH3 contribute to correct insertion-deletion loops. It is known that additional homologues of human MMR protein are necessary for functions other than MMR. These proteins include MSH4 and MSH5, which are essential for meiotic (and possibly mitotic) recombination, but cannot be assumed to be involved in MMR.

Germline mutations in the human MMR gene result in a predisposition to hereditary non-polyposis colon cancer (HNPCC), one of the most common cancer syndromes in humans. Excess colon cancer and a specific range of extra-colon cancers that are diagnosed at an early stage and spread with an autosomal dominant signature constitute the clinical definition of the syndrome. MSI is a marker of HNPCC, which also occurs in approximately 15% to 25% of sporadic tumors of the colorectal and other organs. According to international standards, a high MSI (MSI-H) is defined as the instability of two or more of the five loci, or ≧ 30% to 40% of all microsatellite loci studied, while the instability of fewer loci is termed MSI Low (MSI-L). In non-HNPCC cancers (e.g., breast, prostate and lung cancers), MSI occurs in a high proportion (2% to 50% of the tumor). Depending on the proportion of unstable markers, MSS, MSI-L and MSI-H can be distinguished in these cancers similarly to HNPCC cancers. In one embodiment, is a method of treating cancer that is deficient in mismatched DNA repair pathways. In another embodiment, is a method of treating a cancer that exhibits microsatellite instability due to a reduction or impairment of DNA repair pathways. In another embodiment, is a method of treating a cancer that exhibits genomic instability due to a reduction or impairment of a DNA repair pathway.

In certain embodiments, the compounds or compositions described herein may be used in the preparation of a medicament for treating a cancer that lacks Homologous Recombination (HR) -dependent DNA Double Strand Break (DSB) repair activity, or for treating a patient having a cancer that lacks HR-dependent DNA DSB repair activity, the treatment comprising administering to the patient a therapeutically effective amount of the compound or composition.

The HR-dependent DNA DSB repair pathway repairs double-strand breaks (DSBs) in DNA via a homologous mechanism to reform a continuous DNA helix. Components of HR-dependent DNA DSB repair pathways include, but are not limited to, ATM (NM _000051), RAD51(NM _002875), RAD51L1(NM _002877), RAD51C (NM _002876), RAD51L3(NM _002878), DMC1(NM _007068), XRCC2(NM _005431), XRCC3(NM _005432), RAD52(NM _002879), RAD54 002879 (NM _012415), BRCA 002879 (NM _002879), RAD 002879 (NM _002879), and NBS11 002879 _00248_ 5). Other proteins involved in the HR-dependent DNA DSB repair pathway include regulatory factors such as EMSY (Wood et al, Science, 291,1284-1289 (2001); Khanna et al, Nature genetics, Nature, Genet 27(3):247-254 (2001); and Hughes-Davies, et al, Cell (Cell), 115, 523-535).

In some embodiments, a cancer that lacks HR-dependent DNA DSB repair comprises one or more cancer cells that have a reduced or eliminated ability to repair DNA DSBs via a pathway relative to normal cells, i.e., the activity of the HR-dependent DNA DSB repair pathway is reduced or eliminated in the one or more cancer cells.

In certain embodiments, the activity of one or more components of the HR dependent DNA DSB repair pathway is abolished in one or more cancer cells of an individual having a cancer that lacks HR dependent DNA DSB repair. The components of the HR dependent DNA DSB repair pathway include those listed above.

In some embodiments, the cancer cell has a BRCA1 and/or BRCA2 deficient phenotype, i.e., BRCA1 and/or BRCA2 activity is reduced or eliminated in the cancer cell. In certain embodiments, the expression and/or activity of BRCA1 and/or BRCA2, i.e., BRCA1 and/or BRCA2, is reduced or eliminated in cancer cells, e.g., by virtue of a mutation or polymorphism in the encoding nucleic acid, or by virtue of an amplification, mutation or polymorphism in a gene encoding a regulatory factor, e.g., the EMSY gene encoding a BRCA2 regulatory factor, or by an epigenetic mechanism, e.g., gene promoter methylation.

BRCA1 and BRCA2 are tumor suppressors whose wild type alleles are often lost in tumors heterozygous for the vector. BRCA1 and/or BRCA2 mutations are associated with breast cancer. Amplification of the EMSY gene encoding the BRCA2 binding factor is associated with breast and ovarian Cancer (Jasin M., Oncogene (Oncogene), 21(58),8981-93(2002), Tutt et al, Trends in molecular medicine (Med., 8(12),571-6 (2002), and Radice, P.J., journal of Experimental and clinical Cancer research (Exp Clin Cancer Res.), 21(3 supple.), 9-12 (2002)).

Mutated vectors of BRCA1 and/or BRCA2 are also at higher risk for ovarian, prostate, and pancreatic cancers.

In some embodiments, the individual is heterozygous for one or more variations (e.g., mutations and polymorphisms) in BRCA1 and/or BRCA2, or a modulator thereof. Detection of variation in BRCA1 and BRCA2 is described, for example, in EP 699754, EP 705903, Neuhausen, s.l. and Ostrander, e.a., "genetic testing (genet.test"), 1,75-83 (1992); janatova M., et al, "tumors (Neoplama)," 50(4), "246-50" (2003). The determination of the amplification of the BRCA2 binding factor EMSY is described in Hughes-Davies, et al, cell 115, 523-535.

In some cases, cancer-associated mutations and polymorphisms are detected at the nucleic acid level by detecting the presence of a variant nucleic acid sequence, or at the protein level by detecting the presence of a variant (i.e., a mutant or allelic variant) polypeptide.

In certain embodiments, the compounds described herein are expected to be suitable for patients who have relapsed or become refractory. The term "recurrence" refers to the reoccurrence or regrowth of the disease (or cancer) after a period of remission. The term "refractory" is used to describe a condition in which the cancer does not respond to treatment or does not respond to treatment for a prolonged period of time. For example, the compounds herein may be useful for treating cancer in patients who have been previously treated with the cancer therapies described herein (e.g., enzalutamide).

Composition comprising a metal oxide and a metal oxide

The present disclosure encompasses compositions, including pharmaceutical compositions, of any of the compounds detailed herein. Accordingly, provided herein are pharmaceutical compositions comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions provided herein can be in a form suitable for oral, buccal, parenteral (e.g., intravenous, intramuscular, infusion, or subcutaneous injection), nasal, topical, or rectal administration, or in a form suitable for administration by inhalation.

In one aspect, a compound as described herein can be in a purified form. Compositions, e.g., compositions of substantially pure compounds, comprising a compound or salt thereof as described herein are provided. In some embodiments, a composition comprising a compound or salt thereof as described herein is in a substantially pure form. Unless otherwise indicated, "substantially pure" refers to a composition that does not contain more than 35% impurities, wherein the impurities represent compounds other than the desired compound or salt thereof that comprises the majority of the composition. In one variation, a composition of substantially pure compound or salt thereof is provided, wherein the composition contains no more than 25% impurities. In another variation, a composition of a substantially pure compound or salt thereof is provided, wherein the composition contains or does not exceed 20% impurities. In yet another variation, a composition of a substantially pure compound or salt thereof is provided, wherein the composition contains or does not exceed 10% impurities. In another variation, a composition of a substantially pure compound or salt thereof is provided, wherein the composition contains or does not exceed 5% impurities. In another variation, a composition of a substantially pure compound or salt thereof is provided, wherein the composition contains or does not exceed 3% impurities. In yet another variation, a composition of a substantially pure compound or salt thereof is provided, wherein the composition contains or does not exceed 1% impurities. In another variation, a composition of a substantially pure compound or salt thereof is provided, wherein the composition contains or does not exceed 0.5% impurities.

In certain embodiments, the pharmaceutical compositions are formulated in any manner, including the use of one or more physiologically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of the active compounds into pharmaceutical compositions. In some embodiments, the appropriate formulation depends on the route of administration selected. In various embodiments, any technique, carrier, and excipient is suitably used.

Provided herein are pharmaceutical compositions comprising a compound described herein and a pharmaceutically acceptable diluent, excipient, and/or carrier. In addition, in some embodiments, the compounds described herein are administered as pharmaceutical compositions, wherein the compounds described herein are mixed with other active ingredients, as in combination therapy.

As used herein, a pharmaceutical composition refers to a mixture of a compound described herein with other chemical ingredients, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and/or excipients. In certain embodiments, the pharmaceutical composition facilitates administration of the compound into an organism. In some embodiments, the methods of treatment or use provided herein are practiced comprising administering or using a pharmaceutical composition comprising a therapeutically effective amount of a compound provided herein. In particular embodiments, the methods of treatment provided herein comprise administering such pharmaceutical compositions to a mammal having a disease or condition to be treated. In one embodiment, the mammal is a human. In some embodiments, the therapeutically effective amount will vary greatly depending on the severity of the disease, the age and relative health of the individual, the potency of the compound used, and other factors. In various embodiments, the compounds described herein are used alone or in combination with one or more therapeutic agents as components of a mixture.

In certain embodiments, the pharmaceutical compositions provided herein are formulated for intravenous injection. In certain aspects, the intravenous injection formulations provided herein are formulated as aqueous solutions, and in some embodiments, as physiologically compatible buffers, such as Hank's solution, Ringer's solution, or saline buffer. In certain embodiments, the pharmaceutical compositions provided herein are formulated for transmucosal administration. In certain aspects, the transmucosal formulation includes a penetrant appropriate to the barrier to be permeated. In certain embodiments, the pharmaceutical compositions provided herein are formulated for other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, and in one embodiment, together with physiologically compatible buffers or excipients.

In certain embodiments, the pharmaceutical compositions provided herein are formulated for oral administration. In certain aspects, oral formulations provided herein comprise a compound described herein, formulated with a pharmaceutically acceptable carrier or excipient. Such carriers enable the compounds described herein to be formulated as tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

In some embodiments, the pharmaceutical composition for oral use is obtained by: the process comprises mixing one or more solid excipients with one or more compounds described herein, optionally grinding the resulting mixture, and, if necessary, after adding suitable auxiliaries, processing the mixture of granules to obtain tablets or dragee cores. Suitable excipients include, inter alia, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, microcrystalline cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; or other excipients, such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents are optionally added, such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar or alginic acid, or salts thereof, such as sodium alginate.

In certain embodiments, provided herein are pharmaceutical compositions formulated as dragee cores with suitable coatings. In certain embodiments, concentrated sugar solutions are used to form suitable coatings and optionally contain acacia (gum arabic), talc, polyvinylpyrrolidone, carbopol gel (carbopol gel), polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. In some embodiments, dyes and/or pigments are added to the tablets, dragees and/or their coatings for use, for example, in identifying or characterizing different combinations of active compound doses.

In certain embodiments, pharmaceutical compositions for oral use include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. In some embodiments, push-fit capsules contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In certain embodiments, in soft capsules, the active compound is dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, a stabilizer is optionally added. In certain embodiments, formulations for oral administration are in dosages suitable for such administration.

In certain embodiments, the pharmaceutical compositions provided herein are formulated for buccal or sublingual administration. In certain embodiments, the buccal or sublingual composition is in the form of a tablet, lozenge, or gel formulated in a conventional manner. In certain embodiments, parenteral injection involves rapid injection or continuous infusion. In some embodiments, formulations for injection are provided in unit dosage form, e.g., in ampoules or multi-dose containers with an added preservative. In some embodiments, the pharmaceutical compositions described herein are in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and optionally contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In some embodiments, suspensions of the active compounds are prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes. In certain embodiments, the aqueous injection suspension contains a substance that increases the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension also contains suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. In an alternative embodiment, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

In some embodiments, the compounds described herein are administered topically. In particular embodiments, the compounds described herein are formulated into a variety of topically administrable compositions, such as solutions, suspensions, emulsions, gels, pastes, medicated sticks, balms, creams, or ointments. Such pharmaceutical compounds optionally contain solubilizers, stabilizers, tonicity enhancing agents, buffers and/or preservatives.

In certain embodiments, the pharmaceutical compositions provided herein are formulated for transdermal administration of a compound described herein. In some embodiments, administering such compositions employs transdermal delivery devices and transdermal delivery patches. In certain embodiments, the composition is a lipophilic emulsion or buffered aqueous solution, dissolved and/or dispersed in a polymer or binder. Such patches include patches configured to deliver a pharmaceutical agent continuously, in pulses, or on demand. In some embodiments, transdermal delivery of a compound described herein is achieved through the use of iontophoresis patches and the like. In certain embodiments, the rate of absorption is slowed by the use of a rate controlling membrane or by entrapping the compound within a polymer matrix or gel. Instead, absorption enhancers are optionally used to increase absorption. The absorption enhancer or carrier includes absorbable pharmaceutically acceptable solvents that aid in passing the compound through the skin. For example, a transdermal device is in the form of a bandage comprising a backing element, a reservoir containing a compound and optionally having a carrier, optionally having a rate control barrier for delivering the compound to the skin of a subject at a controlled and predetermined rate over an extended period of time, and means for securing the device to the skin.

In certain embodiments, the pharmaceutical compositions provided herein are formulated for administration by inhalation. In certain embodiments, in such pharmaceutical compositions formulated for inhalation, the compounds described herein are in the form of an aerosol, mist, or powder. In some embodiments, the pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In certain aspects of pressurized aerosols, the dosage unit is determined by providing a valve for delivering a metered amount. In certain embodiments, capsules and cartridges of, for example, gelatin (by way of example only) suitable for use in an inhaler or insufflator are formulated containing a powder mix of a compound described herein and a suitable powder base such as lactose or starch.

In some embodiments, the compounds described herein are formulated in rectal compositions, such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, colloidal suppositories, or retention enemas. In certain embodiments, rectal compositions optionally contain conventional suppository bases such as cocoa butter (cocoa butter) or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In certain suppository forms of the composition, a low melting wax, such as, but not limited to, a mixture of fatty acid glycerides, is first melted, optionally in combination with cocoa butter.

In various embodiments provided herein, pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers (including excipients and auxiliaries) that facilitate processing of the active compounds into pharmaceutically acceptable formulations. In certain embodiments, the appropriate formulation depends on the chosen route of administration. In various embodiments, any of techniques, carriers, and excipients are suitably used. In some embodiments, pharmaceutical compositions comprising a compound described herein are manufactured in a conventional manner, such as, for example only, by means of conventional mixing, dissolving, granulating, dragee-making, watermilling, emulsifying, encapsulating, enrobing or compressing processes.

In certain embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier, diluent or excipient described herein and a compound, which is described herein as an active ingredient in free acid or free base form or in pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also referred to as polymorphs), and active metabolites of these compounds that have the same type of activity. In some cases, the compounds described herein exist in tautomeric forms. All tautomers are included within the scope of the compounds presented herein. Additionally, both solvated and unsolvated forms of the compounds described herein are included herein. Solvated compounds include compounds that are solvated with pharmaceutically acceptable solvents such as water, ethanol, and the like. Solvated forms of the compounds presented herein are also considered disclosed herein. In some embodiments, the pharmaceutical compositions described herein include other pharmaceutical agents or agents, carriers, adjuvants, such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters, salts for regulating osmotic pressure, and/or buffers. In additional embodiments, the pharmaceutical compositions described herein also contain other therapeutically valuable substances.

Methods for preparing compositions containing the compounds described herein include formulating the compounds with one or more inert pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions in which the compounds are dissolved, emulsions comprising the compounds, or solutions containing liposomes, micelles, or nanoparticles comprising the compounds as disclosed herein. Semi-solid compositions include, but are not limited to, gels, suspensions, and creams. In various embodiments, the compositions are in liquid solutions or suspensions, in solid form suitable for solution or suspension in a liquid prior to use, or in the form of an emulsion. These compositions optionally contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like.

In some embodiments, a combination comprising a compound described herein is in the form of a liquid, wherein the agent is present in solution, in suspension, or both. In some embodiments, when the composition is applied in solution or suspension, a first portion of the agent is present in solution and a second portion of the agent is present in particulate form in suspension in the liquid matrix. In some embodiments, the liquid composition comprises a gel formulation. In other embodiments, the liquid composition is aqueous.

Useful aqueous suspensions optionally contain one or more polymers as suspending agents. Useful polymers include water soluble polymers, such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; and water-insoluble polymers, such as polymers containing cross-linked carboxyl groups. Useful compositions optionally comprise mucoadhesive polymers selected from, for example, carboxymethylcellulose, carbomers (acrylic acid polymers), poly (methyl methacrylate), polyacrylamides, polycarbophil (polycarbophil), acrylic acid/butyl acrylate copolymers, sodium alginate, and dextran.

Useful compositions optionally include a solubilizing agent that aids in the solubility of the compounds described herein. The term "solubilizing agent" generally includes agents that cause the formation of a micellar or true solution of the agent. Solubilizers include certain acceptable nonionic surfactants (e.g., polysorbate 80) and ophthalmically acceptable glycols (e.g., polyethylene glycol 400) and glycol ethers.

Useful compositions optionally include one or more pH adjusting agents or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris; and buffers such as citrate/dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

Useful compositions optionally include one or more salts in an amount necessary to maintain the osmotic pressure of the composition within an acceptable range. Such salts include those having a sodium, potassium or ammonium cation and a chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anion; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

Certain useful compositions optionally include one or more preservatives that inhibit microbial activity. Suitable preservatives include mercury-containing materials, such as phenylmercuric borate and thimerosal; stabilizing the chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

Some useful compositions optionally include one or more surfactants that enhance physical stability for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol (octoxynol)10, octoxynol 40.

Certain useful compositions are optionally one or more antioxidants that enhance chemical stability when desired. By way of example only, suitable antioxidants include ascorbic acid and sodium metabisulfite.

In some embodiments, the aqueous suspension composition is packaged in a single-dose non-reclosable container. In an alternative embodiment, a multi-dose reclosable container is used, in which case a preservative is typically included in the composition.

In various embodiments, any delivery system for a hydrophobic drug compound is employed. Liposomes and emulsions are examples of delivery vehicles or carriers for hydrophobic drugs. In certain embodiments, certain organic solvents, such as N-methylpyrrolidone, are employed. In some embodiments, the compound is delivered using a sustained release system, such as a semipermeable matrix of a solid hydrophobic polymer containing the therapeutic agent. Various sustained release materials are used in the examples herein. In certain embodiments, the sustained release capsule releases the compound for several weeks to over 100 days. In some embodiments, other strategies for protein stabilization are employed depending on the chemical nature and biological stability of the therapeutic agent.

In certain embodiments, the formulations or compositions described herein benefit from and/or optionally comprise antioxidants, metal chelators, thiol-containing compounds, and other general stabilizers. Examples of such stabilizers include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1mM to about 10mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) a combination thereof.

Dosing and treatment regimens

In certain embodiments, compositions containing the compounds described herein are administered for prophylactic and/or therapeutic treatment. In certain therapeutic applications, the composition is administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. In some embodiments, an amount effective for such use will depend on the severity and course of the disease or condition, previous therapy, the patient's health, weight and response to the drug, and the diagnosis of the treating physician. In certain instances, the caregiver is deemed appropriate to determine such therapeutically effective amounts by routine experimentation (including, but not limited to, dose escalation clinical trials).

In certain prophylactic applications, compositions containing compounds described herein are administered to patients susceptible to or otherwise at risk of a particular disease, disorder, or condition. In some embodiments, the amount administered is defined as a "prophylactically effective amount or dose. In certain embodiments of this use, the precise amount of the compound administered will depend on the patient's health, weight, and the like. In some embodiments, the caregiver is deemed appropriate to determine such a prophylactically effective amount by routine experimentation (including, but not limited to, dose escalation clinical trials). In certain embodiments, when used in a patient, an amount effective for such use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health and response to the drug, and the diagnosis of the treating physician.

In certain instances, the condition of the patient is not improved or not significantly improved following administration of a compound or composition described herein, and the compound is optionally administered for an extended period of time, i.e., for an extended period of time, including during the entire duration of the patient's life, to improve or control or limit the symptoms of the disease or condition in the patient, at the discretion of the physician.

In certain cases where the patient's condition does or does not substantially improve, optionally continuously administering the compound, at the discretion of the physician; alternatively, the dose of drug administered is optionally temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). In certain embodiments, the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reduction during the drug holiday includes a reduction of about 10% to about 100%, including by way of example only, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

In certain embodiments, once the condition of the patient improves, a maintenance dose is administered as necessary. In some embodiments, the dose, e.g., maintenance dose or frequency of administration, or both, is reduced symptomatically to a level that maintains improvement in the disease, disorder, or condition. However, in certain embodiments, the patient is optionally given chronic intermittent treatment based on any recurrence of symptoms.

In certain embodiments, the amount of agent administered corresponding to an effective amount varies depending on factors such as the particular compound, the disease or condition and its severity, identity (e.g., body weight) of the individual or subject in need of treatment. However, in some embodiments, an effective amount is determined by the particular circumstances surrounding the case, including, for example, the particular agent administered, the route of administration, the condition being treated, and the individual or subject being treated. However, in certain embodiments, the dosage employed for treatment of an adult human is in the range of about 0.02 to about 5000mg per day, and in one particular embodiment about 1 to about 1500mg per day. In various embodiments, the desired dose is conveniently provided in a single dose form, or in divided dose forms administered simultaneously (or over a short period of time) or at appropriate intervals, e.g., two, three, four or more sub-doses per day.

In some embodiments, the pharmaceutical compositions described herein are in unit dosage forms suitable for single administration of precise dosages. In some cases, in unit dosage forms, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. In certain embodiments, the unit dose is in the form of a package containing discrete amounts of the formulation. Non-limiting examples are packed tablets or capsules and powders in vials or ampoules. In some embodiments, the aqueous suspension composition is packaged in a single-dose non-reclosable container. In an alternative embodiment, a multi-dose reclosable container is used, in which case a preservative is typically included in the composition. By way of example only, in some embodiments, formulations for parenteral injection are provided in unit dosage forms including, but not limited to, ampoules or multi-dose containers with a preservative added thereto.

In certain embodiments, a daily dose of about 0.01 to about 2.5mg/kg per body weight is suitable for use with the compounds described herein. In some embodiments, the specified daily dose in a larger individual, including but not limited to humans, is in the range of about 0.5mg to about 100mg, conveniently administered in divided doses, including but not limited to up to four times a day or in an extended release form. In certain embodiments, a unit dosage form suitable for oral administration contains from about 1 to about 50mg of the active ingredient. Due to the large number of variables for each treatment regimen, the above ranges are merely suggestive, and significant shifts from these recommended values are not uncommon. In certain embodiments, the dosage will vary according to a number of variables not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the needs of the individual, the severity of the disease or condition being treated, and the diagnosis of the physician.

In certain embodiments, byStandard pharmaceutical procedures in cell cultures or experimental animals to determine toxicity and therapeutic efficacy of such treatment regimens, including but not limited to determining LD₅₀(lethal dose for 50% of the population) and ED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD ₅₀With ED₅₀To each other. In certain embodiments, compounds exhibiting high therapeutic indices are preferred. In some embodiments, data obtained from cell culture assays and animal studies is used to formulate a range of dosage for human use. In particular embodiments, the dose of such compounds is within a range of circulating concentrations that include ED with minimal toxicity₅₀. In certain embodiments, the dosage varies within this range depending on the dosage form used and the route of administration used.

In certain embodiments, the disclosed compounds exhibit increased affinity, increased potency, or increased therapeutic index for a nuclear target as compared to an unmodified nuclear payload from which the compound is derived. In certain embodiments, such higher affinity, potency, or therapeutic index may provide benefits, such as allowing for lower doses to be administered and thus reducing toxicity potential, improving therapeutic index, and reducing overall cost of treatment. In certain embodiments, a daily dose suitable for administration of a compound described herein is less than 100% of the recommended daily dose for the unmodified nuclear payload, or less than about 90%, or less than about 80% or less than about 70% of the recommended daily dose for the unmodified nuclear payload, or less than about 60%, or less than about 50%, or less than about 40%, or about 20% to about 90%, or about 30% to about 90%, or about 40% to about 90%, or about 50% to about 90%, or about 60% to about 90%, or about 70% to about 90%, or about 20% to about 80%, or about 30% to about 80%, or about 40% to about 80%, or about 50% to about 80%, or about 60% to about 80%, or about 70% to about 80%, or about 20% to about 70%, or about 30% to about 70%, or about 40% to about 70%, or about 50% to about 70%, or about 60% to about 70%.

In certain embodiments, the compounds described herein are used in the preparation or manufacture of a medicament for treating a disease or condition mediated by the enzyme poly (ADP-ribose) polymerase (PARP) or wherein inhibiting the enzyme poly (ADP-ribose) polymerase (PARP) ameliorates the disease or condition. In some embodiments, the method for treating any of the diseases or conditions described herein in a subject in need of such treatment involves administering to the subject a pharmaceutical composition containing at least one compound described herein, or a pharmaceutically acceptable salt, pharmaceutically acceptable N-oxide, pharmaceutically active metabolite, pharmaceutically acceptable prodrug, or pharmaceutically acceptable solvate thereof, in a therapeutically effective amount.

Combination therapy

The compounds described herein (e.g., the compounds of table 1, formula I or formula II, or any other compound of the formulae disclosed herein) may also be used in combination with other active ingredients. Such combinations are selected based on the condition to be treated, the cross-reactivity of the ingredients, and the pharmacological properties of the combination. In one embodiment, the present disclosure provides the use of a compound as described herein in combination with another agent or therapy, e.g., another cancer treatment. For example, in the treatment of cancer, the composition may be combined with other anti-cancer compounds, such as paclitaxel (paclitaxel) or rapamycin.

It is also possible to combine a compound of the present disclosure with one or more other active ingredients in a unitary dosage form for simultaneous or sequential administration to a patient. The combination therapy may be administered in a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administration forms.

Combination therapy may provide "synergy" and "synergy", i.e., the effect obtained when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. When the active ingredients are: (1) co-formulated in a combined formulation and administered or delivered simultaneously; (2) delivered as separate formulations, alternately or in parallel; or (3) when delivered by some other protocol, synergy may be achieved. When delivered in alternation therapy, synergy may be obtained when the compounds are administered or delivered sequentially, e.g., in separate tablets, pills or capsules, or by different injections in separate syringes. Generally, during alternation therapy, the effective dose of each active ingredient is administered sequentially (i.e., consecutively), while in combination therapy, the effective doses of two or more active ingredients are administered together. Synergistic anticancer effect means an anticancer effect which is greater than the predicted purely additive effect of the individual compounds in combination.

Administration of the compounds and compositions of the present disclosure to patients will follow the general protocol for administration of chemotherapeutic agents, taking into account toxicity, if any. It is expected that the treatment cycle will be repeated as needed. It is also contemplated that various standard therapies or adjuvant cancer therapies as well as surgical intervention may be used in combination with the active agent. These therapies include, but are not limited to, chemotherapy, radiation therapy, immunotherapy, gene therapy, and surgery.

In some embodiments, provided herein is a method of treating cancer comprising administering to an individual in need of treatment a therapeutically effective amount of a compound or composition described herein and ionizing radiation or one or more chemotherapeutic agents. In some embodiments, the compounds described herein are administered concurrently with ionizing radiation or one or more chemotherapeutic agents. In other embodiments, the compounds described herein are administered sequentially with ionizing radiation or one or more chemotherapeutic agents.

In certain embodiments, provided herein is a method of treating cancer comprising administering to an individual in need thereof a therapeutically effective amount of a compound or composition described herein, and ionizing radiation and one or more chemotherapeutic agents. In some embodiments, the compounds described herein are administered concurrently with ionizing radiation and one or more chemotherapeutic agents. In other embodiments, the compounds described herein are administered sequentially with ionizing radiation and one or more chemotherapeutic agents.

In certain embodiments, provided herein is a method of treating cancer comprising administering to an individual in need thereof a therapeutically effective amount of a compound or composition described herein and ionizing radiation. In certain embodiments, radiation is administered at a dose of less than about 2.5Gy per day, or about 2.0Gy per day, or about 1.8Gy per day, or about 1.6Gy per day, or about 1.4Gy per day, or about 1.2Gy per day. In certain embodiments, a dose of less than about 2.5Gy, or about 2.0Gy, or about 1.8Gy, or about 1.6Gy, or about 1.4Gy, or about 1.2Gy is administered about 5 times per week. In certain embodiments, radiation is administered at a dose of less than about 2.5Gy per day, or about 2.0Gy per day, or about 1.8Gy per day, or about 1.6Gy per day, or about 1.4Gy per day, or about 1.2Gy per day. In certain embodiments, a dose of less than about 2.5Gy, or about 2.0Gy, or about 1.8Gy, or about 1.6Gy, or about 1.4Gy, or about 1.2Gy is administered about 6 times per week. It is expected that by administering radiation and the compounds or compositions described herein, prostate specific chemical prostatectomy can be achieved while avoiding deleterious side effects such as impotence and incontinence from surgical prostatectomy due to vascular and neural disruption.

Cancer therapy may also include a variety of combination therapies with chemical and radiation-based treatments. Combination chemotherapy involves the use of chemotherapeutic agents such as cisplatin (cisptin) or etoposide, irinotecan, camptotheca (camptostar), topotecan, paclitaxel, docetaxel (docetaxel), epothilone (epothilone), tacrolite (taxotere), tamoxifen, 5-fluorouracil, methotrexate (methoxtrexate), temozolomide (temozolomide), cyclophosphamide (cyclophosphamide), SCH 66336, R115777, L778,123, BMS 214662, etoposide, irinotecan, camptothecan (camptothecan), topotecan (paclitaxel), and combinations thereof,(gefitinib)), (gefitinib),(erlotinib hydrochloride), an anti-EGFR antibody,(imatinib), intron, ara-C, adriamycin (adriamycin), cyclophosphamide (cytoxan), gemcitabine (gemcitabine), uracil mustard (uracilmusard), mechlorethamine (chlormethine), ifosfamide (ifosfamide), melphalan (melphalan), chlorambucil (chlorembucil), pipobroman (pipobroman)Triethylenemelamine (triethylenemelamine), triethylenethiophosphoramide (triethylenethiophosphoramide), busulfan (busufan), carmustine (carmustine), lomustine (lomustine), streptozocin (streptozocin), dacarbazine (dacarbazine), floxuridine (floxuridine), cytarabine (cytarabine), 6-mercaptopurine, 6-thioguanine, fludarabine phosphate (fludarabine phosphate), pentostatin (pentostatin), vinblastine (vinblastine), vincristine (vincristine), vindesine (vindesine), bleomycin (bleomycin), doxorubicin, dactinomycin (dactinomycin), daunorubicin (daunorubicin), epirubicin, daldamycin (mithramycin), mithramycin (gentamycin), Mitomycin (alfa, Mitomycin (C), Mitomycin (gentamycin), Mitomycin (17-C), Mitomycin (gentamycin), Mitomycin (C), Mitomycin (Mitomycin, Mitomycin, Fludroxymethyltestosterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone, leuprolide, flutamide, toremifene, sertraline, carboplatin, amsacrine, procarbazine, mitotane, levofloxacin, ketoprofen, levofloxacin, levo, Velcade (Velcade), zerewin (Zevalin), arsenic trioxide (Trisenox), hiloda (Xeloda), Vinorelbine (Vinorelbine), porphine (porfmer), (cetuximab (cet)uximab), liposomes, Thiotepa (Thiotepa), Altretamine (Altretamine), melphalan, Trastuzumab (Trastuzumab), letrozole (Lerozole), fulvestrant (fulvestrant), exemestane (exemestane), eformide (ifomid), rituximab (rituximab), C225, camphos (Campath), carboplatin, procarbazine, dichloromethyldiethylamine (meclorethamine), cyclophosphamide, camptothecin (camptothecin), ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea (nitrourea), dactinomycin D, daunomycin, doxorubicin, bleomycin, plicamycin (plicamycin), mitomycin, etoposide VP (16), tamoxifen, raloxifene, paclitaxel receptor, paclitaxel receptor, daunomycin, gemcitabine (paclitaxel), nonprofecox (5-farnesyl), nonprofecox (5-one), flunomide (fosfamide), rituximab (rituximab), C, fludarabine, and the like, Vincristine, vinblastine and methotrexate or any analogue or derivative variant of the foregoing.

Causing DNA damage (e.g., radiation therapy), other factors that have been fully utilized include factors commonly referred to as gamma-rays, X-rays, and/or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves and UV irradiation, are also contemplated. Most likely these factors all inflict extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose of X-rays ranges from a daily dose of 50 to 200 roentgens for a long period of time (e.g. 3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dose range of the radioisotope varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by neoplastic cells. As used herein, the terms "contacting" and "exposing," when applied to a cell, describe the process of delivery of a therapeutic construct and a chemotherapeutic or radiotherapeutic agent to or in direct juxtaposition with a target cell. To achieve cell killing or stasis, the two agents are delivered to the cells in a combined amount effective to kill the cells or prevent their division.

Immunotherapeutics generally rely on the use of immune effector cells and molecules that target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may be used as an effector of the therapy or it may recruit other cells to actually effect cell killing. Antibodies can also bind to drugs or toxins (chemotherapeutics, radionuclides, ricin a chain (ricin a chain), cholera toxin, pertussis toxin, etc.) and serve only as targeting agents. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target. A variety of effector cells include cytotoxic T cells and NK cells.

Thus, immunotherapy can be used as part of a combination therapy in combination with gene therapy. General approaches to combination therapy are discussed below. In general, tumor cells must carry some marker that is susceptible to targeting, i.e., not present on most other cells. There are many tumor markers and any of these may be suitable for targeting in the context of the present disclosure. Common tumor markers include carcinoembryonic Antigen, prostate specific Antigen, urinary tumor associated Antigen, embryonic Antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialylated Lewis Antigen (Sialyl Lewis Antigen), MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p 155.

In yet another embodiment, the secondary treatment is secondary gene therapy, wherein the therapeutic polynucleotide is administered before, after, or simultaneously with the first chemotherapeutic therapeutic agent. Delivery of chemotherapeutic agents in combination with vectors encoding gene products will have a combined anti-hyperproliferative effect on the target tissue.

About 60% of people with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies (e.g., the treatments of the present disclosure, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and/or alternative therapies). Curative surgery includes resection in which all or part of the cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present disclosure may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

Administration of a compound or composition as described herein may be preceded or followed by another anti-cancer agent or treatment with a time interval in the range of minutes to weeks. In the example of the separate application of the additional anti-cancer agent and expression construct, we will generally ensure that a significant amount of time does not elapse between the time of each delivery, so that the agent and expression construct will still be able to exert a favorable combined effect on the cell. For example, in such cases, it is contemplated that one can contact the cell, tissue, or organism with the active agent in two, three, four, or more modes substantially simultaneously (i.e., in less than about one minute). In other aspects, the one or more agents can be administered within about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 28 hours, about 31 hours, about 35 hours, about 38 hours, about 42 hours, about 45 hours to about 48 hours, or more, before and/or after administration of the active agent. In certain other embodiments, the agent may be administered within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 8 days, about 9 days, about 12 days, about 15 days, about 16 days, about 18 days, about 20 days to about 21 days before and/or after administration of the active agent. In some cases, it may be desirable to significantly extend the treatment period, but with several weeks (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, or about 8 weeks) between corresponding administrations.

Reagent kit

Kits for achieving an anti-cancer effect are provided comprising a compound or composition described herein. In certain embodiments, a kit comprises a unit dose of a compound or composition described herein and instructions for administering the same. In certain aspects, the kit further comprises a second drug suitable for anti-cancer therapy or instructions for co-administering additional anti-cancer therapy (e.g., radiation or gene therapy). In another aspect, a kit for achieving an anti-cancer effect comprises a low dose (e.g., less than about 500 mg/day, or less than about 400 mg/day, or less than about 300 mg/day, or less than about 200 mg/day) of a compound or composition described herein and a second agent suitable for anti-cancer therapy. In yet another variation, a kit for achieving an anti-cancer effect comprises a high dose (e.g., greater than about 500 mg/day) of a compound or composition as described herein and a second drug suitable for anti-cancer therapy.

Method for manufacturing a medicament

In another aspect of the disclosure, there is provided the use of the compounds and compositions described herein in the manufacture of a medicament. In particular, the manufacture of a medicament for treating a cancer, or a disease or condition that may be mediated, at least in part, by blocking DNA repair and/or transcriptional activation, for example, by inhibiting PARP, is provided. In addition, pharmaceutical compositions of the compounds described herein are also intended for use in the manufacture of a medicament for the treatment of a disease or condition that may be mediated, at least in part, by the inhibition of PARP.

Examples of the invention

The disclosure is further illustrated by the following examples. The following examples are non-limiting and are merely representative of various aspects of the present disclosure. The solid and dotted wedges within the structures disclosed herein illustrate the relative stereochemistry, with absolute stereochemistry depicted only when explicitly illustrated or recited.

The compounds as disclosed herein or any of the compounds of formula (la) or (lb) described herein may be synthesized using standard synthetic techniques known to those skilled in the art. The disclosed compounds can be synthesized using the general synthetic procedures set forth in the general methods 1-5 synthetic examples.

When it is desired to obtain a particular enantiomer of a compound, this may be achieved from the corresponding enantiomeric mixture using conventional procedures suitable for separating or resolving the enantiomer. Thus, for example, diastereomeric derivatives may be produced by reacting a mixture of enantiomers, such as a racemate, with an appropriate chiral compound. The diastereomers may then be separated by any convenient means, for example by crystallization, and the desired enantiomer recovered. In another resolution procedure, chiral high performance liquid chromatography can be used to separate the racemates. Alternatively, if desired, a particular enantiomer may be obtained in one of the processes by using an appropriate chiral intermediate.

Chromatography, recrystallization, and other conventional separation procedures may also be used for intermediates or final products where it is desired to obtain a particular isomer of a compound or otherwise purify the reaction product.

General information

¹H NMR Spectroscopy and¹³c NMR spectra were recorded on a Varian 400MHz or Bruker Avance III500MHz spectrometer. Unless otherwise indicated, the spectra were referenced to residual chloroform (delta 7.26,¹H)、DMSO(δ2.54，¹H) or methanol (delta 3.34,¹H) in that respect Chemical shifts are reported in ppm (δ); multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sext (sext), m (multiplet) and br (broad). Coupling constants J are reported in hertz. Silica gel chromatography Using Teledyne IscoRf + instruments were performed using Hi-Purit Silica Flash cards (National Chromatography) or RediSep Rf Gold C18 cards (Teledyne Isco). Analytical HPLC was performed on a Waters ACQUITY UPLC with photodiode array detector using a Waters ACQUITY BEH Shield RPC18 (2.1X 50mm, 1.7 μm) column. Analytical LCMS was performed on a Waters ACQUITY UPLC with a Waters 3100 mass detector. Chiral HPLC on Waters Alliance e2695 with photodiode array detector using Daicel AD-H、IA、IB、IC、OD-H orOJ-H column. Optical rotations were obtained on a Jasco P-2000 digital polarimeter and reported as [ alpha ]]_D ^TTemperature (T), concentration (c ═ g/100mL), and solvent. Commercially available reagents and solvents were used unless otherwise indicated.

General procedure

General procedure 1

Olaparib-containing analogs can be prepared according to the methods described by Menear et al (Menear, k.a. et al, journal of pharmaceutical chemistry 2008,51, 6581-containing 6591).

Scheme 1. proposed route to olaparib-containing analogues: (i) et (Et)₃N, THF, RT; (ii) NaOH aqueous solution, THF, 100 ℃; 2M HCl; (iii) h₂NNH₂、H₂O; (iv) boc-piperazine, HATU, DIPEA, DMA; (v) HCl, dioxane; (vi) using intermediate A or intermediate C, HATU, HOBt, DMF or using intermediate B or intermediate D, Et₃N、DMF。

General procedure 2

Analogs containing akapani can be prepared as described by Gillmore et al (Gillmore, a.t. et al organic processing research and development (org. process res. dev.2012, 16, 1897-

Scheme 2. proposed route to rukapanib-containing analogs: (i) HNO₃、H₂SO₄An aqueous solution; MeOH, H₂SO₄；(ii)DMF、DMA、Et₃N、120℃；(iii)H₂Pd/C, AcONa, MeOH; (iv) phthalamidoacetaldehyde, TFA, TES, CH₂Cl₂；(v)MeNH₂An aqueous solution; (vi) pyr HBr₃、THF、CH₂Cl₂；(vii)Ar-B(OR)₂，Pd(dppf)Cl₂·CH₂Cl₂、Na₂CO₃Aqueous solution, DMA; (viii) using intermediate A or intermediate C, HATU, HOBt, DMF or using intermediate B or intermediate D, Et ₃N、DMF。

General procedure 3

Talaropanib-containing analogs can be prepared as described by Wang et al (Wang, B. et al J. Pharmacol. Chem. 2016, 59, 335-357).

Scheme 3. proposed pathway containing tazobactam analogs: (i) 1-methyl-1H-1, 2, 4-triazole-5-carbaldehyde, Et₃N、Ac₂O, 2-Me-THF, room temperature to 80 ℃, 2 hours; (ii) MeOH, room temperature, overnight; (iii) aminobenzaldehyde, TiCl₃THF, MeOH, 0 ℃ to room temperature; (iv) h₂NNH₂Aqueous MeOH at room temperature overnight; b. chiral separation (v) using intermediate A or intermediate C, HATU, HOBt, DMF or using intermediate B or intermediate D, Et₃N、DMF。

General procedure 4

DHT-containing intermediates can be prepared by treating commercially available DHT (xx) with a desired tethering group to provide intermediates such as XXI. When R ═ CO₂Me, the ester group may be slightly saponified to provide the carboxylate intermediate A. When R ═ OAc, mild saponification liberates the primary alcohol, which can then be treated with methanesulfonyl chloride to give intermediate B.

Scheme 4. proposed route to DHT containing intermediates: (i) et (Et)₃N, DMF, respectively; (ii) LiOH, aqueous MeOH; (iii) MsCl, Et₃N、DMF。

General procedure 5

Enzalutamide containing intermediates can be prepared starting with carboxylic acid XXII (Jadhavar, et al, Bio-organic and medicinal chemistry 2016, 26, 5222-5228 in bioorg.Med.chem.Lett.) and coupling with the desired tethering group to give intermediates, such as XXIII. When R ═ CO ₂Me, the ester group may be slightly saponified to provide carboxylate intermediate C. When R ═ OAc, mild saponification liberates the primary alcohol, which can then be treated with methanesulfonyl chloride to give intermediate D.

Scheme 5. route to proposed enzalutamide containing intermediates: (i) HATU, HOBt, DMF; (ii) LiOH, aqueous MeOH; (iii) MsCl, Et₃N、DMF。

General procedure 6

CC-115 containing analogs can be prepared as described in Mortensen et al (Mortensen, D.S. et al journal of medicinal chemistry 2015, 58, 5599-5608).

(A)

(B)

(C)

Scheme 6. proposed route to CC-115 containing intermediate: (i) bromoacetic acid ethyl ester, Cs₂CO₃、DMF；ii)DIPEA、NMP；iii)AcOH、MeOH；iv)Pd(dppf)Cl₂、K₂CO₃、DMF；v)NH₂NH₂.H₂O, EtOH or H₂Pd/C; vi) use of intermediates XXII, HATU, DIPEA, DMF or MsCl, intermediates XX, Et₃N, DMF and 2M HCl/MeOH; vii) K₂CO₃DMF; viii) tris (o-tolyl) phosphine, Pd₂(dba)₃、Et₃N、DMF；ix)EtNH₂.HCl、DIPEA、NMP。

Synthesis method

EXAMPLE S-1 preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexylbenzamide (Compound 1.1)

Step 1: preparation of (Z) -2-fluoro-5- ((3-oxoisobenzofuran-1 (3H) -ylidene) methyl) benzonitrile

To a stirred solution of dimethyl phosphonate 3-oxo-1, 3-dihydroisobenzofuran-1-yl ester (10g, 41.3mmol) and 2-fluoro-5-formylbenzonitrile (6.15g, 41.3mmol) in THF (50mL) was slowly added triethylamine (5.76mL, 41.3mmol) at 0 deg.C. The resulting mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, water (200mL) was added and the resulting precipitate was filtered through a Buchner funnel (Buchner tunnel). The product obtained was washed with water (50mL), hexane (50mL), diethyl ether (30mL) and dried under vacuum to give (Z) -2-fluoro-5- ((3-oxoisobenzofuran-1 (3H) -ylidene) methyl) benzonitrile (50: 50 mixture of E and Z isomers) which was used in the next step without further purification. LC-MS: 266[ M + H]⁺。¹H NMR(400MHz，CDCl₃)δ8.13(1H，m)，8.05(1H，m)，7.98(1H，m)，7.79(2H，m)，7.61(1H，m)，7.30(1H，m)，6.35(1H，s)。

Step 2: preparation of 2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoic acid

To a stirred suspension of (Z) -2-fluoro-5- ((3-oxoisobenzofuran-1 (3H) -ylidene) methyl) benzonitrile (3.7g, 13.9mmol) in water (20mL) was added aqueous 13N NaOH solution (5mL), and the mixture was heated at 90 ℃ under nitrogen for 16 hours. The reaction mixture was then cooled to 70 ℃ and hydrazine hydrate (10mL) was added and stirred at 70 ℃ for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction was cooled to room temperature and acidified at 0-5 ℃ with 2N aqueous HCl (pH 1-2) to give a precipitate. The slurry was filtered through a buchner funnel, washed with water (50mL), hexane (50mL), diethyl ether (30mL) and dried under vacuum to give 2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoic acid. LC-MS 299[ M + H ] ]⁺。¹H NMR(400MHz，DMSO-d₆)δ13.22(1H，br.s)，12.61(1H，s)，8.27(1H，m)，7.99-7.81(4H，m)，7.59(1H，m)，7.25(1H，m)，4.36(2H，s)。

And step 3: preparation of tert-butyl 4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazine-1-carboxylate

To a stirred suspension of 2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoic acid (0.1g, 0.335mmol) in DMA (4mL) at 0 ℃ was added HBTU (0.15g, 0.402mmol, 1.2 eq) and the mixture was stirred for 10 min. DIPEA (0.17mL, 1mmol, 3 equivalents) and piperazine-1-carboxylic acid tert-butyl ester (0.075g, 0.402mmol, 1.2 equivalents) were then added to the reaction mixture in that order at 0 ℃ and the resulting reaction mixture was stirred at room temperature for 75 minutes. The reaction was monitored by TLC. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The obtained product was washed with water (10 mL. times.2) and n-pentane (10 mL. times.2) and dried under reduced pressure to give tert-butyl 4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazine-1-carboxylate without further treatmentAnd one-step purification is carried out, namely the next step is carried out. LC-MS 467[ M + H [ ]]⁺。

And 4, step 4: preparation of 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To a stirred solution of tert-butyl 4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazine-1-carboxylate (0.150g, 0.321mmol) was added MeOH (5mL) with 2N aqueous HCl at room temperature and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by LC-MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one as HCl salt. LC-MS 367[ M + H ]⁺。

Step 1 a: preparation of methyl 6-aminocaproate

To 6-aminocaproic acid (10.0g, 76.23mmol) was added MeOH (30mL) containing 2N aqueous HCl at room temperature, and the mixture was stirred at room temperature for 16 h. By passing¹The reaction was monitored by H NMR. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give methyl 6-aminocaproate, which was wet-milled with diethyl ether and pentane (1: 3) to give the title compound as the hydrochloride salt.¹H NMR(400MHz，MeOD-d₄)δ3.66(s，3H)，2.92(t，J＝7.67Hz，2H)，2.37(t，J＝7.45Hz，2H)，1.62-1.71(m，6H)，1.37-1.45(m，2H)。

Step 1 b: preparation of methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl) -2-fluorobenzamido) hexanoate

To a stirred solution of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzoic acid (3g, 6.64mmol) in DMF (25mL) was added HATU (5.05g, 13.29mmol, 2 equivalents) at 0 ℃ and the mixture was stirred for 30 min. DIPEA (5.78mL, 33.2mmol, 5 equivalents) and 6-aminocaproate ester (1.81g, 9.96mmol, 1.5 equivalents) were then added to the reaction mixture in that order, and the mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC and LC-MS. Upon completion, the reaction was diluted with EtOAc (250 mL). The organic layer was washed with NaHCO ₃Saturated aqueous solution (100mL), NH₄Saturated aqueous Cl solution (100mL)Washed with water (100mL), brine (50mL) and dried over anhydrous Na₂SO₄Dried, filtered and concentrated under reduced pressure. The crude material was purified by CombiFlash to give methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzamido) hexanoate. LC-MS 579[ M + H ]]⁺。

Step 1 c: preparation of 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl) -2-fluorobenzamido) hexanoic acid

To a stirred solution of methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzamido) hexanoate (3.5g, 6.05mmol) in THF (30mL) and MeOH (15mL) was added LiOH 10H₂O (2.53g, 60.5mmol, 10 equiv.). The solid was dissolved in water (5mL) at room temperature, and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion, the reaction mixture was concentrated under reduced pressure. The residue obtained was diluted with water (30mL) and acidified with 2N aqueous HCl (pH about 2). The aqueous layer was then extracted with EtOAc (300 mL. times.3). The combined organic layers were washed with brine (200mL) and Na ₂SO₄Drying, filtration and concentration under reduced pressure gave 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzamido) hexanoic acid, which was taken to the next step without further purification. LC-MS 565[ M + H]⁺。¹H NMR(400MHz，DMSO-d₆)δ8.51(br.s.，1H)，8.41(d，J＝7.83Hz，1H)，8.30(s，1H)，8.08(d，J＝8.31Hz，1H)，7.75(t，J＝7.83Hz，1H)，7.42(d，J＝10.27Hz，1H)，7.33(d，J＝7.83Hz，1H)，3.25(d，J＝6.36Hz，2H)，2.72-66(m，4H)，2.51-2.4(m，2H)，2.21(t，J＝7.58Hz，2H)，1.54(s，6H)，1.34(d，J＝6.85Hz，2H)。

And 5: preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexyl) benzamide (Compound 1.1)

To a stirred solution of 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzamido) hexanoic acid (0.150g, 0.265mmol) in DMA (4mL) at 0 ℃ was added HBTU (0.12g, 0.319mmol, 1.2 eq) and the mixture was stirred for 10 minutes. DIPEA (0.16mL, 0.93mmol, 3.5 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloride (0.128g, 0.319mmol, 1.2 equivalents) were then added to the reaction mixture in that order, and the mixture was stirred at room temperature for 1.5 hours. The reaction was monitored by TLC and LC-MS. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The obtained product was washed with water (10mL × 2) and n-pentane (10mL × 2) and dried under reduced pressure to give a crude product, which was purified by reverse phase HPLC to give compound 1.1. LC-MS 913[ M + H ]⁺。¹H NMR(400MHz，CD₃OD-d₄)δ8.36(d，J＝7.8Hz，1H)，8.16(d，J＝7.8Hz，2H)，7.99(dd，J＝7.9，2.1Hz，1H)，7.94(dd，J＝8.3，3.4Hz，1H)，7.91-7.78(m，3H)，7.47(p，J＝4.5，4.0Hz，1H)，7.36(td，J＝9.2，8.8，5.1Hz，3H)，7.15(td，J＝9.0，2.4Hz，1H)，4.37(s，2H)，3.83-3.69(m，2H)，3.69-3.62(m，2H)，3.50(dt，J＝9.2，4.9Hz，2H)，3.42(q，J＝6.4，6.0Hz，2H)，3.35(m，2H)，3.28(m，2H)，2.48(t，J＝7.3Hz，1H)，2.41(t，J＝7.5Hz，1H)，1.67(q，J＝7.5Hz，4H)，1.59(s，6H)，1.53-1.40(m，2H)。

EXAMPLE S-2 preparation of 4- (3- (4- (6- (((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) oxy) hexanoyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (Compound 1.2)

Step 1: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ] phenanthrene-3, 2' - [1, 3] dioxolane ] -17-ol

In the roomTo (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] at room temperature]To a stirred solution of phenanthren-3 (2H) -one (3g, 10.3mmol) in benzene (100mL) was added PTSA (0.982g, 5.1mmol, 0.5 equiv.), followed by ethylene glycol (2.88g, 51.61mmol, 5 equiv.). The resulting mixture was heated to 120 ℃ using a Dean-Stark apparatus for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (200mL) and extracted with EtOAc (350 mL). The organic layer was washed with saturated NaHCO3 solution (100mL), water (200mL), brine (100mL), over Na₂SO₄Drying, filtering and concentrating under reduced pressure to give (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ] ]Phenanthrene-3, 2' - [1, 3]]Dioxolanes]-17-ol. LC-MS 335[ M + H [ ]]⁺。¹H NMR(400MHz，DMSO-d₆)δ4.40(d，J＝5.26Hz，1H)，3.81(s，3H)，3.37-3.45(m，2H)，1.82(br.s.，2H)，1.70(d，J＝12.72Hz，2H)，1.59(d，J＝13.15Hz，2H)，1.52(d，J＝10.52Hz，3H)，1.35(br.s.，1H)，1.23-1.33(m，3H)，1.03-1.21(m，5H)，0.87(dt，J＝7.89，19.95Hz，4H)，0.76(s，3H)，0.61(s，3H)。

Step 2 a: preparation of 2- (6-Chlorohexyloxy) tetrahydro-2H-pyran

To a stirred solution of 6-chlorohex-1-ol (10g, 73.158mmol) in diethyl ether (100mL) was added pTSA (0.05g, 0.365mmol, 0.005 equiv.), followed by 3, 4-dihydro-2H-pyran (8.4g, 102.48mmol, 1.4 equiv.). The resulting reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with diethyl ether (350 mL). The organic layer was washed with 20% KOH solution (100mL), water (300mL), brine (200mL) and Na₂SO₄Drying, filtration and concentration under reduced pressure gave 2- (6-chlorohexyloxy) tetrahydro-2H-pyran.¹H NMR(400MHz，DMSO-d₆)δ4.50(br.s.，1H)，4.25(t，J＝6.80Hz，2H)，4.20(s，1H)，4.00-4.08(m，2H)，3.66-3.74(m，2H)，3.59(dd，J＝6.36，16.01Hz，2H)，2.66(s，2H)，1.81-1.90(m，2H)，1.67(br.s.，1H)，1.38-1.45(m，2H)，1.20-1.27(m，2H)，1.17(t，J＝7.02Hz，2H)。

Step 2: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (6- (tetrahydro-2H-pyran-2-yloxy) hexyloxy) hexadecahydrospiro [ cyclopenta [ a ] phenanthrene-3, 2' - [1, 3] dioxolane ]

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ]]Phenanthrene-3, 2' - [1, 3]]Dioxolanes]To a stirred solution of-17-ol (2g, 5.97mmol) in xylene (50mL) was added NaNH₂(50% suspension in toluene, 1.4mL) and the mixture was heated at 150 ℃ for 1 hour. The reaction mixture was gradually cooled to room temperature, 2- (6-chlorohexyloxy) tetrahydro-2H-pyran (2.8mL) was added thereto, and the resulting mixture was heated again to 150 ℃ for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature, quenched slowly with ice-cold water (250mL), and extracted with EtOAc (300 mL). The organic layer was washed with water (100 mL. times.2), brine (100mL) and Na ₂SO₄Drying, filtering and concentrating under reduced pressure to give a crude product which is purified by CombiFlash chromatography (Teledyne Isco Rf +); eluting the compound with 30% EtOAc/hexanes to give (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (6- (tetrahydro-2H-pyran-2-yloxy) hexyloxy) hexadecahydrospiro [ cyclopenta [ a ] o]Phenanthrene-3, 2' - [1, 3 ]]Dioxolanes]。¹H NMR(400MHz，CDCl3)δ4.57(d，J＝3.95Hz，1H)，3.91-3.95(m，4H)，3.82-3.91(m，2H)，3.73(td，J＝6.80，9.65Hz，2H)，3.42-3.53(m，3H)，3.33-3.42(m，3H)，3.26(t，J＝8.55Hz，2H)，1.91-2.01(m，2H)，1.75-1.90(m，3H)，1.46-1.74(m，13H)，1.31-1.42(m，6H)，1.07-1.28(m，4H)，0.83-0.97(m，4H)，0.81(s，4H)，0.70-0.76(m，4H)，0.07(s，3H)，0.00(s，3H)。

And step 3: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (6-hydroxyhexyloxy) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] phenanthren-3 (2H) -one

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (6- (tetrahydro-2H-pyran-2-yloxy) hexyloxy) hexadecahydrospiro [ cyclopenta [ a ] at room temperature]Phenanthrene-3, 2' - [1, 3 ]]Dioxolanes](1g，1.92mmol) in THF (25mL), water (5mL) was added 6N HCl (15mL) and the resulting reaction mixture was stirred at room temperature for 16 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (150mL) and NaHCO was used₃The saturated solution (pH about 8) was basified. The aqueous layer was then extracted with EtOAc (200 mL. times.3). The organic layer was washed with NaHCO₃Saturated solution (100mL), water (100mL), brine (100mL), washed with Na₂SO₄Drying, filtering and concentrating under reduced pressure to give (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (6-hydroxyhexyloxy) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] ]Phenanthren-3 (2H) -one, which was used in the next step without further purification.¹H NMR(400MHz，CDCl3)δ4.90(br.s.，1H)，4.12(d，J＝7.45Hz，1H)，4.00(br.s.，1H)，3.64(t，J＝6.58Hz，3H)，3.49-3.58(m，1H)，3.37-3.47(m，3H)，3.27(t，J＝8.55Hz，1H)，2.22-2.43(m，3H)，2.10(br.s.，1H)，1.93-2.07(m，2H)，1.90(d，J＝11.84Hz，2H)，1.82(d，J＝7.89Hz，2H)，1.70(d，J＝10.09Hz，2H)，1.48(d，J＝4.38Hz，2H)，1.30-1.42(m，8H)，1.22-1.29(m，2H)，1.01(s，3H)，0.77(s，3H)。

And 4, step 4: preparation of 6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoic acid

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (6-hydroxyhexyloxy) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] over a period of 20 minutes at 0 deg.C]To a stirred solution of phenanthren-3 (2H) -one (0.1g, 0.256mmol) in acetone (5mL) was added dropwise Jones reagent (Jones reagent) (0.5 mL). The resulting mixture was stirred at 0 ℃ for 5 minutes. The reaction was monitored by TLC. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5 mL. times.2) and n-pentane (5 mL. times.2) and dried under vacuum to give 6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ]]Phenanthren-17-yloxy) hexanoic acid, which was carried on to the next step without further purification.¹H NMR(400MHz，DMSO-d6)δ3.19-3.28(m，3H)，2.24-2.35(m，2H)，2.18(t，J＝7.24Hz，2H)，2.08(d，J＝13.59Hz，2H)，1.84-1.95(m，3H)，1.79(d，J＝10.52Hz，2H)，1.61(d，J＝13.59Hz，2H)，1.40-1.54(m，8H)，1.22-1.38(m，8H)，1.19(br.s.，1H)，1.09-1.18(m，2H)，0.97(s，3H)，0.69(s，3H)。

And 5: preparation of 4- (3- (4- (6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (Compound 2)

To 6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred solution of phenanthrene-17-yloxy) hexanoic acid (0.1g, 0.247mmol) in DMA (5mL) was added HBTU (0.112g, 0.296mmol, 1.2 eq) and the resulting reaction mixture was stirred for 10 min. DIPEA (0.94mL, 0.544mmol, 2.2 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (0.108g, 0.296mmol, 1.2 equivalents) were then added to the mixture in that order, and the mixture was stirred at room temperature for 1 hour. The reaction was monitored by TLC and LC-MS. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give the crude product, which was purified by reverse phase HPLC to give compound 1.2. LC-MS 753[ M + H ]]⁺。¹H NMR(400MHz，DMSO-d6)δ8.37(d，J＝7.6Hz，1H)，7.99-7.91(m，1H)，7.91-7.79(m，2H)，7.48(t，J＝6.4Hz，1H)，7.36(d，J＝6.1Hz，1H)，7.21-7.11(m，1H)，4.38(s，2H)，3.82-3.61(m，4H)，3.47(ddt，J＝22.1，14.2，6.3Hz，4H)，3.37-3.33(m，2H)，2.53-2.30(m，4H)，2.26-2.16(m，1H)，2.08-1.95(m，3H)，1.88(t，J＝11.6Hz，1H)，1.75-1.50(m，10H)，1.50-1.13(m，12H)，1.05(s，3H)，1.01-0.86(m，2H)，0.76(d，J＝9.0Hz，4H)。

EXAMPLE S-3 preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- ((4- ((8S, 9R) -5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -3-oxo-2, 7, 8, 9-tetrahydro-3H-pyrido [4, 3, 2-de ] phthalazin-8-yl) phenyl) amino) -6-oxohexyl) benzamide (Compound 1.3)

Step 1 a: preparation of methyl 5-fluoro-2- (2- (1-methyl-1H-1, 2, 4-triazol-5-yl) acetyl) -3-nitrobenzoate

(Z) -6-fluoro-3- ((1-methyl-1H-1, 2, 4-triazol-5-yl) methylene) -4-nitroisobenzofuran-1 (3H) -one (10g, 34.48mmol) was added to a reaction vessel containing MeOH (50mL) containing 2N aqueous HCl at room temperature and the mixture was stirred at room temperature for 16H. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a viscous solid, which was further lyophilized to give methyl 5-fluoro-2- (2- (1-methyl-1H-1, 2, 4-triazol-5-yl) acetyl) -3-nitrobenzoate as the hydrochloride salt. LC-MS 323[ M + H [ ]]⁺。

Step 1 b': preparation of 4-aminobenzaldehyde

To a stirred solution of 4-nitrobenzaldehyde (5g, 33.1mmol) in ethanol (50mL) at 0 deg.C was added SnCl₂(37.35g, 165.5mmol, 5 equiv.) and the mixture is heated at 80 ℃ for 1 hour. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a crude residue, which was suspended in water (100mL) and NaHCO was used₃The solution (pH about 8) was basified. The aqueous layer was then extracted with EtOAc (500 mL. times.3). The combined organic layers were washed with NaHCO ₃Saturated solution (300mL), water (200mL), brine (150mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave 4-aminobenzaldehyde, which was used in the next step without further purification. LC-MS 122[ M + H [ ]]⁺。

Step 1 b: preparation of methyl 2- (4-aminophenyl) -7-fluoro-3- (1-methyl-1H-1, 2, 4-triazol-5-yl) -4-oxo-1, 2, 3, 4-tetrahydroquinoline-5-carboxylate

To 5-fluoro-2- (2- (1-methyl-1H-1, 2, 4-triazol-5-yl) acetyl) -3-nitrobenzoate methyl hydrochloride (5.6g, 15.6mmol) in THF (60mL), MeOH (10mL) with stirringTo the stirred solution was added 4-aminobenzaldehyde (3.8g, 31.2mmol, 2 equivalents). Titanium (III) chloride (20% w/v solution in 2N-HCl) (50mL) was then added dropwise to the mixture at room temperature over a period of 20 minutes, and the mixture was stirred at 50 ℃ for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the solvent was removed under reduced pressure to give a crude residue, which was dissolved in water (300mL) and NaHCO was used₃The saturated solution (pH about 8) was basified. The aqueous layer was then extracted with EtOAc (400 mL. times.3). The combined organic layers were washed with NaHCO₃Saturated solution (300mL), water (300mL), brine (100mL), washed with Na₂SO₄Drying, filtering and concentrating under reduced pressure to give a crude product which is purified by CombiFlash chromatography (Teledyne Isco Rf +); the compound was eluted with 40% EtOAc/hexanes to give methyl 2- (4-aminophenyl) -7-fluoro-3- (1-methyl-1H-1, 2, 4-triazol-5-yl) -4-oxo-1, 2, 3, 4-tetrahydroquinoline-5-carboxylate. LC-MS 396[ M + H ]⁺。

Step 1 c: preparation of 8- (4-aminophenyl) -5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -8, 9-dihydro-2H-pyrido [4, 3, 2-de ] phthalazin-3 (7H) -one

To a stirred suspension of methyl 2- (4-aminophenyl) -7-fluoro-3- (1-methyl-1H-1, 2, 4-triazol-5-yl) -4-oxo-1, 2, 3, 4-tetrahydroquinoline-5-carboxylate (1.5g, 37.9mmol) in methanol (20mL) at 0 ℃ was added hydrazine hydrate (3mL), and the resulting mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC. Upon completion, the reaction mixture was concentrated under reduced pressure and water (20mL) was added. The resulting precipitate was filtered through a buchner funnel. The obtained product was washed with water (10mL × 2) and n-pentane (10mL × 2) and dried under reduced pressure to give a crude product, which was purified by reverse phase chromatography to give 8- (4-aminophenyl) -5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -8, 9-dihydro-2H-pyrido [4, 3, 2-de ] phthalazin-3 (7H) -one as a racemic mixture. The obtained compound was further separated by chiral chromatography to give 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- (4- ((8S, 9R) -5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -3-oxo-3, 7, 8, 9-tetrahydro-2H-pyrido [4, 3, 2-de ] phthalazin-8-yl) phenylamino) -6-oxohexyl) benzamide (9PK-1) (0.300g) and 4- (3- (4-cyano- 3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- (4- ((8S, 9R) -5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -3-oxo-3, 7, 8, 9-tetrahydro-2H-pyrido [4, 3, 2-de ] phthalazin-8-yl) phenylamino) -6-oxohexyl) benzamide (PK-2).

(PK-1 for Compound 1.3, and PK-2 for enantiomeric Compound 1.3)

PK-1：LC-MS 378[M+H]⁺。¹H NMR(400MHz，DMSO-d₆)δ12.30(s，1H)，7.78(s，1H)，7.56(s，1H)，7.02(t，J＝6.7Hz，3H)，6.90(d，J＝11.2Hz，1H)，6.45(d，J＝7.9Hz，2H)，5.10(s，2H)，4.85(d，J＝11.4Hz，1H)，4.72(d，J＝11.4Hz，1H)，3.62(s，3H)。

PK-2：LC-MS 378[M+H]⁺。¹H NMR(400MHz，DMSO-d₆)δ12.29(s，1H)，7.78(s，1H)，7.56(s，1H)，7.02(t，J＝6.7Hz，3H)，6.89(d，J＝11.2Hz，1H)，6.44(d，J＝7.9Hz，2H)，5.10(s，2H)，4.85(d，J＝11.4Hz，1H)，4.71(d，J＝11.4Hz，1H)，3.61(s，3H)。

Step 1: preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- (4- (5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -3-oxo-3, 7' 8, 9-tetrahydro-2H-pyrido [4, 3, 2-de ] phthalazin-8-yl) phenylamino) -6-oxohexyl) benzamide (Compound 1.3)

To a stirred solution of 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzamido) hexanoic acid (0.125g, 0.221mmol) in DMF (7mL) at 0 ℃ was added HATU (0.168g, 0.442mmol, 2 equivalents) and the resulting mixture was stirred for 30 min. DIPEA (0.19mL, 1.11mmol, 5 equiv.) and 8- (4-aminophenyl) -5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -8, 9-dihydro-2H-pyrido [4, 3, 2-de]Phthalazin-3 (7H) -one (PK-1) (0.10g, 0.265mmol, 1.2 equiv.) is added to the reaction mixture in succession and the resulting mixture isStirred at room temperature for 4 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (50mL) and extracted with EtOAc (100 mL. times.2). The combined organic layers were washed with NaHCO ₃Saturated solution (100mL), NH₄Saturated Cl solution (100mL), water (100mL), brine (50mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the crude product, which was purified by reverse phase HPLC to give compound 1.3. LC-MS 924[ M + H]⁺。¹H NMR(400MHz，CD₃OD-d₄)δ8.15(d，J＝9.1Hz，2H)，7.97(d，J＝8.5Hz，1H)，7.87(d，J＝3.0Hz，1H)，7.82(t，J＝8.8Hz，1H)，7.56-7.48(m，2H)，7.34(d，J＝8.4Hz，4H)，7.19(dd，J＝7.1，4.7Hz，1H)，6.90(d，J＝10.4Hz，1H)，3.58(d，J＝3.0Hz，3H)，3.46-3.36(m，2H)，2.38(d，J＝7.6Hz，2H)，1.93(s，2H)，1.80-1.64(m，6H)，1.59(d，J＝2.8Hz，6H)，1.48(ddt，J＝14.6，10.8，4.6Hz，2H)。

Example S-4. preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- ((4- ((8R, 9S) -5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -3-oxo-2, 7, 8, 9-tetrahydro-3H-pyrido [4, 3, 2-de ] phthalazin-8-yl) phenyl) amino) -6-oxohexyl) benzamide (compound enantiomer-1.3).

Step 1: preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- (4- (5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -3-oxo-3, 7, 8, 9-tetrahydro-2H-pyrido [4, 3, 2-de ] phthalazin-8-yl) phenylamino) -6-oxohexyl) benzamide

To a stirred solution of 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl) -2-fluorobenzamido) hexanoic acid (0.210g, 0.371mmol) in DMF (8mL) at 0 deg.C was added HATU (0.282g, 0.743mmol, 2 equivalents) and the mixture was added The resulting mixture was stirred for 30 minutes. DIPEA (0.32mL, 1.86mmol, 5 equiv.) and 8- (4-aminophenyl) -5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -8, 9-dihydro-2H-pyrido [4, 3, 2-de]Phthalazin-3 (7H) -one (PK-2) (0.17g, 0.446mmol, 1.2 equiv.) is added to the reaction mixture in turn and the resulting mixture is stirred at room temperature for 4 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (50mL) and extracted with EtOAc (100 mL. times.2). The combined organic layers were washed with NaHCO₃Saturated solution (100mL), NH₄Saturated Cl solution (100mL), water (100mL), brine (50mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the crude product, which was purified by reverse phase HPLC to give compound enantiomer-1.3. LC-MS 924[ M + H]⁺。¹H NMR(400MHz，CD₃OD-d₄)δ8.15(d，J＝9.1Hz，2H)，7.98(dd，J＝8.1，2.1Hz，1H)，7.90-7.79(m，2H)，7.52(d，J＝8.9Hz，2H)，7.34(d，J＝8.4Hz，4H)，7.21(dd，J＝9.0，2.5Hz，1H)，6.90(dd，J＝10.8，2.5Hz，1H)，3.59(d，J＝3.2Hz，3H)，3.46-3.36(m，2H)，2.40(t，J＝7.4Hz，2H)，1.93(s，2H)，1.72(dp，J＝26.6，7.3Hz，4H)，1.60(s，6H)，1.48(ddt，J＝14.6，10.8，4.6Hz，2H)。

EXAMPLE S-5 preparation of 6- (((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) oxy) -N- (4- (5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -3-oxo-2, 7, 8, 9-tetrahydro-3H-pyrido [4, 3, 2-de ] phthalazin-8-yl) phenyl) hexanamide (Compound 1.4)

Step 1: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ] phenanthrene-3, 2' - [1, 3] dioxolane ] -17-ol

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] at room temperature]Phenanthren-3 (2H) -one (3g, 10.3 mmo)l) to a stirred solution in benzene (100mL) was added PTSA (0.982g, 5.1mmol, 0.5 equiv.), followed by ethylene glycol (2.88g, 51.61mmol, 5 equiv.). The resulting mixture was heated at 120 ℃ for 16 hours using a dean-Stark apparatus. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (200mL) and extracted with EtOAc (350 mL). The organic layer was washed with NaHCO₃Saturated solution (100mL), brine (200mL), brine (100mL) washed over Na₂SO₄Drying, filtering and concentrating under reduced pressure to give (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ]]Phenanthrene-3, 2' - [1, 3 ]]Dioxolanes]-17-ol. LC-MS 335[ M + H [ ]]⁺。¹H NMR(400MHz，DMSO-d₆)δ4.40(d，J＝5.26Hz，1H)，3.81(s，3H)，3.37-3.45(m，2H)，1.82(br.s.，2H)，1.70(d，J＝12.72Hz，2H)，1.59(d，J＝13.15Hz，2H)，1.52(d，J＝10.52Hz，3H)，1.35(br.s.，1H)，1.23-1.33(m，3H)，1.03-1.21(m，5H)，0.87(dt，J＝7.89，19.95Hz，4H)，0.76(s，3H)，0.61(s，3H)。

Step 2 a: preparation of 2- (6-Chlorohexyloxy) tetrahydro-2H-pyran

To a stirred solution of 6-chlorohex-1-ol (10g, 73.158mmol) in diethyl ether (100mL) was added pTSA (0.05g, 0.365mmol, 0.005 equiv.), followed by 3, 4-dihydro-2H-pyran (8.4g, 102.48mmol, 1.4 equiv.). The resulting reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with diethyl ether (350 mL). The organic layer was washed with 20% KOH solution (100mL), water (300mL), brine (200mL) and Na ₂SO₄Drying, filtration and concentration under reduced pressure gave 2- (6-chlorohexyloxy) tetrahydro-2H-pyran.¹H NMR(400MHz，DMSO-d₆)δ4.50(br.s.，1H)，4.25(t，J＝6.80Hz，2H)，4.20(s，1H)，4.00-4.08(m，2H)，3.66-3.74(m，2H)，3.59(dd，J＝6.36，16.01Hz，2H)，2.66(s，2H)，1.81-1.90(m，2H)，1.67(br.s.，1H)，1.38-1.45(m，2H)，1.20-1.27(m，2H)，1.17(t，J＝7.02Hz，2H)。

Step 2: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (6- (tetrahydro-2H-pyran-2-yloxy) hexyloxy) hexadecahydrospiro [ cyclopenta [ a ] phenanthrene-3, 2' - [1, 3] dioxolane ]

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ]]Phenanthrene-3, 2' - [1, 3]]Dioxolanes]To a stirred solution of-17-ol (2g, 5.97mmol) in xylene (50mL) was added NaNH₂(50% suspension in toluene, 1.4mL) and the mixture was heated at 150 ℃ for 1 hour. The reaction mixture was gradually cooled to room temperature, 2- (6-chlorohexyloxy) tetrahydro-2H-pyran (2.8mL) was added thereto, and the resulting mixture was heated again to 150 ℃ for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature, quenched slowly with ice-cold water (250mL), and extracted with EtOAc (300 mL). The organic layer was washed with water (100 mL. times.2), brine (100mL) and Na₂SO₄Drying, filtering and concentrating under reduced pressure to give a crude product which is purified by CombiFlash chromatography (Teledyne Isco Rf +); eluting the compound with 30% EtOAc/hexanes to give (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (6- (tetrahydro-2H-pyran-2-yloxy) hexyloxy) hexadecahydrospiro [ cyclopenta [ a ] o ]Phenanthrene-3, 2' - [1, 3 ]]Dioxolanes]。¹H NMR(400MHz，CDCl3)δ4.57(d，J＝3.95Hz，1H)，3.91-3.95(m，4H)，3.82-3.91(m，2H)，3.73(td，J＝6.80，9.65Hz，2H)，3.42-3.53(m，3H)，3.33-3.42(m，3H)，3.26(t，J＝8.55Hz，2H)，1.91-2.01(m，2H)，1.75-1.90(m，3H)，1.46-1.74(m，13H)，1.31-1.42(m，6H)，1.07-1.28(m，4H)，0.83-0.97(m，4H)，0.81(s，4H)，0.70-0.76(m，4H)，0.07(s，3H)，0.00(s，3H)。

And step 3: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (6-hydroxyhexyloxy) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] phenanthren-3 (2H) -one

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (6- (tetrahydro-2H-pyran-2-yloxy) hexyloxy) hexadecahydrospiro [ cyclopenta [ a ] at room temperature]Phenanthrene-3, 2' - [1, 3 ]]Dioxolanes](1g, 1.92mmol) to a stirred solution in THF (25mL), water (5mL) was added 6N HCl (15mL) and the resulting reaction mixture was stirred at room temperature for 16 h. By passingThe reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (150mL) and NaHCO was used₃The saturated solution (pH about 8) was basified. The aqueous layer was then extracted with EtOAc (200 mL. times.3). The organic layer was washed with NaHCO₃Saturated solution (100mL), water (100mL), brine (100mL), washed with Na₂SO₄Drying, filtering and concentrating under reduced pressure to give (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (6-hydroxyhexyloxy) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ]]Phenanthren-3 (2H) -one, which was used in the next step without further purification.¹H NMR(400MHz，CDCl3)δ4.90(br.s.，1H)，4.12(d，J＝7.45Hz，1H)，4.00(br.s.，1H)，3.64(t，J＝6.58Hz，3H)，3.49-3.58(m，1H)，3.37-3.47(m，3H)，3.27(t，J＝8.55Hz，1H)，2.22-2.43(m，3H)，2.10(br.s.，1H)，1.93-2.07(m，2H)，1.90(d，J＝11.84Hz，2H)，1.82(d，J＝7.89Hz，2H)，1.70(d，J＝10.09Hz，2H)，1.48(d，J＝4.38Hz，2H)，1.30-1.42(m，8H)，1.22-1.29(m，2H)，1.01(s，3H)，0.77(s，3H)。

And 4, step 4: preparation of 6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoic acid

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (6-hydroxyhexyloxy) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] over a 20 minute period at 0 deg.C]To a stirred solution of phenanthren-3 (2H) -one (0.1g, 0.256mmol) in acetone (5mL) was added dropwise jones reagent (0.5 mL). The resulting mixture was stirred at 0 ℃ for 5 minutes. The reaction was monitored by TLC. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5 mL. times.2) and n-pentane (5 mL. times.2) and dried under vacuum to give 6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ]]Phenanthren-17-yloxy) hexanoic acid, which was carried on to the next step without further purification.¹H NMR(400MHz，DMSO-d6)δ3.19-3.28(m，3H)，2.24-2.35(m，2H)，2.18(t，J＝7.24Hz，2H)，2.08(d，J＝13.59Hz，2H)，1.84-1.95(m，3H)，1.79(d，J＝10.52Hz，2H)，1.61(d，J＝13.59Hz，2H)，1.40-1.54(m，8H)，1.22-1.38(m，8H)，1.19(br.s.，1H)，1.09-1.18(m，2H)，0.97(s，3H)，0.69(s，3H)。

And 5: preparation of 6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) -N- (4- (5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -3-oxo-3, 7, 8, 9-tetrahydro-2H-pyrido [4, 3, 2-de ] phthalazin-8-yl) phenyl) hexanamide

To 6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred solution of phenanthrene-17-yloxy) hexanoic acid (0.080g, 0.197mmo |) in THF (5mL) was added T ₃Solution of P (50% in EtOAc, 0.25mL, 0.435mmol, 2.2 eq) and the resulting mixture stirred for 30 min. DIPEA (0.24mL, 1.38mmol, 7 equiv.) then was added sequentially to 8- (4-aminophenyl) -5-fluoro-9- (1-methyl-1H-1, 2, 4-triazol-5-yl) -8, 9-dihydro-2H-pyrido [4, 3, 2-de]Phthalazin-3 (7H) -one (PK-1) (0.074g, 0.197mmol, 1 eq) was added to the reaction mixture and the resulting mixture was stirred at room temperature for 16H. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (50mL) and extracted with EtOAc (100 mL. times.3). The combined organic layers were washed with NaHCO₃Saturated solution (80mL), NH₄Saturated Cl solution (60mL), water (80mL), brine (60mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the crude product, which was purified by reverse phase HPLC to give compound 1.4. LC-MS 764[ M + H ]]⁺。¹H NMR(400MHz，MeOD-d4)δ7.88(s，1H)，7.56-7.49(m，2H)，7.35(d，J＝8.5Hz，2H)，7.21(dd，J＝9.0，2.4Hz，1H)，6.92(dd，J＝2.41，10.74Hz，1H)，3.60(s，3H)，3.42-3.50(m，3H)，2.31-2.40(m，2H)，1.91-2.12(m，5H)，1.88(br.s.，2H)，1.64-1.78(m，4H)，1.57(dd，J＝6.36，13.81Hz，4H)，1.46(dd，J＝7.45，15.79Hz，4H)，1.13-1.40(m，10H)，0.82-0.97(m，4H)，0.71-0.82(m，3H)。

EXAMPLE S-6 preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- ((4- (8-fluoro-1-oxo-2, 3, 4, 6-tetrahydro-1H-azepino [5, 4, 3-cd ] indol-5-yl) benzyl) amino) hexyl) benzamide (Compound 1.5)

Step 1: preparation of 4- (8-fluoro-6-oxo-3, 4, 5, 6-tetrahydro-1H-azepino [5, 4, 3-cd ] indol-2-yl) benzaldehyde

To 2-bromo-8-fluoro-4, 5-dihydro-1H-azepino [5, 4, 3-cd at room temperature]To a stirred solution of indol-6 (3H) -one (2g, 7.06mmol, 1 eq) in DMA (37.5mL) was added Na₂CO₃(1.48g, 14.1mmol, 2 equiv.) in H₂A solution in O (18.5mL) and 4-formylphenylboronic acid (1.27g, 8.4mmol, 1.2 equiv.). The resulting mixture was degassed at room temperature under nitrogen for 30 minutes, followed by the addition of Pd (dppf) Cl₂DCM (0.142g, 0.18mmol, 0.025 eq). The mixture was further degassed with nitrogen for 15 minutes. The reaction mixture was then heated at 95 ℃ for 4 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature, water (30mL) was added, and the resulting precipitate was filtered through a buchner funnel. The obtained crude material was washed with water (20 mL. times.2) and n-pentane (10 mL. times.2), and dried under reduced pressure to give 4- (8-fluoro-6-oxo-3, 4, 5, 6-tetrahydro-1H-azepino [5, 4, 3-cd-e]Indol-2-yl) benzaldehyde. LC-MS 309[ M + H [ ]]⁺。¹H NMR(400MHz，DMSO-d₆)δ11.90(s，1H)，10.06(s，1H)，8.31(br.s.，1H)，8.02-8.08(m，J＝7.89Hz，2H)，7.81-7.89(m，J＝7.89Hz，2H)，7.46(d，J＝10.96Hz，1H)，7.37(d，J＝9.21Hz，1H)，3.41(br.s.，2H)，3.11(br.s.，2H)。

Step 2 a: preparation of tert-butyl 6-aminohexylcarbamate

To a stirred solution of hexane-1, 6-diamine (10g, 86.05mmol) in DCM (400mL) was added di-tert-butyl dicarbonate (4.69g, 21.5mmol, 0.25 eq) dissolved in DCM (100mL) dropwise over a period of 1 hour, and the resulting mixture was stirred at room temperature for 16 hours. The reaction was then filtered through a buchner funnel to remove unreacted hexane-1, 6-diamine. Will obtain Was dissolved in water (200mL) and then extracted with DCM (200mL × 3). The combined organic layers were washed with NaHCO₃Saturated solution (300mL), water (200mL), brine (150mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave 6-aminohexylcarbamic acid tert-butyl ester, which was used in the next step without further purification.¹H NMR(400MHz，CDCl₃)δ4.70(br，1H)，3.02(m，2H)，2.62(m，2H)，1.37(s，15H)，1.26(m，4H)。

Step 2: preparation of tert-butyl 6- (4- (8-fluoro-6-oxo-3, 4, 5, 6-tetrahydro-1H-azepino [5, 4, 3-cd ] indol-2-yl) benzyl-amino) hexylcarbamate

To 4- (8-fluoro-6-oxo-3, 4, 5, 6-tetrahydro-1H-azepino [5, 4, 3-cd]To a stirred solution of indol-2-yl) benzaldehyde (0.6g, 1.94mmol) in MeOH (36mL) and THF (18mL) was added tert-butyl 6-aminohexylcarbamate (1.26g, 5.8mmol, 3 equiv.), and the reaction mixture was stirred at room temperature for 2 hours. Then NaBH is added at 0 DEG C₄(0.22g, 5.8mmol, 3 equiv.) was slowly added to the mixture and the resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (100mL) and extracted with EtOAc (150 mL. times.2). The combined organic layers were washed with water (75mL) and brine (75mL) and washed with Na ₂SO₄Dried, filtered and concentrated under reduced pressure to give a crude material which is wet milled using DCM/hexane (1: 4) to give 6- (4- (8-fluoro-6-oxo-3, 4, 5, 6-tetrahydro-1H-azepino [5, 4, 3-cd)]Indol-2-yl) benzyl-amino) hexylcarbamic acid tert-butyl ester. LC-MS 509[ M + H]⁺。¹H NMR(400MHz，MeOD-d₄)7.60(d，J＝7.89Hz，2H)，7.46-7.56(m，3H)，7.30(br.s.，1H)，3.83(s，2H)，3.54(br.s.，2H)，3.14(m，3H)，3.02(s，1H)，2.62(t，J＝7.24Hz，2H)，1.57(br.s.，4H)，1.46(m，4H)，1.42(s，5H)，1.36(br.s.，4H)。

And step 3: preparation of 2- (4- ((6-aminohexylamino) methyl) phenyl) -8-fluoro-4, 5-dihydro-1H-azepino [5, 4, 3-cd ] indol-6 (3H) -one

At 0 ℃ in the direction of6- (4- (8-fluoro-6-oxo-3, 4, 5, 6-tetrahydro-1H-azepino [5, 4, 3-cd)]To a stirred solution of tert-butyl indol-2-yl) benzyl-amino) hexylcarbamate (0.3g, 0.58mmol, 1 eq) in MeOH (1mL) was added 4N-HCl in 1, 4 dioxane (10 mL). The mixture was stirred at room temperature for 1 hour. The reaction was monitored by TLC. After completion of the reaction, the solvent was removed under reduced pressure to give a residue, which was wet-milled using diethyl ether to give 2- (4- ((6-aminohexylamino) methyl) phenyl) -8-fluoro-4, 5-dihydro-1H-azepino [5, 4, 3-cd-as the hydrochloride salt]Indol-6 (3H) -ones. LC-MS 445[ M + H ]]⁺。

And 4, step 4: preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl) -2-fluoro-N- (6- (4- (8-fluoro-6-oxo-3, 4, 5, 6-tetrahydro-1H-azepino [5, 4, 3-cd ] indol-2-yl) benzylamino) hexyl) benzamide

To a stirred solution of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzoic acid (0.15g, 0.32mmol) in DMA (6mL) at 0 ℃ was added HBTU (0.15g, 0.39mmol, 1.2 eq) and the resulting mixture was stirred at the same temperature for 30 minutes. DIPEA (0.128g, 0.99mmol, 3 equivalents) and 2- (4- ((6-aminohexylamino) methyl) phenyl) -8-fluoro-4, 5-dihydro-1H-azepino [5, 4, 3-cd]Indol-6 (3H) -one (0.221g, 0.43mmol, 1.5 eq) was added to the reaction mixture in succession and the resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature, water (30mL) was added, and the resulting precipitate was filtered through a buchner funnel. The crude material obtained was purified by reverse phase HPLC to give compound 1.5. LC-MS 842[ M + H]⁺。¹H NMR(400MHz，DMSO-d₆)δ11.67(s，1H)，8.50(br.s.，1H)，8.40(d，J＝8.33Hz，1H)，8.29(s，1H)，8.24(br.s.，1H)，8.09(s，1H)，7.74(s，1H)，7.54-7.61(m，J＝7.89Hz，2H)，7.44-7.51(m，J＝7.89Hz，2H)，7.40(s，1H)，7.43(s，1H)，7.27-7.36(m，2H)，3.5-3.3(m，4H)，3.26(d，J＝5.70Hz，2H)，3.04(br.s.，2H)，1.89(s，2H)，1.48-1.58(m，6H)，1.47(br.s.，2H)，1.33(br.s.，4H)。

Example S-7 preparation of 4- [ [ 4-fluoro-3- (piperazine-1-carbonyl) phenyl ] methyl ] -2H-phthalazin-1-one

Step 1: preparation of (Z) -2-fluoro-5- ((3-oxoisobenzofuran-1 (3H) -ylidene) methyl) benzonitrile

To a stirred solution of dimethyl phosphonate 3-oxo-1, 3-dihydroisobenzofuran-1-yl ester (10g, 41.28mmol) and 2-fluoro-5-formylbenzonitrile (6.15g, 41.28mmol, 1 eq) in THF (50mL) was slowly added triethylamine (5.76mL, 41.28mmol, 1 eq) at 0 deg.C. The resulting mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, water (200mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (50mL), hexane (50mL), diethyl ether (30mL) and dried under vacuum to give the title compound as a mixture of E-and Z-isomers which was used in the next step without further purification. LC-MS 266[ M + H ]⁺。¹H NMR(400MHz，CDCl₃)δ8.13(1H，m)，8.05(1H，m)，7.98(1H，m)，7.79(2H，m)，7.61(1H，m)，7.30(1H，m)，6.35(1H，s)。

Step 2: preparation of 2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoic acid

To a stirred suspension of (Z) -2-fluoro-5- ((3-oxoisobenzofuran-1 (3H) -ylidene) methyl) benzonitrile (3.7g, 13.94mmol) in water (20mL) was added a 13N NaOH solution (5mL), and the mixture was heated at 90 ℃ under nitrogen for 16 hours. The reaction mixture was cooled to 70 ℃ and hydrazine hydrate (10mL) was added and stirred at 70 ℃ for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction was cooled to room temperature and acidified at 0-5 ℃ with 2N HCl (pH 1-2) to give a precipitate. The precipitated solid was filtered through a buchner funnel, washed with water (50mL), n-hexane (50mL), diethyl ether (30mL) and dried under vacuum to give the title compound. LC-MS 299[ M + H ]]⁺。¹H NMR(400MHz，DMSO-d₆)δ13.22(1H，br s)，12.61(1H，s)，8.27(1H，m)，7.99-7.81(4H，m)，7.59(1H，m)，7.25(1H，m)，4.36(2H，s)。

And step 3: preparation of tert-butyl 4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazine-1-carboxylate

To a stirred suspension of 2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoic acid (0.100g, 0.335mmol) in DMA (4mL) at 0 ℃ was added HBTU (0.15g, 0.402mmol, 1.2 eq) and the mixture was stirred for 10 min. DIPEA (0.17mL, 1.00mmol, 3 equivalents) and piperazine-1-carboxylic acid tert-butyl ester (0.075g, 0.402mmol, 1.2 equivalents) were then added to the reaction mixture in that order at 0 ℃ and the resulting reaction mixture was stirred at room temperature for 75 minutes. The reaction was monitored by TLC. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (10mL × 2) and n-pentane (10mL × 2) and dried under reduced pressure to give the title compound, which was taken to the next step without further purification. LC-MS 467[ M + H [ ] ]⁺。

And 4, step 4: preparation of 4- [ [ 4-fluoro-3- (piperazine-1-carbonyl) phenyl ] methyl ] -2H-phthalazin-1-one

To tert-butyl 4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazine-1-carboxylate (10.0g, 76.23mmol) was added MeOH (30mL) with 2N HCl at room temperature and the mixture was stirred at room temperature for 16 hours. By monitoring the reaction.¹H NMR. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a compound, which was wet-milled with diethyl ether and pentane to give the title compound as a hydrochloride salt. LC-MS 403[ M + H]⁺。¹H NMR(400MHz，MeOD-d₄)δ3.66(s，3H)，2.92(t，J＝7.67Hz，2H)，2.37(t，J＝7.45Hz，2H)，1.62-1.71(m，6H)，1.37-1.45(m，2H)。

EXAMPLE S-8 preparation of (R) -methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropylsulfonyl) phenylamino) hexanoate

Step 1: preparation of (R) -3-bromo-N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide

To a solution of (R) -3-bromo-2-hydroxy-2-methylpropionic acid (13g, 71.04mmol) in THF (250mL) cooled to 0 deg.C was added SOCl dropwise₂(14.1g, 118.53mmol) and a catalytic amount of DMF. The solution was stirred at this temperature for 1.5 hours. A solution of 4-amino-2- (trifluoromethyl) -benzonitrile (7.4g, 39.78mmol) and TEA (15.8g, 156.29mmol) in THF (50mL) was then added dropwise at this temperature. The mixture was allowed to warm to room temperature overnight. TLC showed the reaction was complete. The mixture is washed with NaHCO ₃Quenched and extracted with EtOAc. The organic layer was washed with Na₂SO₄Dried and concentrated under reduced pressure. The crude product was purified by silica gel column to give the title compound.

Step 2: preparation of (R) -3- (4-aminophenylthio) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide

At 0 ℃ under N₂Next, 4-aminobenzenethiol (712mg, 5.68mmol) in THF (30mL) was added dropwise to a solution of NaH (251mg, 6.26mmol) in THF (20 mL). The mixture was allowed to warm to room temperature and stirred for 1 hour. A solution of (R) -3-bromo-N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (2g, 5.68mmol) in THF (20mL) was then added dropwise to the mixture at 0 ℃. The reaction mixture was stirred at room temperature overnight. TLC showed the reaction was complete. Water was added cautiously and the mixture was extracted with EtOAc and Na₂SO₄Dried and concentrated under reduced pressure. The compound was purified by silica gel column to give the title compound.

And step 3: preparation of (R) -methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropanylthio) phenylamino) hexanoate

To a solution of (R) -3- (4-aminophenylthio) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (2g, 5mmol) in DMF (100mL) was added K ₂CO₃(2.1g，15mmol)、KI(100mg，0.5mmol) and methyl 6-bromohexanoate (10g, 50 mmol). The mixture was stirred at 60 ℃ overnight. TLC showed the reaction was complete. The mixture was washed with water and extracted with EtOAc over Na₂SO₄And (5) drying. The organic layer was concentrated under reduced pressure and purified by flash chromatography to give the title compound.

And 4, step 4: preparation of (R) -methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropylsulfonyl) phenylamino) hexanoate

To a solution of (R) -methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropylthio) phenylamino) hexanoate (2g, 2.8mmol) in DCM (60mL) was added m-CPBA (1.83g, 8.4mmol) and the mixture was stirred at room temperature for 2 h. The mixture is washed with NaHCO₃The aqueous solution was quenched and washed with water and extracted with DCM. Subjecting the organic layer to Na₂SO₄Dried and purified by flash chromatography to give the title compound.¹H NMR(400MHz，DMSO-d₆)δ10.27(s，1H)，8.41(s，1H)，8.16(d，J＝8.6Hz，1H)，8.04(d，J＝8.6Hz，1H)，7.45(d，J＝8.9Hz，2H)，6.63-6.37(m，3H)，6.30(s，1H)，3.79(d，J＝14.5Hz，1H)，3.58(s，3H)，3.47(d，J＝14.5Hz，1H)，2.89(dq，J＝11.8，7.0，6.3Hz，2H)，2.31(t，J＝7.4Hz，2H)，1.55(p，J＝7.5Hz，2H)，1.47(q，J＝7.2Hz，2H)，1.38(s，3H)，1.36-1.26(m，2H)。

EXAMPLE S-9 preparation of (S) -methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropoxy) phenylamino) hexanoate

Step 1: preparation of (S) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methyl-3- (4-nitrophenoxy) propionamide

To 4-nitrophenol (1.2g, 8.55mmol) and K₂CO₃(3.9g, 28.50mmol) to a stirred suspension in isopropanol (40mL) was added (R) -3-bromo-N- (4-cyano-3- (trifluoromethyl)) Phenyl) -2-hydroxy-2-methylpropanamide (2g, 5.70 mol). The reaction was refluxed for 2 hours. TLC indicated the reaction was complete. The reaction mixture was concentrated in vacuo, diluted with water, and extracted with EtOAc. The combined organic phases were washed with saturated NaHCO₃Washed with brine and over anhydrous Na₂SO₄Dried and concentrated in vacuo to give a residue as an oil. The residue was purified by silica gel column chromatography to give the title compound.

Step 2: preparation of (S) -3- (4-aminophenoxy) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide

To (S) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methyl-3- (4-nitrophenoxy) propionamide 3(2.0g, 4.64mmol) in EtOH (20mL) and H₂To a stirred suspension in O (20mL) was added NH₄Cl (2.5g, 46.42mmol) and iron powder (2.1g, 3.71 mmol). Reacting with N₂Degassed and stirred at 90 ℃ for 1 hour. TLC indicated the reaction was complete. The mixture was diluted with EtOAc, filtered through a pad of celite, washed with brine, and dried over anhydrous Na₂SO₄Dried and concentrated in vacuo to give the title compound.

And step 3: preparation of (S) -methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropoxy) phenylamino) hexanoate

To a solution of (S) -3- (4-aminophenoxy) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (1.7g, 4.48mmol) and methyl 6-bromohexanoate (0.7g, 3.45mmol) in DMF (30mL) was added K₂CO₃(1.0g, 7.56mmol) and KI (0.6g, 3.80 mmol). The reaction was stirred at 100 ℃ for 4 hours. TLC indicated about 30% of (S) -3- (4-aminophenoxy) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide remained. The mixture was diluted with water and extracted with EtOAc. The combined organic phases were washed with brine and over anhydrous Na₂SO₄Dried and concentrated in vacuo to give a residue. The residue was purified by silica gel column chromatography to give the title compound. LCMS 508.6[ M +1 ]]⁺。¹H NMR(400MHz，DMSO-d₆)δ10.54(s，1H)，8.57(d，J＝2Hz，1H)，8.32(dd，J＝2Hz，8.4Hz 1H)，8.11(d，J＝8.8Hz，1H)，6.70-6.68(m，1H)，6.48-6.46(m，1H)，6.17(s，1H)，5.18(s，1H)，4.10(d，J＝9.6Hz，1H)，3.87(d，J＝9.6Hz，1H)，3.58(s，1H)，2.32(t，J＝7.2Hz)，1.57-1.49(m，4H)，1.41(s，3H)，1.36-1.33(m，2H)。

EXAMPLE S-10 preparation of (R) -3- (4- (6-bromohexyloxy) phenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide

Step 1: preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-3- (4-hydroxyphenylthio) -2-methylpropanamide

(R) -3-bromo-N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methyl-propionamide (1.5g, 4.27mmol), 4-mercaptophenol (0.539g, 4.27mmol) and K ₂CO₃(0.649g, 4.69mmol) in acetone (15 mL). The reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction mixture was complete. The solution was concentrated in vacuo. The crude product was purified by column chromatography to give the title compound.

Step 2: preparation of (R) -3- (4- (6-bromohexyloxy) phenylthio) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide

(R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-3- (4-hydroxyphenylthio) -2-methylpropanamide (1.34g, 3.38mmol), 1, 6-dibromohexane (8.25g, 33.81mmol) and K₂CO₃(2.34g, 16.9mmol) in CAN (20 mL). The reaction mixture was stirred at room temperature for 3 hours. TLC showed the mixture was complete. The solution was concentrated in vacuo and purified by column chromatography to give the title compound.

And step 3: preparation of (R) -3- (4- (6-bromohexyloxy) phenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide

(R) -3- (4- (6-bromohexyloxy) phenylthio) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (1).A mixture of 5g, 2.68mmol) in DCM (15mL) was cooled to 0 deg.C and m-CPBA (1.39g, 8.04mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. TLC showed the mixture was complete. The solution was poured into 1N NaOH and extracted with DCM (50mL × 3). The organic layer was washed with brine, over Na ₂SO₄Drying, concentration and purification by column chromatography gave the title compound.¹H NMR(400MHz，CDCl₃)δ8.94(s，1H)，7.88(d，J＝2.8Hz 1H)，7.74-7.68(m，2H)，7.34-7.30(m，2H)，6.66-6.62(m，2H)，3.81-3.71(m，4H)，3.43(t，J＝6.8Hz 2H)，2.99(d，J＝14.4Hz 1H)，1.93-1.86(m，2H)，1.78-1.71(m，2H)，1.57-1.43(m，7H)。

EXAMPLE S-11 preparation of 4- (1- (4- (2-bromoethoxy) phenyl) -2-phenylbut-1-enyl) pivalic acid phenyl ester

Step 1: preparation of 4- (4-hydroxybenzoyl) phenyl

Part 1: to a solution of bis (4-hydroxyphenyl) methanone 1(2g, 9.34mmol) in 100mL THF was added Et₃N (3.8g, 37.36 mmol). PivCl (2.82g, 23.35mmol) was then added dropwise to the mixture at 0 ℃. The mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was quenched with water and extracted with EtOAc. The organic layer was washed with 1N HCl and brine, over Na₂SO₄Dried and concentrated under reduced pressure to give the crude product.

Section 2: to a solution of the crude product (3.5g) in THF (45mL) and MeOH (3mL) was added LiOH (290mg, 12.14mmol) and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and purified by silica gel chromatography to give the title compound.

Step 2: preparation of (E) -4- (1- (4-hydroxyphenyl) -2-phenylbut-1-enyl) pivalic acid

At 0 ℃ under N₂To a suspension of Zn (1.7g, 26.8mmol) in dry THF (150mL) was added TiCl₄(2.5g, 13.4 mmol). Returning the mixture to Flow for 2 hours and cool at 40 ℃. A mixture of 4- (4-hydroxybenzoyl) phenyl pivalate (1g, 3.35mmol) and propiophenone (1.5g, 10.72mmol) in anhydrous THF (50mL) was immediately added and the mixture was refluxed for 1 hour. The reaction mixture was taken up with 10% K₂CO₃Quench, extract with EtOAc, wash with brine, and wash over Na₂SO₄Drying, purification by silica gel chromatography gave the crude product, which was then added MeOH (10mL) and stirred for 20 min to give the title compound.

And step 3: preparation of 4- (1- (4- (2-bromoethoxy) phenyl) -2-phenylbut-1-enyl) pivalic acid phenyl ester

To a solution of (E) -4- (1- (4-hydroxyphenyl) -2-phenylbut-1-enyl) phenyl pivalate (0.4g, 1mmol) in acetone (6mL) was added K₂CO₃(0.56g, 4mmol), 1, 2-dibromoethane 5(1.9g, 10 mmol). The mixture was stirred at 110 ℃ overnight. TLC showed the reaction was complete. The mixture was washed with water and extracted with EtOAc over Na₂SO₄And (5) drying. The organic layer was concentrated under reduced pressure and purified by silica gel column to give the title compound.¹H NMR(400MHz，CDCl₃，E/Z＝1∶1)δ7.23(d，J＝8.7Hz，2H)，7.20-7.14(m，6H)，7.14-7.07(m，6H)，7.05(d，J＝8.6Hz，2H)，6.87(dd，J＝15.3，8.8Hz，4H)，6.77(d，J＝8.9Hz，2H)，6.70(d，J＝8.8Hz，2H)，6.55(d，J＝8.9Hz，2H)，4.31(t，J＝6.3Hz，2H)，4.15(t，J＝6.3Hz，2H)，3.66(t，J＝6.3Hz，2H)，3.55(t，J＝6.3Hz，2H)，2.57-2.39(m，4H)，1.37(s，9H)，1.28(s，9H)，0.97-0.89(m，6H)。

EXAMPLE S-12 preparation of (R) -3- (4- (2-bromoethylamino) phenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) -phenyl) -2-hydroxy-2-methylpropanamide

Step 1: preparation of (R) -3- (4-aminophenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide

At 0 ℃ to (R)) To a solution of (E) -3- (4-aminophenylthio) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide 1(3.0g, 7.59mmol) in residual DCM (20mL) was added 85% mCPBA (4.6g, 22.76 mmol). The reaction was stirred at room temperature for 4 hours. TLC indicated the reaction was complete. The mixture was washed with saturated NaHCO₃And (6) washing. The organic phase was washed with brine, over anhydrous Na₂SO₄Drying, concentration under reduced pressure, and purification by column chromatography gave the title compound.

Step 2: synthesis of 2-bromoacetaldehyde

A mixture of 2-bromo-1, 1-diethoxyethane (20g, 0.101mol) in HBr (47%, 32mL) was stirred at 100 ℃ for 2 hours. The mixture was washed with Et₂And (4) extracting. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄Dried and concentrated under reduced pressure to give the title compound, which was used directly in the next step.

And step 3: synthesis of (R) -3- (4- (2-bromoethylamino) phenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) -phenyl) -2-hydroxy-2-methylpropanamide

To a solution of (R) -3- (4-aminophenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (2g, 4.68mmol) and 2-bromo-1, 1-diethoxyethane (5.2g, 42.11mmol) in MeOH (40mL) was added AcOH (0.18g, 3.04mmol), followed by BH ₃NMe₃(4.8g, 65.51 mmol). The reaction was monitored by TLC and an additional portion of 2-bromo-1, 1-diethoxyethane was added until the conversion was complete. The mixture was diluted with EtOAc and saturated NaHCO₃Washed with brine and over anhydrous Na₂SO₄Dried and concentrated under reduced pressure. The crude material was purified by column chromatography to give the title compound. LCMS 536.1[ M +1 ]]⁺。¹H NMR(600MHz，DMSO-d₆)δ10.33(s，1H)，8.44(d，J＝3Hz，1H)，8.20(dd，J＝3Hz，12.6Hz，1H)，8.08(d，J＝12.6Hz，1H)，7.51(d，J＝13.8Hz，2H)，6.60(d，J＝13.8Hz，2H)，6.33(s，1H)，2.98(s，3H)，3.82(d，J＝21.6Hz，1H)，3.53(m，6H)，1.39(s，3H)。

EXAMPLE S-13 preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide (Compound 1.68)

Step A: preparation of (S) -6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylhydro) -2-hydroxy-2-methyl-3-oxopropylsulfonyl) phenylamino) hexanoic acid

To a solution of (S) -methyl 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropylsulfonyl) phenylamino) hexanoate (0.20g, 0.360mmol) in THF MeOH H₂To O (6 mL: 3 mL: 1mL) was added lithium hydroxide monohydrate (0.151mg, 3.6mmol, 10 equivalents), and the mixture was heated at 50 ℃ for 2 hours. After completion, the mixture was concentrated under reduced pressure. The residue obtained was acidified with 1N HCl (pH about 2) to give a precipitate, which was filtered through a buchner funnel to give the title compound. LC-MS 542[ M + H ]⁺。

Preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide (Compound 1.68)

To a stirred solution of (S) -6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropylsulfonyl) phenylamino) hexanoic acid (0.20g, 0.369mmol) in DMF (5mL) at 0 ℃ was added HATU (0.21g, 0.554mmol, 1.5 eq) and the mixture was stirred at 0 ℃ for 30 min. DIPEA (0.34mL, 1.84mmol, 5 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloride (0.297g, 0.739mmol, 2.0 equivalents) were then added to the mixture in succession and the resulting mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC and LC-MS. Upon completion, the reaction was diluted with EtOAc (250 mL). The organic layer was washed with water (100mL), brine (50mL) and Na₂SO₄Drying, filtering andconcentration under reduced pressure gave a crude residue which was purified by reverse phase HPLC to give compound 1.68. LC-MS 890[ M + H [)]⁺。¹H NMR(400MHz，CD₃OD-d₄)δ8.36(d，J＝7.8Hz，1H)，8.21(s，1H)，7.98-7.77(m，5H)，7.56-7.43(m，3H)，7.38(d，J＝6.4Hz，1H)，7.15(t，J＝9.0Hz，1H)，6.42(dd，J＝9.0，5.2Hz，2H)，4.38(s，2H)，4.00(d，J＝14.5Hz，1H)，3.80-3.60(m，4H)，3.55-3.38(m，3H)，3.28(s，1H)，2.89(tt，J＝12.3，6.0Hz，2H)，2.43(dt，J＝28.6，7.4Hz，2H)，1.60(ddt，J＝36.4，15.2，7.6Hz，4H)，1.44(d，J＝14.4Hz，5H)。

EXAMPLE S-14 preparation of (S) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexylamino) phenoxy) -2-hydroxy-2-methylpropanamide (Compound 1.83)

Step A: preparation of (R) -6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropoxy) phenylamino) hexanoic acid

To 6- (4- (3- (4-cyano-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropoxy) phenylamino) hexanoic acid (R) -methyl ester (0.20g, 0.360mmol) in THF: MeOH: H₂To O (6 mL: 3 mL: 1mL) was added lithium hydroxide monohydrate (0.151mg, 3.6mmol, 10 equivalents), and the mixture was heated at 50 ℃ for 2 hours. After completion, the mixture was concentrated under reduced pressure. The residue obtained was acidified with 1N-HCl (pH about 2) to give a precipitate, which was filtered through a buchner funnel to give the title compound. LC-MS494[ M + H ]]⁺。

Preparation of (S) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexylamino) phenoxy) -2-hydroxy-2-methylpropanamide (Compound 1.83)

To (R) -6- (4- (3- (4-cyano) group at 0 deg.CTo a stirred solution of-3- (trifluoromethyl) phenylamino) -2-hydroxy-2-methyl-3-oxopropoxy) phenylamino) hexanoic acid (0.20g, 0.405mmol) in DMF (5mL) was added HATU (0.23g, 0.608mmol, 1.5 eq) and the mixture was stirred at 0 ℃ for 30 min. DIPEA (0.37mL, 2.02mmol, 5 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.24g, 0.608mmol, 1.5 equivalents) were then added to the reaction mixture in that order, and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was diluted with EtOAc (250 mL). The organic layer was washed with water (100mL), brine (50mL) and Na ₂SO₄Drying, filtration and concentration under reduced pressure gave a crude residue which was purified by reverse phase HPLC to give compound 1.83. LC-MS 842.8[ M + H [ ]]⁺。¹H NMR(400MHz，DMSO-d₆)δ8.55(d，J＝2.9Hz，1H)，8.28(d，J＝10.5Hz，2H)，8.08(d，J＝8.6Hz，1H)，7.96(d，J＝8.1Hz，1H)，7.89(t，J＝7.4Hz，1H)，7.83(t，J＝7.5Hz，1H)，7.44(t，J＝6.6Hz，1H)，7.36(s，1H)，7.23(t，J＝9.0Hz，1H)，7.06(d，J＝8.5Hz，1H)，6.89(s，1H)，6.67(d，J＝7.8Hz，2H)，6.45(d，J＝8.1Hz，2H)，5.06(t，J＝5.5Hz，1H)，4.33(s，2H)，4.06(d，J＝9.5Hz，1H)，3.86(t，J＝9.9Hz，1H)，3.62(s，2H)，3.51(t，J＝4.9Hz，2H)，3.15(d，J＝14.4Hz，2H)，2.91(d，J＝17.5Hz，2H)，2.28(d，J＝7.9Hz，1H)，1.69(s，2H)，1.51(q，J＝6.5，5.7Hz，4H)，1.38(d，J＝12.8Hz，6H)。

EXAMPLE S-15 preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) hexyloxy) phenylsulfonyl) -2-hydroxy-2-methylpropanamide (Compound 1.96)

To a stirred solution of 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloride (0.15g, 0.254mmol) in EtOH (4mL) was added DIPEA (0.234mL, 1.27 mm)ol, 5 eq), followed by addition of (S) -3- (4- (6-bromohexyloxy) phenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (0.153g, 0.381mmol, 2.0 eq), and the mixture was stirred at 80 ℃ for 16 h. The reaction was monitored by TLC and LC-MS. After completion, the reaction was concentrated under reduced pressure to give a crude residue which was purified by reverse phase HPLC to give compound 1.96. LC-MS 877[ M + H ]]⁺。¹H NMR(400MHz，DMSO-d₆)δ12.53(d，J＝39.4Hz，1H)，10.47(s，1H)，8.39(d，J＝2.1Hz，1H)，8.26(d，J＝7.8Hz，1H)，8.18-8.10(m，1H)，8.05(d，J＝8.6Hz，1H)，7.96(d，J＝8.0Hz，1H)，7.88(t，J＝7.5Hz，1H)，7.82(t，J＝7.4Hz，1H)，7.73(d，J＝8.4Hz，2H)，7.42(ddd，J＝8.2，5.1，2.4Hz，1H)，7.30(dd，J＝6.4，2.4Hz，1H)，7.21(t，J＝9.0Hz，1H)，6.94(d，J＝8.5Hz，2H)，4.32(s，2H)，3.85(dt，J＝15.8，11.9Hz，3H)，3.65-3.56(m，3H)，3.13(t，J＝5.0Hz，2H)，2.37(t，J＝5.0Hz，2H)，2.27(t，J＝7.3Hz，2H)，2.24-2.17(m，2H)，1.78(s，2H)，1.66(p，J＝7.2Hz，2H)，1.50-1.27(m，7H)。

EXAMPLE S-16 preparation of (S) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (2- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) methyl) -1H-1, 2, 3-triazol-1-yl) ethylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide (Compound 1.97)

Step 1: preparation of (S) -3- (4- (2-azidoethylamino) phenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide

To a stirred solution of (S) -3- (4- (2-bromoethylamino) phenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (0.30g, 0.56mmol) in DMF (5mL) was added sodium azide (0.073, 0.112mmol, 2 eq) at room temperature and the mixture was heated at 80 ℃ for 2 hours. The reaction was monitored by TLC. After the reaction is completed, the reaction solution is added,the mixture was diluted with EtOAc (250 mL). The organic layer was washed with water (100 mL. times.2), brine (100mL), and anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave the crude product, which was purified by CombiFlash chromatography to give the title compound. LC-MS 497[ M + H]⁺。

Step 2 a: preparation of 4- (4-fluoro-3- (4- (prop-2-ynyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To a stirred solution of 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.40g, 0.99mmol) in EtOH (5mL) was added DIPEA (0.91mL, 4.97mmol, 5 equivalents), followed by bromoacetylene (0.176g, 1.49mmol, 2.0 equivalents), and the mixture was heated at 80 ℃ for 16 hours. The reaction was monitored by TLC and LC-MS. After completion, the mixture was concentrated under reduced pressure to give a crude product which was purified by CombiFlash chromatography to give the title compound. LC-MS 405[ M + H [ ] ]⁺。

Step 2: preparation of (S) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (2- (4- ((4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) methyl) -1H-1, 2, 3-triazol-1-yl) ethylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide (Compound 1.97)

To (S) -3- (4- (2-azidoethylamino) phenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (0.150g, 0.301mmol) and 4- (4-fluoro-3- (4- (prop-2-ynyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (0.182g, 0.452mmol, 15 equivalents) in MeCN: H₂To a stirred solution in O (2: 1) (6mL) was added copper sulfate pentahydrate (0.024g, 0.15mmol, 0.5 equiv.) and hydrazine hydrate (0.011mg, 1.00mmol, 1 equiv.) in that order, and the resulting mixture was heated at 60 ℃ for 2 hours. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was diluted with water (50mL) and extracted with ethyl acetate (50mL × 2). The combined organic layers were washed with 10% NH₄OH solution washing, anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave the crude product, which was purified by CombiFlash chromatography to give compound 1.97. LC-MS 901[ M + H [ ]]⁺。¹H NMR(400MHz，DMSO-d₆)δ12.59(s，1H)，10.35(s，1H)，8.43(d，J＝2.1Hz，1H)，8.29-8.15(m，2H)，8.06(d，J＝8.6Hz，1H)，8.00(s，1H)，7.96(d，J＝8.0Hz，1H)，7.88(t，J＝7.5Hz，1H)，7.82(t，J＝7.5Hz，1H)，7.48(d，J＝8.5Hz，2H)，7.42(s，1H)，7.32(d，J＝6.1Hz，1H)，7.22(t，J＝8.9Hz，1H)，6.72(t，J＝6.0Hz，1H)，6.56(d，J＝8.5Hz，2H)，6.32(s，1H)，4.47(s，2H)，4.32(s，2H)，3.78(d，J＝14.5Hz，1H)，3.52(dt，J＝19.4，11.7Hz，6H)，3.14(s，2H)，2.40(s，2H)，2.28(s，1H)，1.38(s，3H)，1.23(s，1H)。

EXAMPLE S-17 preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -2-oxoethylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide (Compound 1.98)

Step 1: preparation of 4- (3- (4- (2-bromoacetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one

To 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (1.0g, 2.48mmol) in DCM (5mL) at room temperature was added Et₃N (3.58mL, 24.8mmol) and the mixture was added slowly to a previously stirred solution of 2-bromoacetyl chloride (3.85g, 24.8mmol) in DCM (20mL) at 0 deg.C. The resulting mixture was stirred at room temperature for 10 minutes and monitored by TLC and LC-MS. After completion, the reaction was quenched with NaHCO₃The saturated solution (30mL) was quenched and extracted with DCM (200 mL. times.2). The combined organic layers were washed with H₂O (100mL), brine (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude residue which was purified by CombiFlash chromatography to afford the title compound. LC-MS 487[ M + H ]]⁺

Step 2: preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -2-oxoethylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide

To a stirred solution of (R) -3- (4-aminophenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (0.105g, 0.246mmol, 1 eq) in DMF (5ml) was added 4- (3- (4- (2-bromoacetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (0.12g, 0.246mmol, 1 eq) and the reaction mixture was stirred at 80 ℃ for 16H. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the mixture was diluted with EtOAc (100 mL). Subjecting the organic layer to ice-cold H ₂O (50mL × 2), brine (30mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude residue which is purified by reverse phase HPLC to give the title compound. LC-MS: 487[ M + H ]]⁺。¹HNMR(400MHz，DMSO-d₆)δ12.60(s，1H)，10.33(s，1H)，8.43(d，J＝2.1Hz，1H)，8.31-8.23(m，1H)，8.21-8.15(m，1H)，8.06(d，J＝8.5Hz，1H)，7.97(t，J＝5.2Hz，1H)，7.90(d，J＝6.6Hz，1H)，7.85(d，J＝7.9Hz，1H)，7.48(t，J＝9.7Hz，1H)，7.42-7.33(m，3H)，7.25(q，J＝7.6Hz，2H)，6.64(dd，J＝11.7，8.2Hz，2H)，6.32(s，1H)，4.34(s，2H)，3.90(d，J＝19.0Hz，2H)，3.79(d，J＝14.5Hz，1H)，3.70(s，1H)，3.63(s，1H)，3.57(s，2H)，3.52(s，1H)，3.42(s，2H)，3.26(s，2H)，3.20(s，1H)，1.39(s，3H)。

EXAMPLE S-18 preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) ethylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide (Compound 1.99)

Step 1: preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) ethylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide

To 4- (4-fluoro-3- (piperazine-1)-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloride (0.075g, 0.187mmol, 1.0 equiv) to a stirred solution in EtOH (10mL) was added DIPEA (0.201mL, 1.12mmol, 6 equiv) followed by (R) -3- (4- (2-bromoethylamino) phenylsulfonyl) -N- (4-cyano-3- (trifluoromethyl) phenyl) -2-hydroxy-2-methylpropanamide (0.10g, 0.187mmol) and the mixture was stirred at 80 ℃ for 16H. The reaction was monitored by TLC and LC-MS. After completion, the reaction was concentrated under reduced pressure to give a crude residue which was purified by reverse phase HPLC to give the title compound. LC-MS-820 [ M + H ═ M ]⁺。¹HNMR(400MHz，DMSO-d₆)δ12.61(br.s.，1H)，8.41(br.s.，1H)，8.26(d，J＝7.5Hz，1H)，8.14(d，J＝7.0Hz，1H)，8.03(d，J＝7.9Hz，1H)，7.96(d，J＝7.5Hz，1H)，7.91-7.80(m，2H)，7.50-7.38(m，3H)，7.32(d，J＝4.4Hz，1H)，7.22(t，J＝9.0Hz，1H)，6.58-6.48(m，2H)，6.40(br.s.，1H)，3.76(d，J＝14.0Hz，1H)，3.62(br.s.，2H)，3.50(d，J＝14.5Hz，1H)，3.16(br.s.，2H)，3.07(d，J＝5.3Hz，2H)，2.49-2.4(m，4H)，2.31(br.s.，2H)，1.68(s，2H)，1.37(s，3H)。

EXAMPLE S-19 preparation of 4- (3- (4- (2- (2- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) ethoxy) acetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (Compound 1.104)

Step 1: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ] phenanthrene-3, 2' - [1, 3] dioxolane ] -17-ol

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] at room temperature]To a stirred solution of phenanthren-3 (2H) -one (15g, 51.65mmol) in benzene (500mL) was added PTSA (4.85g, 25.8mmol, 0.5 equiv.), followed by ethylene glycol (14.5mL, 258mmol, 5 equiv.). The resulting mixture was refluxed for 16 hours using a dean-Stark apparatus. The reaction was monitored by TLC. Inverse directionAfter completion, the reaction mixture was diluted with water (500mL) and extracted with EtOAc (500 mL. times.2). The combined organic layers were washed with NaHCO₃The saturated solution (250mL), water (400mL), brine (200mL) was washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound.¹HNMR(400MHz，DMSO-d₆)δ4.40(d，J＝5.26Hz，1H)，3.81(s，3H)，3.45-3.37(m，2H)，1.82(br.s.，2H)，1.70(d，J＝12.72Hz，2H)，1.59(d，J＝13.15Hz，2H)，1.52(d，J＝10.52Hz，3H)，1.35(br.s.，1H)，1.33-1.23(m，3H)，1.21-1.03(m，5H)，0.87(dt，J＝7.89，19.95Hz，4H)，0.76(s，3H)，0.61(s，3H)

Step 2 a: preparation of 2- (2- (2-chloroethoxy) ethoxy) tetrahydro-2H-pyran

To a stirred solution of 2- (2-chloroethoxy) ethanol (10g, 80.6mmol) in diethyl ether (150mL) was added PTSA (1.54g, 0.806mmol, 0.1 equiv.), followed by 3, 4-dihydro-2H-pyran (8.12g, 96.7mmol, 1.2 equiv.). The resulting reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with diethyl ether (700 mL). The organic layer was washed with 20% KOH solution (300mL), water (600mL), brine (300mL) and Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound which was taken to the next step without further purification.¹HNMR(400MHz，DMSO-d₆)δ4.62-4.56(m，1H)，3.75-3.66(m，6H)，3.63-3.56(m，2H)，3.53-3.46(m，1H)，3.46-3.39(m，1H)，1.75-1.67(m，1H)，1.65-1.57(m，1H)，1.52-1.39(m，4H)。

Step 2: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (2- (2- (tetrahydro-2H-pyran-2-yloxy) ethoxy) hexadecahydrospiro [ cyclopenta [ a ] phenanthrene-3, 2' - [1, 3] dioxolane ]

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ]]Phenanthrene-3, 2' - [1, 3]]Dioxolanes]To a stirred solution of-17-ol (1.2g, 3.59mmol) in xylene (12mL) was added NaNH₂(50% suspension in toluene, 3.5mL) and the mixture was brought to 150 deg.CThe mixture was heated for 1 hour. The reaction mixture was gradually cooled to 25 ℃, 2- (6-chlorohexyloxy) tetrahydro-2H-pyran (7.47g, 35.9mmol) was added thereto, and the resulting mixture was further heated at 150 ℃ for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature, quenched slowly with ice-cold water (500mL), and extracted with EtOAc (300mL × 2). The combined organic layers were washed with water (250 mL. times.2), brine (200mL) and Na ₂SO₄Drying, filtration and concentration under reduced pressure gave the crude product, which was purified by CombiFlash chromatography to give the title compound.¹HNMR(400MHz，CDCl3)δ4.64(t，J＝3.5Hz，1H)，3.93(s，4H)，3.90-3.81(m，4H)，3.74(dd，J＝5.5，3.7Hz，1H)，3.70-3.64(m，4H)，3.64-3.54(m，4H)，3.54-3.44(m，2H)，3.32(t，J＝8.3Hz，2H)，2.02-1.93(m，2H)，1.90-1.80(m，2H)，1.66-1.60(m，6H)，1.44-1.33(m，4H)，1.28-1.16(m，6H)，0.98-0.84(m，4H)，0.84-0.77(m，2H)，0.76-0.69(m，2H)。

And step 3: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (2- (2-hydroxyethoxy) ethoxy) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] phenanthren-3 (2H) -one

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (2- (2- (tetrahydro-2H-pyran-2-yloxy) ethoxy) hexadecahydrospiro [ cyclopenta [ a ] at room temperature]Phenanthrene-3, 2' - [1, 3 ]]Dioxolanes](0.5g, 0.988mmol) in THF (30mL) -H₂To a stirred solution in O (10mL) was added 6N HCl (20mL), and the resulting reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (30mL) and NaHCO was used₃The saturated solution (pH about 8) was basified. The aqueous layer was then extracted with EtOAc (40 mL. times.3). The organic layer was washed with NaHCO₃Saturated solution (30mL), water (30mL), brine (20mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound which was used in the next step without further purification.¹HNMR(400MHz，CDCl3)δ3.75-3.69(m，2H)，3.69-3.60(m，4H)，3.36(t，J＝8.3Hz，1H)，2.66(t，J＝6.4Hz，1H)，2.37(dd，J＝13.8，6.4Hz，1H)，2.33-2.25(m，2H)，2.12-2.08(m，1H)，2.07-1.96(m，2H)，1.96-1.87(m，2H)，1.70(dd，J＝13.4，3.3Hz，2H)，1.47-1.40(m，2H)，1.27-1.28(m，4H)，1.27-1.15(m，4H)，1.04-0.99(m，4H)，0.97(d，J＝7.9Hz，2H)，0.88-0.81(m，2H)，0.79(s，2H)。

And 4, step 4: preparation of 2- (2- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) ethoxy) acetic acid

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (2- (2-hydroxyethoxy) ethoxy) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] over a period of 30 minutes at 0 deg.C]To a stirred solution of phenanthren-3 (2H) -one (0.40g, 0.947mmol) in acetone (25mL) was added dropwise jones reagent (2.4 mL). The resulting mixture was stirred at 0 ℃ for 10 minutes. The reaction was monitored by TLC. Upon completion, water (100mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (50mL × 2) and n-pentane (50mL × 2) and dried under vacuum to give the title compound which was taken to the next step without further purification.¹HNMR(400MHz，DMSO-d₆)δ12.51(br.s.，1H)，4.02(s，2H)，3.58-3.46(m，3H)，2.39(dd，J＝6.8，14.7Hz，2H)，2.34-2.23(m，2H)，2.08(d，J＝13.6Hz，2H)，1.97-1.85(m，3H)，1.80(d，J＝12.7Hz，1H)，1.61(d，J＝13.2Hz，1H)，1.51(br.s.，3H)，1.42-1.33(m，2H)，1.32-1.06(m，8H)，1.00-0.92(m，2H)，0.70(s，2H)

And 5: preparation of 4- (3- (4- (2- (2- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) ethoxy) acetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one

To 2- (2- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred solution of phenanthren-17-yloxy) ethoxy) acetic acid (0.05g, 0.127mmol) in DMF (5mL) was added HATU (0.058g, 0.153mmol, 1.2 eq) and the resulting reaction mixture was stirred for 10 min. DIPEA (0.114mL, 0.637mmol, 5 equiv.) and 4- (4-fluoro-3- (piperazine-1-carbonyl) were then added ) Benzyl) phthalazin-1 (2H) -one hydrochloride (0.035g, 0.089mmo |, 0.7 eq) was added to the mixture in succession, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC and LC-MS. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (25mL × 2) and n-pentane (25mL × 2) and dried under vacuum to give a crude residue which was purified by reverse phase HPLC to give the title compound. LC-MS 741[ M + H [ ]]⁺。¹HNMR(400MHz，MeOD-d₄)δ8.37(d，J＝7.8Hz，1H)，7.96(t，J＝7.5Hz，1H)，7.86(dt，J＝18.5，7.4Hz，2H)，7.48(t，J＝7.0Hz，1H)，7.45-7.33(m，1H)，7.16(td，J＝9.0，3.5Hz，1H)，4.39(s，2H)，4.28(s，1H)，4.21(s，1H)，3.85-3.72(m，2H)，3.71-3.48(m，9H)，3.39-3.32(m，2H)，3.17(s，2H)，3.12(s，2H)，2.53-2.30(m，1H)，2.09-1.95(m，1H)，1.88(ddd，J＝13.8，10.0，6.3Hz，2H)，1.79-1.20(m，11H)，1.04(dd，J＝14.2，8.7Hz，2H)，0.90(td，J＝11.9，11.3，6.7Hz，1H)，0.79(dd，J＝13.2，6.4Hz，4H)，0.60(d，J＝11.1Hz，2H)。

EXAMPLE S-20 preparation of 4- (4-fluoro-3- (4- (2- (4- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) -1H-1, 2, 3-triazol-1-yl) acetyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 1.107)

Step 1: preparation of 4- (3- (4- (2-bromoacetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one

To 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (1.0g, 2.48mmol) in DCM (5mL) at room temperature was added Et₃N (3.58mL, 24.8mmol) and the mixture was added slowly to a previously stirred solution of 2-bromoacetyl chloride (3.85g, 24.8mmol) in DCM (20mL) at 0 deg.C. The resulting mixture was stirred at room temperature for 10 minutes and monitored by TLC and LC-MS. After completion, the reaction mass was used NaHCO₃The saturated solution (30mL) was quenched and extracted with DCM (200 mL. times.2). The combined organic layers were washed with H₂O (100mL), brine (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude residue which was purified by CombiFlash chromatography to afford the title compound. LC-MS 487[ M + H ]]⁺

Step 2: preparation of 4- (3- (4- (2-azidoacetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one

To a stirred solution of 4- (3- (4- (2-bromoacetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (0.20g, 0.411mmol, 1 eq) in DMF (10ml) was added sodium azide (0.053, 0.83mmol, 2 eq) at room temperature and the mixture was heated at 80 ℃ for 2H. The reaction was monitored by TLC. Upon completion, the mixture was diluted with EtOAc (100mL) the organic layer was washed with ice cold water (80mL × 2), brine (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude residue which was purified by CombiFlash chromatography to give the title compound. LC-MS 450[ M + H [ ]]⁺

And step 3: preparation of 4- (4-fluoro-3- (4- (2- (4- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) -1H-1, 2, 3-triazol-1-yl) acetyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To 4- (3- (4- (2-azidoacetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (0.120g, 0.27mmol, 1 eq.) and (5S, 8R, 9S, 10S, 13S, 14S, 17R) -17-ethynyl-17-hydroxy-10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a]Phenanthren-3 (2H) -one (0.093g, 0.32mmol, 1.2 equiv.) in MeCN: H₂To a stirred solution of O (2: 1) (6mL) was added CuSO₄.5H₂O (0.021g, 0.13mmol, 0.5 equiv) and hydrazine hydrate (0.0085g, 0.26mmol, 1 equiv) and the resulting reaction mixture was heated at 80 ℃ for 16 h. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was diluted with water (30mL) and extracted with EtOAc (40X 2 mL). The combined organic layers were washed with brine (30mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by reverse phase HPLCTo yield the title compound. LC-MS 764[ M + H ]]⁺。¹HNMR(400MHz，DMSO-d₆)δ12.60(s，1H)，8.30-8.23(m，1H)，8.01-7.79(m，3H)，7.68(s，1H)，7.48-7.33(m，2H)，7.25(td，J＝9.2，5.9Hz，1H)，5.46(s，1H)，5.40(s，1H)，5.05(s，1H)，4.34(s，2H)，3.71(s，1H)，3.68-3.53(m，3H)，3.46(s，1H)，3.40(s，2H)，3.28(d，J＝7.5Hz，1H)，3.20(s，1H)，2.42-2.21(m，2H)，2.10-1.99(m，1H)，1.95-1.78(m，3H)，1.71(dd，J＝22.7，10.8Hz，2H)，1.47-1.31(m，5H)，1.30-1.12(m，4H)，0.96(s，3H)，0.91(s，3H)，0.84(d，J＝105Hz，1H)，0.48-0.38(m，2H)。

EXAMPLE S-21 preparation of 4- (3- (4- (2- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) acetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (Compound 1.110)

Step 1: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ] phenanthrene-3, 2' - [1, 3] dioxolane ] -17-ol

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] at room temperature]To a stirred solution of phenanthren-3 (2H) -one (10g, 34.48mmol) in benzene (330mL) was added PTSA (1.54g, 17.27mmol, 0.1 equiv.), followed by ethylene glycol (9.63g, 172.7mmol, 5.0 equiv.). The resulting mixture was heated at 150 ℃ for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (200mL) and extracted with EtOAc (350 mL). The organic layer was washed with NaHCO₃Saturated solution (100mL), water (200mL), brine (100mL), washed with Na₂SO₄Drying, filtering and concentrating under reduced pressure to give (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ]]Phenanthrene-3, 2' - [1, 3 ]]Dioxolanes]-17-ol.¹H NMR(400MHz，DMSO-d₆)δ4.40(d，J＝5.26Hz，1H)，3.81(s，3H)，3.37-3.45(m，2H)，1.82(brs，2H)，1.70(d，J＝12.72Hz，2H)，1.59(d，J＝13.15Hz，2H)，1.52(d，J＝10.52Hz，3H)，1.35(br.s.，1H)，1.23-1.33(m，3H)，1.03-1.21(m，5H)，0.87(dt，J＝7.89，19.95Hz，4H)，0.76(s，3H)，0.61(s，3H)/

Step 2 a: preparation of 2- (2-Bromoethoxy) tetrahydro-2H-pyran

To a stirred solution of 2-bromoethanol (10g, 80.6mmol) in diethyl ether (150mL) was added PTSA (1.54g, 8.0mmol, 0.1 equiv), followed by 3, 4-dihydro-2H-pyran (8.83g, 96.5mmol, 1.2 equiv), and the resulting mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (200mL) and extracted with EtOAc (350 mL). The organic layer was washed with NaHCO ₃Saturated solution (100mL), water (200mL), brine (100mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound as a pale yellow liquid which was taken to the next step without further purification.¹H NMR(400MHz，DMSO-d₆)δ4.66(t，J＝3.3Hz，1H)，3.93-3.85(m，1H)，3.82-3.66(m，2H)，3.66-3.57(m，2H)，3.49-3.39(m，1H)，1.72(d，J＝9.2Hz，1H)，1.62(d，J＝2.2Hz，1H)，1.53-1.40(m，4H)。

Step 2: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (2- (tetrahydro-2H-pyran-2-yloxy) ethoxy) hexadecahydrospiro [ cyclopenta [ a ] phenanthrene-3, 2' - [1, 3] dioxolane ]

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydrospiro [ cyclopenta [ a ]]Phenanthrene-3, 2' - [1, 3]]Dioxolanes]To a stirred solution of-17-ol (1g, 2.99mmol) in xylene (10mL) was added NaNH₂(50% suspension in toluene, 3mL) and the mixture was heated at 150 ℃ for 1 hour. The reaction mixture was gradually cooled to room temperature, 2- (6-chlorohexyloxy) tetrahydro-2H-pyran (6g, 29.9mmol) was added thereto, and the resulting mixture was heated again to 150 ℃ for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature, quenched slowly with ice-cold water (250mL), and quenched with ice-cold waterEtOAc (300mL) extraction. The organic layer was washed with water (100 mL. times.2), brine (100mL) and Na ₂SO₄Drying, filtration and concentration under reduced pressure gave the crude product, which was purified by CombiFlash chromatography to give the title compound. LC-MS 463[ M + H]⁺

And step 3: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (2-hydroxyethoxy-) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] phenanthren-3 (2H) -one

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-17- (2- (tetrahydro-2H-pyran-2-yloxy) ethoxy) hexadecahydrospiro [ cyclopenta [ a ] at room temperature]Phenanthrene-3, 2' - [1, 3 ]]Dioxolanes](0.635g, 1.37mmol) to a stirred solution in THF (25mL), water (5mL) was added 6N HCl (15mL) and the resulting reaction mixture was stirred at room temperature for 16 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (150mL) and NaHCO was used₃The saturated solution (pH about 8) was basified. The aqueous layer was then extracted with EtOAc (200 mL. times.3). The organic layer was washed with NaHCO₃Saturated solution (100mL), water (100mL), brine (100mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound which was used in the next step without further purification. LC-MS 335[ M + H [ ]]⁺

And 4, step 4: preparation of 2- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) acetic acid

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (2-hydroxyethoxy) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] at 0 ℃ over a period of 20 minutes]To a stirred solution of phenanthren-3 (2H) -one (0.31g, 0.928mmol) in acetone (25mL) was added dropwise jones reagent (2.5 mL). The resulting mixture was stirred at 0 ℃ for 5 minutes. The reaction was monitored by TLC. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give the title compound which was taken to the next step without further purification. LC-MS 349[ M + H]⁺

And 5: preparation of 4- (3- (4- (2- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) acetyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one

To 2- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred solution of phenanthren-17-yloxy) acetic acid (0.025g, 0.071mmol) in DMF (3mL) was added HATU (0.032g, 0.086mmol, 1.2 eq) and the resulting reaction mixture was stirred for 10 min. DIPEA (0.062mL, 0.358mmol, 5 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.020g, 0.05mmol, 07 equivalents) were then added to the mixture in succession, and the mixture was stirred at room temperature for 1 hour. The reaction was monitored by TLC and LC-MS. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give the crude product, which was purified by reverse phase HPLC to give the title compound. LC-MS 697[ M + H ] ]⁺，¹H NMR(400MHz，MeOD-d4)δ8.41-8.34(m，1H)，8.00-7.92(m，1H)，7.86(dtd，J＝18.0，7.3，1.4Hz，2H)，7.49(t，J＝6.3Hz，1H)，7.39(t，J＝6.0Hz，1H)，7.17(t，J＝9.0Hz，1H)，4.61(s，2H)，4.39(s，2H)，4.21(s，1H)，4.15(s，1H)，3.76(d，J＝7.6Hz，2H)，3.66(q，J＝5.9，5.3Hz，2H)，3.51(s，2H)，3.47-3.37(m，1H)，3.35(d，J＝4.2Hz，1H)，2.46(d，J＝15.1Hz，1H)，2.37(t，J＝14.3Hz，1H)，2.22(d，J＝15.0Hz，1H)，2.11-1.80(m，6H)，1.73(d，J＝13.8Hz，1H)，1.63(d，J＝10.8Hz，2H)，1.52(m，3H)，1.39-1.27(m，6H)，1.25(s，1H)，1.06(d，J＝6.6Hz，3H)，0.99-0.88(m，1H)，0.83(s，1H)。

EXAMPLE S-22 preparation of 4- (4-fluoro-3- (4- (6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -3-hydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 1.111)

Step 1: preparation of 4- (3- (4- (6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one

To 6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred solution of phenanthrene-17-yloxy) hexanoic acid (0.10g, 0.247mmol) in DMF (15mL) was added HATU (0.141g, 0.371mmol, 1.5 eq) and the resulting reaction mixture was stirred for 10 min. DIPEA (0.17mL, 0.99mmol, 4 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.119g, 0.297mmol, 0.1.2 equivalents) were then added to the mixture in succession, and the mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC and LC-MS. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (25 mL. times.2) and n-pentane (25 mL. times.2) and dried under vacuum to give the title compound. LC-MS 753[ M + H ] ]⁺

Step 6: preparation of 4- (4-fluoro-3- (4- (6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -3-hydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To 4- (3- (4- (6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred solution of phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (0.070g, 0.093mmol) in methanol (10mL) was slowly added NaBH₄(0.0068g, 0.186mmol, 2.0 equiv.) and the mixture was stirred at room temperature for 30 min. The reaction was monitored by TLC. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel to give a crude residue which was purified by reverse phase HPLC to give the title compound. LC-MS 755[ M + H ]]⁺，¹H NMR(400MHz，MeOD-d₄)δ8.37(d，J＝7.7Hz，1H)，7.95(s，1H)，7.86(dt，J＝15.5，7.3Hz，2H)，7.49(d，J＝7.1Hz，1H)，7.37(d，J＝6.1Hz，1H)，7.16(t，J＝9.0Hz，1H)，4.39(s，2H)，3.82-3.61(m，4H)，3.49(ddd，J＝22.0，12.8，5.5Hz，6H)，3.28(d，J＝7.6Hz，1H)，2.45(t，J＝7.6Hz，1H)，2.38(t，J＝7.6Hz，1H)，2.00-1.91(m，1H)，1.85(t，J＝11.3Hz，1H)，1.78-1.47(m，10H)，1.48-1.19(m，10H)，1.14(d，J＝14.1Hz，2H)，0.98(q，J＝10.6，9.9Hz，2H)，0.88(s，2H)，0.83(s，3H)，0.73(d，J＝8.8Hz，3H)。

EXAMPLE S-23 preparation of 4- (3- (4- (6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -3, 3-difluoro-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (Compound 1.114)

Step 1: preparation of 4- (3- (4- (6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one

To 6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred solution of phenanthren-17-yloxy) hexanoic acid (0.50g, 1.23mmol) in DMF (20mL) was added HATU (0.70g, 1.84mmol, 1.5 equiv.) and the mixture was stirred for 10 min. DIPEA (1.1mL, 6.18mmol, 5 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.497g, 1.23mmol, 1.0 equivalent) were then added to the reaction mixture in succession at 0 ℃ and the resulting reaction mixture was stirred at room temperature for 75 minutes. The reaction was monitored by TLC. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (10mL × 2) and n-pentane (10mL × 2) and dried under reduced pressure to give a crude product which was purified by CombiFlash chromatography to the title compound. LC-MS 753[ M + H ]]⁺

Step 2: preparation of 4- (3- (4- (6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -3, 3-difluoro-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one

To 4- (3- (4- (6- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] ]To a stirred solution of phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) -4-fluorophenylmethyl) phthalazin-1 (2H) -one (0.25g, 0.332mmol) in 1, 2-dichloroethane (10mL) was added DAST (0.535g, 3.32mmol, 10 equivalents) dropwise and the mixture was heated to 50 ℃ for 1 hour. The reaction was monitored by TLC. After completion, the mixture was washed with NaHCO₃The saturated solution (50mL) was quenched slowly and extracted with DCM (100 mL. times.2). The combined organic layers were washed with H₂O (100mL), brine (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude material, which was purified by reverse phase HPLC to give the title compound. LC-MS 775[ M + H]⁺，¹H NMR(400MHz，DMSO-d₆)δ12.59(s，1H)，8.26(d，J＝7.8Hz，1H)，7.96(d，J＝8.0Hz，1H)，7.89(t，J＝7.5Hz，1H)，7.83(t，J＝7.5Hz，1H)，7.44(t，J＝6.7Hz，1H)，7.36(t，J＝6.0Hz，1H)，7.23(t，J＝9.0Hz，1H)，4.33(s，2H)，3.62(s，1H)，3.59-3.47(m，3H)，3.36(d，J＝9.2Hz，3H)，3.24(q，J＝7.5Hz，1H)，3.20-3.10(m，2H)，2.33-2.22(m，2H)，1.87(dd，J＝16.6，6.2Hz，2H)，1.81-1.56(m，6H)，1.48(q，J＝9.3，8.2Hz，6H)，1.39-1.01(m，12H)，0.98-0.81(m，2H)，0.79(s，2H)，0.66(d，J＝6.7Hz，4H)。

EXAMPLE S-24 preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide (Compound 1.115)

Step 1: preparation of (5R, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-ol

At room temperature to(5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ]]Phenanthren-3 (2H) -one (1.0g, 3.4mmol) in acetic acid (20mL) -H ₂To a stirred solution in O (10mL) was added zinc metal (10.0g), and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was diluted with water (20mL) and filtered through celite bed, the crude residue obtained from celite bed was dissolved in methanol (100mL) and concentrated under reduced pressure to give the title compound.¹HNMR(400MHz，DMSO-d₆)δ4.40(d，J＝4.8Hz，1H)，3.45-3.38(m，1H)，1.81(d，J＝7.5Hz，1H)，1.71(d，J＝11.4Hz，1H)，1.66-1.55(m，4H)，1.53-1.41(m，4H)，1.37-1.27(m，4H)，1.25-1.13(m，6H)，1.01(br.s.，1H)，0.93(dd，J＝3.7，12.9Hz，1H)，0.89-0.79(m，4H)，0.76(s，2H)，0.61(s，2H)。

Step 2 a: preparation of 2- (6-Chlorohexyloxy) tetrahydro-2H-pyran

To a stirred solution of 6-chlorohex-1-ol (10g, 73.5mmol) in diethyl ether (100mL) was added PTSA (1.4g, 7.35mmol, 0.1 equiv) at room temperature followed by 3, 4-dihydro-2H-pyran (7.41g, 88.0mmol, 1.2 equiv) and the mixture was stirred at room temperature for 16H. The reaction was monitored by TLC. After completion of the reaction, the mixture was diluted with water (200mL) and extracted with EtOAc (350 mL). The organic layer was washed with NaHCO₃Saturated solution (100mL), water (200mL), brine (100mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound which was taken to the next step without further purification.¹HNMR(400MHz，DMSO-d₆)δ4.50(br.s.，1H)，4.25(t，J＝6.80Hz，2H)，4.20(s，1H)，4.08-4.00(m，2H)，3.74-3.66(m，2H)，3.59(dd，J＝6.36，16.01Hz，2H)，2.66(s，2H)，1.90-1.81(m，2H)，1.67(br.s.，1H)，1.45-1.38(m，2H)，1.27-1.20(m，2H)，1.17(t，J＝7.02Hz，2H)。

Step 2: preparation of 2- (6- ((5R, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexyloxy) tetrahydro-2H-pyran

The reaction is carried out in the direction of (5R,8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a]To a stirred solution of phenanthren-17-ol (1.0g, 2.40mmol) in xylene (10mL) was added NaNH₂(50% suspension in toluene, 3mL) and the mixture was heated at 150 ℃ for 1 hour. The reaction mixture was gradually cooled to room temperature, 2- (6-chlorohexyloxy) tetrahydro-2H-pyran (4.2g, 24.0mmol) was added thereto, and the resulting mixture was heated again to 150 ℃ for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature, quenched slowly with ice-cold water (250mL), and extracted with EtOAc (300 mL). The organic layer was washed with water (100 mL. times.2), brine (100mL) and Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude residue which was purified by CombiFlash chromatography to give the title compound.¹HNMR(400MHz，CDCl3)δ5.82(ddt，J＝17.0，10.3，6.7Hz，1H)，5.05-4.91(m，2H)，4.57(d，J＝4.4Hz，2H)，3.87(ddd，J＝11.2，7.7，3.1Hz，2H)，3.79-3.66(m，2H)，3.55-3.45(m，2H)，3.24-3.32(m，2H)，3.26(t，J＝8.3Hz，1H)，2.12-2.01(m，2H)，2.00-1.90(m，2H)，1.90-1.75(m，2H)，1.68-1.56(m，8H)，1.54-1.41(m，8H)，1.41-1.31(m，4H)，1.26-1.17(m，4H)，0.94-0.8(m，2H)，0.78(s，2H)，0.73(s，2H)。

And step 3: preparation of 6- ((5R, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hex-1-ol

To 2- (6- ((5R, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] at room temperature]Phenanthren-17-yloxy) hexyloxy) tetrahydro-2H-pyran (1.0g, 2.17mmol, 1 eq) in THF (28mL) -H ₂To a stirred solution in O (7mL) was added 6N — HCl (20mL), and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (150mL) and NaHCO was used₃The saturated solution (pH about 8) was basified. The aqueous layer was then extracted with EtOAc (200 mL. times.3). The organic layer was washed with NaHCO₃Saturated solution (100mL), water (100mL), brine (100mL), washed with Na₂SO₄Drying, filtering and concentrating under reduced pressure to obtain the targetThe title compound, which was used in the next step without further purification.¹HNMR(400MHz，DMSO-d₆)δ5.76(s，1H)，4.32(t，J＝5.3Hz，1H)，3.42-3.33(m，4H)，3.24(t，J＝8.3Hz，1H)，2.01(d，J＝7.0Hz，1H)，1.95-1.86(m，1H)，1.77(d，J＝11.8Hz，1H)，1.66-1.54(m，4H)，1.48-1.32(m，8H)，1.32-1.23(m，6H)，1.22-1.09(m，6H)，1.01(br.s.，1H)，0.90-0.79(m，4H)，0.75(s，2H)，0.69-0.61(m，2H)。

And 4, step 4: preparation of 6- ((5R, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoic acid

To 6- ((5R, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] at 0 ℃ over a period of 20 minutes]To a stirred solution of phenanthren-17-yloxy) hex-1-ol (0.40g, 1.09mmol, 1 eq) in acetone (25mL) was added dropwise jones reagent (1.2 mL). The resulting mixture was stirred at 0 ℃ for 5 minutes. The reaction was monitored by TLC. After completion, H was added₂O (20mL), and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give the title compound which was taken to the next step without further purification. ¹HNMR(400MHz，DMSO-d₆)δ11.96(br.s.，1H)，3.28-3.18(m，2H)，2.18(t，J＝7.5Hz，2H)，1.89(d，J＝5.7Hz，2H)，1.77(d，J＝11.4Hz，2H)，1.53-1.40(m，8H)，1.39-1.26(m，6H)，1.23(br.s.，2H)，1.23-1.06(m，10H)，0.96-0.80(m，4H)，0.75(s，2H)，0.71-0.56(m，2H)。

And 5: preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide

To 6- ((5R, 8R, 9S, 10S, 13S, 14S, 17S) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred solution of phenanthrene-17-yloxy) hexanoic acid (0.190g, 0.487mmol, 1 eq) in DMF (12mL) was added HATU (0.277g, 0.73mmol, 1.2 eq) and the resulting reaction mixture was stirred for 10 min.DIPEA (0.36mL, 1.94mmol, 4.0 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.156g, 0.389mmol, 0.8 equivalents) were then added to the mixture in that order, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC and LC-MS. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give a crude residue which was purified by reverse phase HPLC to give the title compound. LC-MS 739[ M + H]⁺。¹HNMR400MHz，DMSO-d6)δ12.59(s，1H)，8.26(d，J＝7.9Hz，1H)，8.01-7.93(m，1H)，7.89(t，J＝7.2Hz，1H)，7.86-7.77(m，1H)，7.44(br.s.，1H)，7.36(br.s.，1H)，7.23(t，J＝9.0Hz，1H)，4.33(s，2H)，3.62(br.s.，1H)，3.56(br.s.，1H)，3.51(br.s.，2H)，3.27-3.02(m，3H)，2.33(br.s.，1H)，2.26(br.s.，1H)，1.90(br.s.，2H)，1.76(br.s.，1H)，1.59-1.47(m，8H)，1.35-1.28(m，6H)，1.20-1.16(m，10H)，1.13(br.s.，2H)，0.83(br.s.，4H)，0.74(br.s.，3H)，0.65(d，J＝6.6Hz，4H)。

EXAMPLE S-25 preparation of 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propynoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 1.116)

Step 1: preparation of 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propynoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To 3- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred suspension of phenanthren-17-yl) propiolic acid (0.50g, 1.39mmol) in DMF (20mL) was added HATU (0.795g, 2.095mmol, 1.5 equiv) and the mixture was stirred for 10 min. Then at 0 ℃ willDIPEA (1.45mL, 8.35mmol, 6 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.503g, 1.25mmol, 0.9 equivalents) were added to the reaction mixture in that order, and the resulting reaction mixture was stirred at room temperature for 75 minutes. The reaction was monitored by TLC. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (10mL × 2) and n-pentane (10mL × 2) and dried under reduced pressure to give a crude material, which was purified by CombiFlash chromatography to give the title compound. LC-MS 707[ M + H]⁺。¹HNMR(400MHz，DMSO-d₆)δ12.58(d，J＝2.6Hz，1H)，8.24(d，J＝7.9Hz，1H)，8.00-7.92(m，1H)，7.87(t，J＝7.2Hz，1H)，7.83-7.73(m，1H)，7.41(d，J＝6.1Hz，1H)，7.33(d，J＝7.0Hz，1H)，7.24-7.14(m，1H)，5.70-5.63(m，1H)，4.30(d，J＝7.9Hz，2H)，3.74(br.s.，1H)，3.63(br.s.，3H)，3.39(br.s.，1H)，3.24-3.10(m，2H)，2.39-2.20(m，2H)，2.15-1.98(m，2H)，1.94-1.75(m，2H)，1.61(br.s.，2H)，1.56(br.s.，4H)，1.48-1.33(m，4H)，1.33-1.11(m，8H)，0.97-0.91(m，3H)，0.76-0.73(m，3H)。

EXAMPLE S-26 preparation of 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17R) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 1.117)

Step 1: preparation of 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propynoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To 3- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] at 0 deg.C]To a stirred suspension of phenanthren-17-yl) propiolic acid (0.50g, 1.39mmol) in DMF (20mL) was added HATU (795g, 2.095mmol, 1.5 equiv.) and the mixture was stirred for 10 min. DIPEA (1.45mL, 8.35mmol, 6 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.503g, 1.25mmol, 0.9 equivalents) were then added to the reaction mixture in succession at 0 ℃ and the resulting reaction mixture was stirred at room temperature for 75 minutes. The reaction was monitored by TLC. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (10mL × 2) and n-pentane (10mL × 2) and dried under reduced pressure to give a crude material, which was purified by CombiFlash chromatography to give the title compound. LC-MS 707[ M + H]⁺。¹HNMR(400MHz，DMSO-d₆)δ12.58(d，J＝2.6Hz，1H)，8.24(d，J＝7.9Hz，1H)，8.00-7.92(m，1H)，7.87(t，J＝7.2Hz，1H)，7.83-7.73(m，1H)，7.41(d，J＝6.1Hz，1H)，7.33(d，J＝7.0Hz，1H)，7.24-7.14(m，1H)，5.70-5.63(m，1H)，4.30(d，J＝7.9Hz，2H)，3.74(br.s.，1H)，3.63(br.s.，3H)，3.39(br.s.，1H)，3.24-3.10(m，2H)，2.39-2.20(m，2H)，2.15-1.98(m，2H)，1.94-1.75(m，2H)，1.61(br.s.，2H)，1.56(br.s.，4H)，1.48-1.33(m，4H)，1.33-1.11(m，8H)，0.97-0.91(m，3H)，0.76-0.73(m，3H)。

Step 2: preparation of 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17R) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a)]To a stirred suspension of phenanthren-17-yl) propynoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (0.100g, 0.141mmol) in methanol (10mL) was added 10% Pd/C (10mg), and the mixture was hydrogenated using a hydrogen balloon for 16 hours. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was filtered through a celite bed, and the filtrate was concentrated under reduced pressure to give a crude material which was purified by reverse phase HPLC to give the title compound. LC-MS 711[ M + H [ ]]⁺。¹H NMR(400MHz，DMSO-d₆)12.59(s，1H)，8.26(d，J＝7.8Hz，1H)，7.96(d，J＝8.0Hz，1H)，7.89(t，J＝7.6Hz，1H)，7.83(t，J＝7.5Hz，1H)，7.44(dd，J＝8.7，5.0Hz，1H)，7.35(d，J＝6.9Hz，1H)，7.24(t，J＝9.0Hz，1H)，4.33(s，2H)，3.98(dd，J＝15.2，8.0Hz，1H)，3.68-3.49(m，2H)，3.40-3.34(m，2H)，3.23-3.11(m，2H)，3.05(s，1H)，3.00(s，1H)，2.44-2.34(m，1H)，2.28(d，J＝14.2Hz，1H)，2.13-2.04(m，1H)，1.97-1.76(m，1H)，1.72-1.51(m，6H)，1.45(d，J＝16.1Hz，6H)，1.36-1.13(m，8H)，0.98(s，2H)，0.91-0.58(m，4H)。

EXAMPLE S-27 preparation of 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) prop-2-ynyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 1.118)

Step 1: preparation of (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (3-bromoprop-1-ynyl) -17-hydroxy-10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] phenanthren-3 (2H) -one

To (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-17- (3-hydroxyprop-1-ynyl) -10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ] at room temperature ]To a stirred suspension of phenanthren-3 (2H) -one (1.0g, 2.90mmol, 1 eq) in DCM (20mL) was added sequentially triphenylphosphine (2.28g, 8.72mmol, 3.0 eq) and carbon tetrabromide (1.98gm, 5.98mmol, 2 eq) and the mixture was stirred at room temperature for 3H. The reaction was monitored by TLC. Upon completion, water (50mL) was added and the aqueous layer was extracted with DCM (100mL × 2). The combined organic layers were washed with water (50mL), brine (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude residue which was purified by CombiFlash chromatography to give the title compound.¹H NMR(400MHz，DMSO-d₆)δ5.36(d，J＝5.3Hz，1H)，4.49(s，2H)，4.30(s，2H)，2.29(s，2H)，1.90(br.s.，6H)，1.55(br.s.，4H)，1.30(br.s.，4H)，1.29-1.20(m，4H)，0.98(s，2H)，0.74(s，4H)。

Step 2: preparation of 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) prop-2-ynyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To a stirred solution of 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloride (0.20g, 0.497mmol) in EtOH (10mL) was added DIPEA (0.44mL, 2.48mmol, 5 equiv.), followed by (5S, 8R, 9S, 10S, 13S, 14S, 17S) -17- (3-bromoprop-1-ynyl) -17-hydroxy-10, 13-dimethyldecatetrahydro-1H-cyclopenta [ a ], a]Phenanthren-3 (2H) -one (0.201g, 0.497mmol, 1.0 eq), and the mixture is heated at 80 ℃ for 1 hour. The reaction was monitored by TLC and LC-MS. After completion, the mixture was concentrated under reduced pressure to give a crude residue which was purified by CombiFlash chromatography to give the title compound. LC-MS 693[ M + H ] ]⁺。¹HNMR(400MHz，DMSO-d₄)δ12.58(s，1H)，8.26(d，J＝7.0Hz，1H)，7.98-7.91(m，1H)，7.90-7.76(m，2H)，7.43(br.s.，1H)，7.25(d，J＝4.8Hz，1H)，7.22-7.13(m，1H)，5.17(s，1H)，4.32(s，2H)，3.63(br.s.，2H)，3.35(s，2H)，3.17(d，J＝5.3Hz，2H)，2.38(br.s.，2H)，2.35-2.30(m，1H)，2.00(d，J＝14.5Hz，2H)，1.89-1.75(m，4H)，1.60-1.52(m，6H)，1.47-1.40(m，6H)，1.22(d，J＝6.6Hz，6H)，1.06-0.95(m，2H)，0.94(s，2H)，0.72(s，2H)。

EXAMPLE S-28 preparation of (R) -N- (4-cyano-3- (trifluoromethyl) phenyl) -3- (4- (6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexylamino) phenylsulfonyl) -2-hydroxy-2-methylpropanamide (Compound 1.119)

Step 1: preparation of 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17R) -17-hydroxy-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To 4- (4-fluoro-3- (4- (3- ((5S, 8R, 9S, 10S, 13S, 14S, 17S) -17-hydroxy)Radical-10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a]To a stirred suspension of phenanthren-17-yl) prop-2-ynyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (0.120g, 0.173mmol, 1 eq) in methanol (10mL) was added 10% Pd/C (12mg) and the mixture was hydrogenated under a hydrogen atmosphere for 16H. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was filtered through a celite bed, and the resulting filtrate was concentrated under reduced pressure to give a crude residue which was purified by CombiFlash chromatography to give the title compound. LC-MS697[ M + H ]]⁺。¹HNMR(400MHz，DMSO-d₆)δ12.59(s，1H)，8.26(d，J＝7.5Hz，1H)，7.96(d，J＝7.9Hz，1H)，7.92-7.76(m，2H)，7.45-7.36(m，1H)，7.30(d，J＝6.1Hz，1H)，7.22(t，J＝9.0Hz，1H)，4.32(s，2H)，3.99(br.s.，1H)，3.59(br.s.，1H)，3.13(br.s.，2H)，2.44-2.36(m，2H)，2.33(br.s.，2H)，2.25(dd，J＝11.6，19.1Hz，4H)，2.09(d，J＝10.1Hz，1H)，1.89(t，J＝13.6Hz，1H)，1.72(br.s.，2H)，1.60(br.s.，2H)，1.55(br.s.，2H)，1.50(br.s.，2H)，1.46-1.42(br.s.，4H)，1.37-1.33(d，J＝12.3Hz，4H)，1.31-1.04(m，8H)，0.98(s，2H)，0.80-0.74(m，2H)。

EXAMPLE S-29 preparation of 4- (4-fluoro-3- (4- (6- ((8R, 9S, 13S, 14S, 17S) -3-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 2.2)

Step 1: preparation of (8R, 9S, 13S, 14S, 17S) -3- (4-methoxyphenylmethyloxy) -13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] phenanthren-17-ol

To (8R, 9S, 13S, 14S, 17S) -13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] at room temperature]To a stirred solution of phenanthrene-3, 17-diol (1.0g, 3.6mmol) in DMF (20mL) was added sodium hydride (60% suspension in mineral oil; 0.22g,5.5mmol) and the mixture is heated at 50 ℃ for 2 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was quenched with ice-cold water (20mL), and extracted with ethyl acetate (100mL × 2). The combined organic layers were washed with chilled water (50mL × 3), brine (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude material which was purified by CombiFlash chromatography to give the title compound.¹HNMR(400MHz，CDCl₃)δ7.39-7.32(m，J＝8.8Hz，2H)，7.26(s，1H)，7.20(d，J＝8.8Hz，1H)，6.95-6.85(m，J＝8.8Hz，2H)，6.77(dd，J＝2.6，8.3Hz，1H)，6.74-6.67(m，1H)，5.30(s，1H)，4.95(s，2H)，3.82(s，3H)，3.73(t，J＝8.6Hz，2H)，3.54(t，J＝6.8Hz，1H)，2.84(d，J＝4.8Hz，2H)，2.31(dd，J＝3.1，13.6Hz，1H)，2.20-2.10(m，2H)，1.99-1.84(m，2H)，1.74-1.67(m，1H)，1.62(d，J＝14.0Hz，2H)，1.51-1.39(m，3H)，1.24-1.14(m，1H)，0.89-0.78(m，2H)。

Step 2 a: preparation of 2- (6-Chlorohexyloxy) tetrahydro-2H-pyran

To a stirred solution of 6-chlorohex-1-ol (10g, 73.5mmol) in diethyl ether (100mL) was added PTSA (1.4g, 7.35mmol, 0.1 equiv) at room temperature followed by 3, 4-dihydro-2H-pyran (7.41g, 88.0mmol, 1.2 equiv) and the mixture was stirred at room temperature for 16H. The reaction was monitored by TLC. After completion of the reaction, the mixture was diluted with water (200mL) and extracted with EtOAc (350 mL). The organic layer was washed with NaHCO ₃Saturated solution (100mL), water (200mL), brine (100mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound as a yellow liquid (12.0g, 74%) which was taken to the next step without further purification.¹HNMR(400MHz，DMSO-d₆)δ4.50(br.s.，1H)，4.25(t，J＝6.80Hz，2H)，4.20(s，1H)，4.08-4.00(m，2H)，3.74-3.66(m，2H)，3.59(dd，J＝6.36，16.01Hz，2H)，2.66(s，2H)，1.90-1.81(m，2H)，1.67(br.s.，1H)，1.45-1.38(m，2H)，1.27-1.20(m，2H)，1.17(t，J＝7.02Hz，2H)。

Step 2: preparation of 2- (6- ((8R, 9S, 13S, 14S, 17S) -3- (4-methoxyphenylmethyloxy) -13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] phenanthren-17-yloxy) hexyloxy) tetrahydro-2H-pyran

To (8R, 9S, 13S, 14S, 17S) -3- (4-methoxybenzyloxy) -13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ]]To a stirred solution of phenanthren-17-ol (0.50g, 1.27mmol) in xylene (5mL) was added NaNH₂(50% suspension in toluene, 1.5mL) and the mixture was heated at 150 ℃ for 1 hour. The reaction mixture was gradually cooled to room temperature, 2- (6-chlorohexyloxy) tetrahydro-2H-pyran (2.8g, 12.5mmol, 10 equivalents) was added thereto, and the resulting mixture was heated again to 150 ℃ for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature, quenched slowly with ice-cold water (250mL), and extracted with EtOAc (300 mL). The organic layer was washed with water (100 mL. times.2), brine (100mL) and Na ₂SO₄Drying, filtration and concentration under reduced pressure gave a crude residue which was purified by CombiFlash chromatography to give the title compound.¹HNMR(400MHz，DMSO-d6)δ7.37-7.31(m，J＝8.3Hz，2H)，7.14(d，J＝8.3Hz，1H)，6.94-6.90(m，J＝8.3Hz，2H)，6.72(d，J＝7.9Hz，1H)，6.67(br.s.，1H)，4.53(br.s.，2H)，3.76-3.73(m，3H)，3.64-3.56(m，2H)，3.45-3.36(m，4H)，2.75(br.s.，2H)，2.26(br.s.，2H)，2.10(d，J＝12.7Hz，2H)，1.99(s，2H)，1.90(d，J＝8.3Hz，2H)，1.80(br.s.，2H)，1.69(br.s.，2H)，1.60(br.s.，2H)，1.55-1.39(m，8H)，1.35-1.27(m，6H)，1.25(br.s.，2H)，0.90-0.81(m，2H)。

And step 3: preparation of 6- ((8R, 9S, 13S, 14S, 17S) -3- (4-methoxybenzyloxy) -13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] phenanthren-17-yloxy) hex-1-ol

To 2- (6- ((8R, 9S, 13S, 14S, 17S) -3- (4-methoxyphenylmethyloxy) -13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] at room temperature]Phenanthren-17-yloxy) hexyloxy) tetrahydro-2H-pyran (0.42g, 0.72mmol) to a stirred solution in THF (12mL), water (3mL) was added 6-NHCl (9mL) and the solution was stirredThe resulting mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (40mL) and NaHCO was used₃The saturated solution (pH about 8) was basified. The aqueous layer was then extracted with EtOAc (50 mL. times.3). The organic layer was washed with NaHCO₃Saturated solution (40mL), water (40mL), brine (50mL), washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound. LC-MS 493[ M + H ]]⁺

And 4, step 4: preparation of 6- ((8R, 9S, 13S, 14S, 17S) -3-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoic acid

To 6- ((8R, 9S, 13S, 14S, 17S) -3- (4-methoxybenzyloxy) -13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] over a period of 20 minutes at 0 deg.C]To a stirred solution of phenanthren-17-yloxy) hex-1-ol (0.18g, 0.365mmol) in acetone (18mL) was added dropwise Jones' reagent (1.2 mL). The resulting mixture was stirred at 0 ℃ for 5 minutes. The reaction was monitored by TLC. Upon completion, water (20mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give the title compound which was taken to the next step without further purification. LC-MS 387[ M + H ]]⁺

And 5: preparation of 4- (4-fluoro-3- (4- (6- ((8R, 9S, 13S, 14S, 17S) -3-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] phenanthren-17-yloxy) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To 6- ((8R, 9S, 13S, 14S, 17S) -3-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [ a ] at 0 DEG C]To a stirred solution of phenanthrene-17-yloxy) hexanoic acid (0.190g, 0.466mmol) in DMF (8mL) was added HATU (0.277g, 0.699mmol, 1.5 equiv) and the resulting reaction mixture was stirred for 10 min. DIPEA (0.24mL, 1.86mmol, 4.0 equiv.) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.149g, 0.373mmol, 0.8 equiv.) are then added to the mixture in that order, and the mixture is mixed The mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC and LC-MS. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give a crude residue which was purified by reverse phase HPLC to give the title compound. LC-MS 735[ M + H]⁺。¹HNMR(400MHz，DMSO-d6)δ12.59(s，1H)，8.99(s，1H)，8.26(d，J＝7.8Hz，1H)，7.96(d，J＝7.9Hz，1H)，7.89(t，J＝7.6Hz，1H)，7.83(t，J＝7.5Hz，1H)，7.48-7.32(m，2H)，7.23(t，J＝9.0Hz，1H)，7.06-6.98(m，1H)，6.49(dd，J＝8.3，2.6Hz，1H)，6.42(d，J＝2.6Hz，1H)，4.32(s，2H)，3.63(d，J＝6.0Hz，1H)，3.54(d，J＝22.1Hz，3H)，3.39(dt，J＝17.2，5.3Hz，4H)，3.15(dd，J＝13.2，8.4Hz，2H)，2.70(d，J＝10.9Hz，2H)，2.35(d，J＝7.7Hz，1H)，2.28(d，J＝7.6Hz，1H)，2.24-2.16(m，1H)，2.12-2.03(m，2H)，2.02-1.81(m，3H)，1.75(t，J＝7.5Hz，1H)，1.64-1.41(m，4H)，1.41-1.10(m，8H)，0.70(d，J＝6.7Hz，3H)。

Example S-30 preparation of 4- (4-fluoro-3- (4- (6- ((2- (4- (6-hydroxy-2- (4-hydroxyphenyl) benzo [ b ] thiophene-3-carbonyl) phenoxy) ethyl) (methyl) amino) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 2.4)

Step 1: preparation of 6- (methylamino) hexanoic acid hydrochloric acid

To N-methylcaprolactam (5g, 39.3mmol) was added concentrated HCl (25mL) -H₂O (36mL) was at room temperature, and the mixture was refluxed at 110 ℃ for 16 hours. The reaction was monitored by TLC. After 16 hours, the mixture was concentrated under reduced pressure, to the residue obtained 10mL of H was added₂O, and concentrated again. The crude material obtained was wet-milled twice with acetone/n-pentane (1: 5) to give the title compound.¹H NMR(400MHz，DMSO-d₆)δ12.05(1H，s，br)，8.70(2H，br)，3.6(s，3H)，2.18-2.23(2H，m)，2.77-2.86(2H，m)，1.44-1.62(2H，m)，1.24-1.34(2H，m)。

Step 2: preparation of 6- (tert-butoxycarbonyl (methyl) amino) hexanoic acid

To a stirred solution of 6- (methylamino) hexanoic acid hydrochloric acid (1.5g, 10.3mmol) in 1, 4-dioxane was added 1N-NaOH (20.7mL, 2.5 equiv.) at 0 deg.C, followed by di-tert-butyl dicarbonate (2.7g, 12.4mmol, 1.2 equiv.), and the mixture was stirred at room temperature for 16 h. The reaction was monitored by TLC. After completion of the reaction, the mixture was concentrated under reduced pressure to give the title compound, which was forwarded without further purification. ¹H NMR(400MHz，DMDO-d₆)δ11.97(br s，1H)，3.57(s，3H)，3.12(t，J＝6.6Hz，2H)，2.29(t，J＝7.1Hz，2H)，2.19(t，J＝7.1Hz，2H)，1.52(d，J＝6.4Hz，2H)，1.38(s，9H)，1.20(br s，2H)。

And step 3: preparation of tert-butyl 6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) -6-oxohexyl (methyl) carbamate

To a stirred suspension of 6- (tert-butoxycarbonyl (methyl) amino) hexanoic acid (0.20g, 0.8mmol) in DMF (4mL) at 0 deg.C was added HATU (0.62g, 1.6mmol, 2 equiv.) and the mixture was stirred at 0 deg.C for 10 min. DIPEA (0.45mL, 2.4mmol, 3 equivalents) and 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (0.36g, 0.89mmol, 1.1 equivalents) were then added to the reaction mixture in succession at 0 ℃ and the resulting reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion, the mixture was washed with H₂O (50mL) was diluted and extracted with EtOAc (50 mL. times.2). The combined organic layers were washed with water (50mL), brine (50mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by CombiFlash chromatography to give the title compound. LC-MS 594[ M + H [ ]]⁺。

And 4, step 4: preparation of 4- (4-fluoro-3- (4- (6- (methylamino) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To a solution containing 6- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) at 0 DEG C Yl) piperazin-1-yl) -6-oxohexyl (methyl) carbamic acid tert-butyl ester (0.23g, 0.38mmol) in DCM (5mL) TFA (0.3mL) was added slowly and the mixture was heated at 60 ℃ for 2 h. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was concentrated under reduced pressure to give a crude residue which was wet-milled with n-pentane (10mL × 2) to give the title compound as the trifluoroacetate salt. LC-MS 494[ M + H ]]⁺。

Step 5 a: preparation of (4-fluorophenyl) (6-methoxy-2- (4-methoxyphenyl) benzo [ b ] thiophen-3-yl) methanone

Reacting 6-methoxy-2- (4-methoxyphenyl) benzo [ b]A mixture of thiophene (8.5g, 31.44mmol) in DCM (100mL) was cooled to 0 deg.C and then 4-fluorobenzoyl chloride (5.73g, 36.16mmol) and AlCl were added₃(4.61g, 34.58 mmol). The reaction mixture was stirred at room temperature for 4 hours. TLC showed the reaction mixture was complete. The solution was poured into water and extracted with DCM/MeOH (100 mL. times.3). The organic layer was washed with brine and over NaSO₄Dried and concentrated in vacuo. The crude product was purified by column chromatography to give the title compound.

And step 5 b: preparation of (4- (2-hydroxyethoxy) phenyl) (6-methoxy-2- (4-methoxyphenyl) benzo [ b ] thiophen-3-yl) methanone

A mixture of ethylene glycol (25.59g, 412.8mmol) in DMF (100mL) was cooled to 0 deg.C and NaH (3.3g, 82.56mmol) was added slowly. The reaction mixture was stirred at room temperature for 1 hour. (4-fluorophenyl) (6-methoxy-2- (4-methoxyphenyl) benzo [ b ] was added dropwise at 0 ℃]Solution of thiophen-3-yl) methanone (10.8g, 27.52mmol) in DMF (20 mL). The reaction mixture was stirred at 90 ℃ for 1 hour. TLC showed the mixture was complete. The solution was concentrated in vacuo and purified by column chromatography to give the title compound. LC-MS 435.3[ M +1 ]]⁺。¹H NMR(400MHz，DMSO-d₆)δ7.70-7.65(m，3H)，7.34-7.31(m，3H)，7.0(dd，J＝6.8，2.4Hz，1H)，6.95-6.88(m，4H)，4.88(t，J＝1.6Hz，1H)，4.01(t，J＝4.8Hz，2H)，3.84(s，3H)，3.72(s，3H)，3.71-3.67(m，2H)。

And step 5 c: preparation of (4- (2-bromoethoxy) phenyl) (6-methoxy-2- (4-methoxyphenyl) benzo [ b ] thiophen-3-yl) methanone

To (4- (2-hydroxyethoxy) phenyl) (6-methoxy-2- (4-methoxyphenyl) benzo [ b ] at 0 DEG C]Add PBr to a stirred solution of thiophen-3-yl) methanone (200mg, 0.460mmol) in DMF (5mL)₃(0.87mL, 0.92mmol, 2 equiv.) and the mixture was stirred at 50 ℃ for 1 hour. The reaction was monitored by TLC. After the reaction was complete, the mixture was washed with NaHCO₃The saturated solution was quenched and extracted with EtOAc (60 mL. times.3). The combined organic layers were washed with water (60mL), brine (60mL), and Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound. LC-MS 497[ M + H ]⁺。

And 5: preparation of 4- (4-fluoro-3- (4- (6- ((2- (4- (6-methoxy-2- (4-methoxyphenyl) benzo [ b ] thiophene-3-carbonyl) phenoxy) ethyl) (methyl) oxy) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To a stirred solution of 4- (4-fluoro-3- (4- (6- (methylamino) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one trifluoroacetate (0.080g, 0.162mmol) in EtOH (2mL) was added DIPEA (0.15mL, 0.81mmol, 5 equivalents) followed by (4- (2-bromoethoxy) phenyl) (6-methoxy-2- (4-methoxyphenyl) benzo [ b]Thien-3-yl) methanone (0.08g, 0.162mmol, 1 eq), and the mixture was heated at 80 ℃ for 16 h. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure to give a crude material, which was purified by CombiFlash chromatography to give the title compound. LC-MS 910[ M + H [ ]]⁺。

Step 6: preparation of 4- (4-fluoro-3- (4- (6- ((2- (4- (6-hydroxy-2- (4-hydroxyphenyl) benzo [ b ] thiophene-3-carbonyl) phenoxy) ethyl) (methyl) amino) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 2.4)

To 4- (4-fluoro-3- (4- (6- ((2- (4- (6-methoxy-2- (4-methoxyphenyl)) benzo [ b) at 0 deg.C]To a stirred solution of thiophene-3-carbonyl) phenoxy) ethyl) (methyl) amino) hexanoyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (100mg, 0.11mmol) in DCM (2mL) was added BBr ₃(1M in DCM; 1.09mL, 1.1mmol, 10 equivalents) and the mixture was cooled at room temperatureStirred for 16 hours. The reaction was monitored by LC-MS and TLC. After the reaction is completed, NaHCO is used₃The mixture was quenched with saturated solution (60 mL). The aqueous layer was then extracted with EtOAc (60 mL. times.3). The combined organic layers were washed with water (60mL), brine (60mL), and Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by reverse phase HPLC to give compound 2.4. LC-MS 882[ M + H ]]⁺。¹H NMR(400MHz，CD₃OD-d₄)δ8.54(s，1H)，8.39-8.31(m，1H)，7.98-7.91(m，1H)，7.83(p，J＝7.3Hz，1H)，7.75-7.66(m，2H)，7.47(d，J＝8.6Hz，2H)，7.42(d，J＝8.7Hz，1H)，7.36(s，1H)，7.25(d，J＝2.3Hz，1H)，7.17(dd，J＝8.6，6.7Hz，2H)，6.91-6.81(m，3H)，6.62(d，J＝8.3Hz，2H)，4.38(s，2H)，4.16(d，J＝4.9Hz，2H)，3.78-3.69(m，2H)，3.63(d，J＝7.0Hz，2H)，3.35(s，2H)，2.93(s，1H)，2.59(s，1H)，2.42(d，J＝6.8Hz，3H)，1.58(d，J＝15.1Hz，4H)。

EXAMPLE S-31 preparation of 4- (4-fluoro-3- (4- (2- (4- (6-hydroxy-2- (4-hydroxyphenyl) benzo [ b ] thiophene-3-carbonyl) phenoxy) ethyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 2.13)

Step 1 a: preparation of (4- (2-bromoethoxy) phenyl) (6-methoxy-2- (4-methoxyphenyl) benzo [ b ] thiophen-3-yl) methanone

To (4- (2-hydroxyethoxy) phenyl) (6-methoxy-2- (4-methoxyphenyl) benzo [ b ] at 0 DEG C]Add PBr to a stirred solution of thiophen-3-yl) methanone (200mg, 0.460mmol) in DMF (5mL)₃(0.87mL, 0.92mmol, 2 equiv.) and the mixture was stirred at 50 ℃ for 1 hour. The reaction was monitored by TLC. After the reaction was complete, the mixture was washed with NaHCO₃The saturated solution was quenched and extracted with EtOAc (60 mL. times.3). The combined organic layers were washed with water (60mL), brine (60mL), and Na ₂SO₄Drying, filtering and concentrating under reduced pressure to give (4- (2-bromoethoxy) phenyl) (6-methyl)Oxy-2- (4-methoxyphenyl) benzo [ b]Thiophen-3-yl) methanone. LC-MS 497[ M + H]⁺

Step 1: preparation of 4- (4-fluoro-3- (4- (2- (4- (6-methoxy-2- (4-methoxyphenyl) benzo [ b ] thiophene-3-carbonyl) phenoxy) ethyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To a stirred suspension of 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloric acid (150mg, 0.372mmol) in DMF (5mL) was added K in order₂CO₃(102mg, 0.744mmol, 2 equiv.), NaI (11.16mg, 0.0743mmol, 0.2 equiv.), and (4- (2-bromoethoxy) phenyl) (6-methoxy-2- (4-methoxyphenyl) benzo [ b]Thien-3-yl) methanone (222mg, 0.444mmol, 1.2 equiv.), and the resulting mixture was stirred at 100 ℃ for 6 hours. The reaction was monitored by TLC. Upon completion, the mixture was diluted with water (20 mL). The resulting precipitate was filtered through a Buchner funnel to give a crude product, which was purified by combi flash chromatography to give 4- (4-fluoro-3- (4- (2- (4- (6-methoxy-2- (4-methoxyphenyl) benzo [ b)]Thiophene-3-carbonyl) phenoxy) ethyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one. LC-MS 783[ M + H ]]⁺

Step 2: preparation of 4- (4-fluoro-3- (4- (2- (4- (6-hydroxy-2- (4-hydroxyphenyl) benzo [ b ] thiophene-3-carbonyl) phenoxy) ethyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To 4- (4-fluoro-3- (4- (2- (4- (6-methoxy-2- (4-methoxyphenyl)) benzo [ b) at 0 DEG C]To a stirred solution of thiophene-3-carbonyl) phenoxy) ethyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (100mg, 0.127mmol) in DCM (5mL) was added BBr₃(1M in DCM; 1.5mL, 1.53mmol, 12 equiv.) and the mixture was stirred at room temperature for 16 h. The reaction was monitored by LC-MS and TLC. After the reaction is completed, NaHCO is used₃The mixture was quenched with saturated solution (60 mL). The aqueous layer was then extracted with EtOAc (60 mL. times.3). The combined organic layers were washed with water (60mL), brine (60mL), and Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by reverse phase HPLC to give 4- (4-fluoro-3- (4- (2- (4- (6-hydroxy-2- (4-hydroxyphenyl) benzo [ b)]Thiophene-3-carbonyl) phenoxy) ethyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one。LC-MS 755[M+H]⁺。¹H NMR(400MHz，DMSO-d₆)δ12.60(s，1H)，9.79(s，1H)，9.74(s，1H)，8.25(d，J＝7.9Hz，1H)，7.96(d，J＝8.1Hz，1H)，7.88(t，J＝7.7Hz，1H)，7.81(t，J＝7.5Hz，1H)，7.65(d，J＝8.5Hz，2H)，7.41(t，J＝6.9Hz，1H)，7.34(d，J＝2.3Hz，1H)，7.31(dd，J＝6.7，2.1Hz，1H)，7.27-7.19(m，2H)，7.19-7.14(m，2H)，6.92(d，J＝8.7Hz，2H)，6.85(dd，J＝8.6，2.3Hz，1H)，6.70-6.64(m，2H)，4.32(s，2H)，4.10(dt，J＝7.6，5.3Hz，2H)，3.59(s，2H)，3.17(d，J＝5.2Hz，1H)，3.13(s，2H)，2.69(t，J＝5.5Hz，2H)，2.44(s，2H)，2.34(s，1H)。

EXAMPLE S-32 Synthesis of 4- (4-fluoro-3- (4- (2- (4- (1- (4-hydroxyphenyl) -2-phenylbut-1-enyl) phenoxy) ethyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one (Compound 2.14)

Step 1: preparation of 4- (1- (4- (2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) piperazin-1-yl) ethoxy) phenyl) -2-phenylbut-1-enyl) pivalic acid phenyl ester

To a stirred solution of 4- (4-fluoro-3- (piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one hydrochloride (0.10g, 0.197mmol) in EtOH (2mL) was added DIPEA (0.91mL, 4.97mmol, 5 equivalents), followed by 4- (1- (4- (2-bromoethoxy) phenyl) -2-phenylbut-1-enyl) pivalate (0.119g, 0.296mmol, 1.5 equivalents), and the mixture was heated at 80 ℃ for 16 hours. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure to give a crude material, which was purified by reverse phase HPLC to give the title compound. LC-MS 793[ M + H ]]⁺。

Step 2: preparation of 4- (4-fluoro-3- (4- (2- (4- (1- (4-hydroxyphenyl) -2-phenylbut-1-enyl) phenoxy) ethyl) piperazine-1-carbonyl) benzyl) phthalazin-1 (2H) -one

To pivalic acid 4- (1- (4- (2- (4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl)) benzoyl) Piperazin-1-yl) ethoxy) phenyl) -2-phenylbut-1-enyl) phenyl ester (0.035g, 0.044mmol) to which was added 1.25N HCl in EtOH (2mL), and the mixture was stirred at room temperature for 24 hours. The reaction was monitored by TLC and LC-MS. After completion, the mixture was concentrated under reduced pressure to give a crude material which was purified by reverse phase HPLC to give compound 2.14. LC-MS 709[ M + H ]⁺。¹H NMR(400MHz，DMSO-d₆)δ12.59(br s，1H)，9.41(s，1H)，9.16(s，1H)，8.21-8.29(m，1H)，7.94-8.00(m，1H)，7.88(d，J＝6.1Hz，1H)，7.81(d，J＝4.8Hz，1H)，7.41(br s，1H)，7.32(br s，1H)，7.12-7.25(m，2H)，7.08(d，J＝8.3Hz，2H)，6.93(d，J＝8.8Hz，3H)，6.66-6.79(m，2H)，6.59(dd，J＝8.8，3.5Hz，2H)，6.39(d，J＝8.8Hz，1H)，4.32(d，J＝4.4Hz，2H)，4.08(d，J＝5.7Hz，1H)，3.92(d，J＝5.3Hz，1H)，3.60(d，J＝18.0Hz，2H)，3.14(d，J＝19.7Hz，2H)，2.73(d，J＝5.7Hz，1H)，2.63(br s，1H)，2.39(t，J＝7.2Hz，4H)，0.84(t，J＝7.5Hz，3H)。

EXAMPLE S-33 preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thione-imidazolidin-1-yl) -N- (6- (3- (5- (8-ethyl-7-oxo-5, 6, 7, 8-tetrahydropyrazino [2, 3-b ] pyrazin-2-yl) -6-methylpyridin-2-yl) -1H-1, 2, 4-triazol-1-yl) hexyl) -2-fluorobenzamide (Compound 3.1)

Step 1: preparation of 3-bromo-2-methyl-6- (1H-1, 2, 4-triazol-3-yl) pyridine

To 3-bromo-2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridine (1.6g, 4.95mmol) was added MeOH (15mL) with 2M-HCl, and the resulting mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC and LC-MS. After completion, the mixture was concentrated under reduced pressure to give the title compound. LC-MS 239[ M + H [ ]]⁺

Step 2 a: preparation of 2- (6-iodohexyl) isoindoline-1, 3-dione

To a solution of potassium 1, 3-dioxoisoindoline-2-oxide (5.0g, 26.99mmol) in DMF (50mL) was added 1, 6-diiodohexane (22.8g, 67.49mmol), and the mixture was heated at 70 ℃ for 2.5 hours. The reaction was monitored by TLC. After completion, the mixture was washed with H₂O (100mL) was diluted and extracted with EtOAc (200 mL. times.2). The combined organic layers were washed with brine (100mL) and dried over anhydrous Na ₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by CombiFlash chromatography to give the title compound. LC-MS 358[ M + H ]]⁺

Step 2: preparation of 2- (6- (3- (5-bromo-6-methylpyridin-2-yl) -1H-1, 2, 4-triazol-1-yl) hexyl) isoindoline-1, 3-dione

To a solution of 3-bromo-2-methyl-6- (1H-1, 2, 4-triazol-3-yl) pyridine (1.4g, 5.08mmol) in DMF (25mL) at 0 deg.C was added K₂CO₃(1.75g, 12.70mmol), and the mixture was stirred at room temperature for 15 minutes. 2- (6-iodohexyl) isoindoline-1, 3-dione (1.99g, 5.58mmol) was then added to the mixture, and the resulting mixture was heated at 70 ℃ for 1.5 hours. The reaction was monitored by TLC. After the reaction is completed, the mixture is washed with H₂O (50mL) was diluted and extracted with EtOAc (100 mL. times.2). The combined organic layers were washed with brine (100mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by CombiFlash chromatography to give the title compound. LC-MS 468[ M + H]⁺

Step 3 a: preparation of 1-ethyl-7- (trimethylstannyl) -3, 4-dihydropiperazino [2, 3-b ] pyrazin-2 (1H) -one

To 7-bromo-1-ethyl-3, 4-dihydropiperazino [2, 3-b]To a stirred solution of pyrazin-2 (1H) -one (0.5g, 1.94mmol) in DMF (8mL) was added hexamethylditin (0.48mg, 2.33mmol, 1.2 eq) at room temperature and the mixture was degassed under nitrogen for 15 min. Then Pd (PPh) ₃)₄(0.224mg, 0.194mmol, 0.1 equiv.) was added to the mixture and the resulting reaction mixture was heated at 100 ℃ for 90 minutes. The reaction was monitored by TLC. Upon completion, the mixture was diluted with water (200mL) and extracted with EtOAC (200 mL. times.2)And (6) taking. The combined organic layers were washed with water (100mL), brine (150mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by CombiFlash chromatography to give the title compound. LC-MS 343[ M + H ]]⁺

And step 3: preparation of 2- (6- (3- (5- (8-ethyl-7-oxo-5, 6, 7, 8-tetrahydropyrazino [2, 3-b ] pyrazin-2-yl) -6-methylpyridin-2-yl) -1H-1, 2, 4-triazol-1-yl) hexyl) isoindoline-1, 3-dione

To a stirred solution of 2- (6- (3- (5-bromo-6-methylpyridin-2-yl) -1H-1, 2, 4-triazol-1-yl) hexyl) isoindoline-1, 3-dione (0.5g, 1.94mmol) in DMF (8mL) at room temperature was added 1-ethyl-7- (trimethylstannyl) -3, 4-dihydropiperazino [2, 3-b]Pyrazin-2 (1H) -one (0.507g, 1.48mmol, 1.7 equivalents) and triethylamine (0.48mL, 3.50mmol, 4.0 equivalents), and the mixture was degassed under nitrogen for 15 minutes. Then tris (o-tolyl) phosphine (0.053g, 0.0175mmol, 0.2 equiv.) and Pd ₂(dba)₃(0.080mg, 0.087mmol, 0.1 equiv.) was added to the mixture in order, and the resulting mixture was heated at 100 ℃ for 16 hours. The reaction was monitored by TLC. Upon completion, the mixture was diluted with water (50mL) and extracted with EtOAc (200 mL. times.2). The combined organic layers were washed with brine (100mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by CombiFlash chromatography to give the title compound. LC-MS 566[ M + H]⁺

And 4, step 4: preparation of 5- (5-amino-2- (p-tolyloxy) phenyl) -1-methyl-3- (methylamino) pyridin-2 (1H) -one

To 2- (6- (3- (5- (8-ethyl-7-oxo-5, 6, 7, 8-tetrahydropyrazino [2, 3-b))]To a stirred solution of pyrazin-2-yl) -6-methylpyridin-2-yl) -1H-1, 2, 4-triazol-1-yl) hexyl) isoindoline-1, 3-dione (0.20g, 0.353mmol) in EtOH (10mL) was added NH₂NH₂.H₂O (0.52mL, 1.06mmol, 3.0 equiv.) and the mixture was heated at 100 ℃ for 4 hours. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was diluted with water (200mL) and extracted with 10% MeOH/DCM (200 mL. times.3).The combined organic layers were washed with water (100mL), brine (150mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound. LC-MS 436[ M + H ]⁺

And 5: preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thione-imidazolidin-1-yl) -N- (6- (3- (5- (8-ethyl-7-oxo-5, 6, 7, 8-tetrahydropyrazino [2, 3-b ] pyrazin-2-yl) -6-methylpyridin-2-yl) -1H-1, 2, 4-triazol-1-yl) hexyl) -2-fluorobenzamide

To a stirred solution of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzoic acid (0.13g, 0.287mmol) in DMA (5mL) at 0 ℃ was added HBTU (0.130g, 0.345mmol, 1.2 eq) and the resulting mixture was stirred for 10 minutes. DIPEA (0.150mL, 0.575mmol, 2.0 equiv.) and 5- (5-amino-2- (p-tolyloxy) phenyl) -1-methyl-3- (methylamino) pyridin-2 (1H) -one (0.150g, 0.345mmol, 1.2 equiv.) were then added to the mixture in that order and the resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC and LC-MS. Upon completion, water (20mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give a crude residue which was purified by reverse phase HPLC to give the title compound. LC-MS 869[ M + H ] ]⁺。¹HNMR(400MHz，MeOD-d₄)δ8.17(dd，J＝5.2，3.1Hz，2H)，8.06-7.95(m，4H)，7.86(s，1H)，7.80(t，J＝8.2Hz，1H)，7.41-7.29(m，2H)，4.27(s，2H)，4.18(q，J＝7.0Hz，2H)，3.38(t，J＝6.9Hz，2H)，2.76(s，3H)，2.06-1.91(m，3H)，1.61(d，J＝8.6Hz，3H)，1.58(s，6H)，1.50-1.43(m，3H)，1.26(m，2H)。

EXAMPLE S-34 preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -N- (6- (4-ethyl-6- (2-methyl-6- (1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -3-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) hexyl) -2-fluorobenzamide (Compound 3.2)

Step 1: preparation of ethyl 2- (3, 5-dibromopyrazin-2-ylamino) acetate

To a stirred solution of 3, 5-dibromopyrazin-2-amine (20.0g, 79.6mmol) in DMF (100mL) at 0 deg.C was added Cs₂CO₃(36.2g, 102.8mmol), and the mixture was stirred for 15 minutes. Ethyl 2-bromoacetate (10.4g, 94.8mmol) was then added to the mixture, and the mixture was heated at 80 ℃ for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was diluted with water (200mL) to give a precipitate, which was filtered through a buchner funnel and dried under vacuum to give the title compound. LC-MS 338[ M + H]⁺

Step 2: preparation of ethyl 2- (5-bromo-3- (ethylamino) pyrazin-2-ylamino) acetate

To a stirred solution of ethyl 2- (3, 5-dibromopyrazin-2-ylamino) acetate (1.1g, 3.22mmol) in NMP (9mL) were added DIPEA (2.82mL, 16.2mmol, 5 equiv.) and eth ethanamine hydrochloric acid (70% aqueous solution; 1.04mL, 12.9mmol, 2.0 equiv.) and the mixture was heated in a closed tube at 120 ℃ for 16 h. The reaction was monitored by TLC and LC-MS. After completion, the mixture was washed with NaHCO ₃The solution (100mL) was diluted and extracted with EtOAc (200 mL. times.2). The combined organic layers were washed with brine (100mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by CombiFlash chromatography to give the title compound. LC-MS 303[ M + H [ ]]⁺

And step 3: preparation of 7-bromo-1-ethyl-3, 4-dihydropiperazino [2, 3-b ] pyrazin-2 (1H) -one

To a stirred solution of ethyl 2- (5-bromo-3- (ethylamino) pyrazin-2-ylamino) acetate (0.97g, 3.20mmol) in methanol (1mL) was added AcOH (5mL) at room temperature and the mixture was heated in a closed tube at 120 ℃ for 16 h. The reaction was monitored by TLC and LC-MS. After completion, the mixture was washed with NaHCO₃The saturated solution (100mL) was diluted and extracted with EtOAc (100 mL. times.3). The combined organic layers are washed withWashed with brine (50mL) over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by CombiFlash chromatography to give the title compound. LC-MS257[ M + H]⁺

And 4, step 4: preparation of 2- (6- (6-bromo-4-ethyl-3-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) hexyl) isoindoline-1, 3-dione

To 7-bromo-1-ethyl-3, 4-dihydropiperazino [2, 3-b ] at room temperature ]To a stirred solution of pyrazin-2 (1H) -one (0.8g, 3.11mmol) in DMF (25mL) was added Cs₂CO₃(2.02g, 6.24mmol, 2 equiv.) and the mixture stirred for 15 minutes. 2- (6-iodohexyl) isoindoline-1, 3-dione (2.2g, 6.24mmol, 2 equiv.) is then added to the mixture, and the resulting mixture is heated at 80 ℃ for 2 hours. The reaction was monitored by TLC. Upon completion, ice-cold water (50mL) was added to the mixture to give a precipitate, which was filtered through a buchner funnel and dried under vacuum to give the title compound. LC-MS486[ M + H]⁺

And 5: preparation of 2- (6- (4-Ethyl-6- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -3-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) hexyl) isoindoline-1, 3-dione

To 2- (6- (6-bromo-4-ethyl-3-oxo-3, 4-dihydropiperazino [2, 3-b)]To a stirred solution of pyrazin-1 (2H) -yl) hexyl) isoindoline-1, 3-dione (1.0g, 2.06mmol) in DMF (20mL) was added 2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) -3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridine (1.52g, 4.12mmol, 2.0 equiv.) and K dissolved in water (3mL) ₂CO₃(0.567mg, 4.11mmol, 2 equiv.) and the mixture degassed under nitrogen for 15 min. Then Pd (dppf) Cl₂(0.032mg, 0.043mmol, 0.1 equiv.) was added to the mixture and the resulting mixture was heated at 70 ℃ for 16 hours. The reaction was monitored by TLC. Upon completion, the mixture was diluted with water (200mL) and extracted with 10% MeOH/DCM (300 mL. times.3). The combined organic layers were washed with water (100mL), brine (150mL) and driedWater Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude residue which was purified by CombiFlash chromatography to give the title compound. LC-MS 650[ M + H ]]⁺

Step 6: preparation of 4- (6-aminohexyl) -1-ethyl-7- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -3, 4-dihydropiperazino [2, 3-b ] pyrazin-2 (1H) -one

To 2- (6- (4-ethyl-6- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -3-oxo-3, 4-dihydropiperazino [2, 3-b)]To a stirred solution of pyrazin-1 (2H) -yl) hexyl) isoindoline-1, 3-dione (0.30g, 1.38mmol) in EtOH (8mL) was added NH₂NH₂.H₂O (0.067.1.38mmol), and the mixture was heated at 100 ℃ for 16 hours. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was diluted with water (200mL) and extracted with 10% MeOH/DCM (300 mL. times.3). The combined organic layers were washed with water (100mL), brine (150mL) and dried over anhydrous Na ₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound. LC-MS 520[ M + H ]]⁺

And 7: preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -4-hydroxy-5, 5-dimethyl-2-thioimidazolidin-1-yl) -N- (6- (4-2-6- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -3-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) hexyl) -2-fluorobenzamide

To a stirred solution of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzoic acid (0.08g, 0.177mmol) in DMA (6mL) at 0 ℃ was added HBTU (0.08g, 0.212mmol, 1.2 eq) and the resulting mixture was stirred for 10 minutes. DIPEA (0.067mL, 0.389mmol, 2.2 equiv.) and 2- (6- (4-ethyl-6- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -3-oxo-3, 4-dihydropiperazino [2, 3-b ] are then added]Pyrazin-1 (2H) -yl) hexyl) isoindoline-1, 3-dione (0.110g, 0.212mmol, 1.2 equivalents) was added to the mixture in turn, and the mixture was stirred at room temperature for 2 hours. By passingThe reaction was monitored by TLC and LC-MS. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give a crude residue which was purified by chromatography to give the title compound. LC-MS 955[ M + H ] ]⁺

And 8: preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -N- (6- (4-ethyl-6- (2-methyl-6- (1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -3-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) hexyl) -2-fluorobenzamide

To 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -N- (6- (4-ethyl-6- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -3-oxo-3, 4-dihydropiperazino [2, 3-b]Pyrazin-1 (2H) -yl) hexyl) -2-fluorobenzamide (0.18g, 0.188mmol) to was added MeOH with 2M-HCl (7mL), and the resulting reaction mixture was stirred at room temperature for 16H. The reaction was monitored by TLC and LC-MS. After completion, the mixture was passed through NaHCO₃The solution (30mL) was quenched and extracted with 10% MeOH/DCM (200 mL. times.2). The combined organic layers were washed with water (100mL), brine (150mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude residue which was purified by reverse phase HPLC to give the title compound. LC-MS 871[ M + H ]]⁺。¹HNMR(400MHz，MeOD-d₄)δ8.31(s，1H)，8.19-8.09(m，2H)，8.04-7.95(m，3H)，7.93(s，1H)，7.83(t，J＝8.1Hz，1H)，7.39-7.29(m，2H)，4.31(s，2H)，4.19(q，J＝7.0Hz，2H)，3.63(t，J＝7.5Hz，2H)，3.43(t，J＝6.9Hz，2H)，2.76(s，3H)，1.93(d，J＝7.4Hz，1H)，1.79-1.60(m，3H)，1.57(s，6H)，1.50(qd，J＝13.4，10.6，5.3Hz，4H)，1.27(q，J＝7.0，5.6Hz，3H)。

EXAMPLE S-35 preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioneylimidazolidin-1-yl) -2-fluoro-N- (5- (7- (2-methyl-6- (1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -2-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) pentyl) benzamide (Compound 3.3)

Step 1: preparation of ethyl 2- (3, 5-dibromopyrazin-2-ylamino) acetate

To a stirred solution of 3, 5-dibromopyrazin-2-amine (20.0g, 79.6mmol) in DMF (100mL) at 0 deg.C was added Cs₂CO₃(36.2g, 102.8mmol), and the mixture was stirred for 15 minutes. Ethyl 2-bromoacetate (10.4g, 94.8mmol) was then added to the mixture, and the mixture was heated at 80 ℃ for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was diluted with water (200mL) to give a precipitate, which was filtered through a buchner funnel and dried under vacuum to give the title compound. LC-MS 338[ M + H]⁺

Step 2': preparation of 2- (6-aminohexyl) isoindoline-1, 3-dione

To the compound 2- (6-iodohexyl) isoindoline-1, 3-dione (6.2g, 17.35mmol) was added liquid NH₃(35mL) and the mixture was heated at 80 ℃ for 3 hours. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was concentrated under reduced pressure and washed with DCM/hexane (30mL/100mL) and dried under vacuum to give the title compound. LC-MS 247[ M + H]⁺

Step 2: preparation of ethyl 2- (5-bromo-3- (5- (1, 3-dioxoisoindolin-2-yl) pentylamino) pyrazin-2-ylamino) acetate

To a stirred solution of 2- (5-aminopentyl) isoindoline-1, 3-dione (3.2g, 9.44mmol) in NMP (15mL) were added DIPEA (8.2mL, 47.20mmol, 5 equiv.) and 2- (6-aminohexyl) isoindoline-1, 3-dione (4.6g, 18.88mmol, 2.0 equiv.), and the mixture was heated in a sealed tube at 120 ℃ for 16 h. The reaction was monitored by TLC and LC-MS. After completion, the mixture was washed with NaHCO ₃The saturated solution (50mL) was diluted and extracted with EtOAc (100 mL. times.2). The combined organic layers were washed with brine (50mL) and dried over anhydrous Na₂SO₄Drying and filteringAnd concentrated under reduced pressure to give a crude material which was purified by column chromatography to give the title compound. LC-MS 490[ M + H [ ]]⁺

And step 3: preparation of 2- (6- (7-bromo-2-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) hexyl) isoindoline-1, 3-dione

To a stirred solution of ethyl 2- (5-bromo-3- (5- (1, 3-dioxoisoindolin-2-yl) pentylamino) pyrazin-2-ylamino) acetate (2.0g, 3.97mmol) in methanol (5mL) was added AcOH (15mL) and the mixture was heated at 120 ℃ for 16 h in a closed tube. The reaction was monitored by TLC and LC-MS. After completion, the mixture was washed with NaHCO₃The saturated solution (50mL) was diluted and extracted with EtOAc (100 mL. times.2). The combined organic layers were washed with brine (50mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude material which was purified by column chromatography to give the title compound. LC-MS 458[ M + H ]]⁺

And 4, step 4: preparation of 2- (6- (7- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -2-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) hexyl) isoindoline-1, 3-dione

To 2- (6- (7-bromo-2-oxo-3, 4-dihydropiperazino [2, 3-b)]To a stirred solution of pyrazin-1 (2H) -yl) hexyl) isoindoline-1, 3-dione (0.20g, 0.43mmol) in DMF (6mL) was added 2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) -3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridine (0.32mg, 0.872mmol, 2.0 equiv.) and K dissolved in water (3mL)₂CO₃(0.12mg, 0.872mmol, 2 equiv.) and the mixture degassed under nitrogen for 15 minutes. Then Pd (dppf) Cl₂(0.032mg, 0.043mmol, 0.1 equiv.) was added to the mixture and the resulting mixture was heated at 70 ℃ for 16 hours. The reaction was monitored by TLC. Upon completion, the mixture was diluted with water (200mL) and extracted with DCM (200mL × 3). The combined organic layers were washed with water (100mL), brine (150mL) and dried over anhydrous Na₂SO₄Drying, filtering and concentrating under reduced pressure to give a crude material which is passed through CombiFlash chromatography gave the title compound. LC-MS 622[ M + H [ ]]⁺

And 5: preparation of 5- (5-amino-2- (p-tolyloxy) phenyl) -1-methyl-3- (methylamino) pyridin-2 (1H) -one

To 2- (6- (7- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -2-oxo-3, 4-dihydropiperazino [2, 3-b ] at room temperature ]To a stirred solution of pyrazin-1 (2H) -yl) hexyl) isoindoline-1, 3-dione (0.21g, 0.427mmol) in EtOH (5mL) was added NH₂NH₂.H₂O (0.80mL), and the mixture was heated at 100 ℃ for 16 hours. The reaction was monitored by TLC and LC-MS. Upon completion, the mixture was diluted with water (200mL) and extracted with 10% MeOH/DCM (200 mL. times.2). The combined organic layers were washed with water (100mL), brine (150mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave the title compound. LC-MS 492[ M + H [ + ]]⁺

Step 6: preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (5- (7- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) 2-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) pentyl) benzamide

To a stirred solution of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluorobenzoic acid (0.12g, 0.265mmol) in DMA (7mL) at 0 ℃ was added HBTU (0.129g, 0.319mmol, 1.2 eq) and the mixture was stirred at 0 ℃ for 10 minutes. DIPEA (0.94mL, 0.524mmol, 2.2 equivalents) and 5- (5-amino-2- (p-tolyloxy) phenyl) -1-methyl-3- (methylamino) pyridin-2 (1H) -one (0.156g, 0.319mmol, 1.2 equivalents) were then added to the mixture in succession, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC and LC-MS. Upon completion, water (10mL) was added and the resulting precipitate was filtered through a buchner funnel. The product obtained was washed with water (5mL × 2) and n-pentane (5mL × 2) and dried under vacuum to give a crude product which was purified by CombiFlash chromatography to give the title compound. LC-MS 925[ M + H ]⁺

And 7: preparation of 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (6- (7- (2-methyl-6- (1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -2-oxo-3, 4-dihydropiperazino [2, 3-b ] pyrazin-1 (2H) -yl) hexyl) benzamide

To 4- (3- (4-cyano-3- (trifluoromethyl) phenyl) -5, 5-dimethyl-4-oxo-2-thioimidazolidin-1-yl) -2-fluoro-N- (5- (7- (2-methyl-6- (1- (tetrahydro-2H-pyran-2-yl) -1H-1, 2, 4-triazol-3-yl) pyridin-3-yl) -2-oxo-3, 4-dihydropiperazino [2, 3-b]Pyrazin-1 (2H) -yl) pentyl) benzamide (0.13g, 0.140mmol) was added MeOH (10mL) with 2M-HCl and the resulting reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC and LC-MS. After completion, the reaction mixture was passed through NaHCO₃The solution (25mL) was quenched and extracted with 10% MeOH × DCM (100mL × 2). The combined organic layers were washed with water (100mL), brine (150mL) and dried over anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a crude residue which was purified by reverse phase HPLC to give the title compound. LC-MS 841[ M + H]⁺。¹H NMR(400MHz，MeOD-d₄)δ8.20-8.13(m，2H)，8.01(dd，J＝10.3，7.6Hz，4H)，7.85(s，1H)，7.79(t，J＝8.1Hz，1H)，7.38-7.28(m，2H)，4.29(s，2H)，4.15(t，J＝7.6Hz，2H)，3.41-3.34(m，2H)，2.76(s，3H)，1.76(q，J＝7.3Hz，2H)，1.67-1.59(m，2H)，1.57(s，6H)，1.51-1.43(m，2H)，1.30(d，J＝8.8Hz，2H)。

The compounds of table 1 were prepared according to the experimental details exemplified in the synthesis examples, using the appropriate starting materials and reagents.

Biological assays

The following methods were used to evaluate the in vitro biological properties of the test articles.

Rbc hot spot poly (ADP-ribose) polymerase 1(PARP1) and poly (ADP-ribose) polymerase 2(PARP2) assays: the assay principle is a filtration binding assay based on radioisotopes, in which a radioisotope-labeled NAD is used⁺Incorporation into Filter captured substrate to elute free NAD⁺And then detecting. Use Excel and GraphPad Prism software analysis of IC₅₀Curve-fitted data. Each assay was performed using PJ34 as a positive control.

Parp1 assay: human recombinant PARP1 at a final concentration of 2.5nM was combined with histone H4 (20. mu.M) and test article at various concentrations in reaction buffer (50mM Tris-HCl, pH 8.0, 50mM NaCl, 10mM MgCl)₂1mM DTT, 1% DMSO, and 20. mu.g/mL of activating DNA). The solution was inoculated for 20 minutes at room temperature and by adding [ adenosine-³²P]-a Nicotinamide Adenine Dinucleotide (NADP),³²P-NAD⁺the reaction is initiated. After 2 hours incubation at room temperature, the reaction mixture was filtered and washed with 0.75% phosphoric acid to detect radioactivity.

Parp2 assay: human recombinant PARP2 at a final concentration of 2.5nM was combined with histone H3 (20. mu.M) and test article at various concentrations in reaction buffer (50mM Tris-HCl, pH 8.0, 50mM NaCl, 10mM MgCl) ₂1mM DTT, 1% DMSO, and 20. mu.g/mL of activating DNA). The solution was inoculated for 20 minutes at room temperature and by adding [ adenosine-³²P]-a Nicotinamide Adenine Dinucleotide (NADP),³²P-NAD⁺the reaction is initiated. After 2 hours incubation at room temperature, the reaction mixture was filtered and washed with 0.75% phosphoric acid to detect radioactivity.

Ar binding assay: the AR in LNCaP cytosol was used to determine the binding affinity of the test and reference compounds progesterone (Sigma), cat # E2785, st. louis, MO). Determination of IC Using 8 concentrations/Compound₅₀. Cytosol was plated at 200 μ g/well (100 μ L) into a 96-well conical polypropylene plate (Agilent, catalog No. 5042-1385, Santa Clara, CA) and mixed with 3 μ L of test compound. Adding 100 μ L³After H-methyltrienolone (PerkinElmer, Cat. No.: NET590250UC, San Jose, Calif.), the plates were sealed and shaken at 300rpm for 24 hours at 4 ℃. After incubation, the cells will contain 10mM Tris-HCl, pH 7.4; 1.5mM EDTA; 1mM DTT; 0.25% carbon; 100 μ L of 0.0025% dextran Radioligand adsorption buffer was added to each well. The plate was shaken at 4 ℃ for 15 minutes and then centrifuged at 3000rpm at 4 ℃ for 30 minutes. mu.L of the supernatant was transferred to a scint tube (Perkin Elmer, cat # 6000192) and mixed with 2mL of Ultima Gold mixture (Perkin Elmer, cat # 6013329). Radioactivity was counted using a TriCarb 2910TR scintillation counter (perkin elmer). The inhibition of radioactivity by the test article was calculated using the following equation:

percent inhibition ═ 1- (assay wells-average _ LC)/(average _ HC-average _ LC)) × 100%.

IC was calculated and plotted using the model "log (inhibitor) versus response- -variable slope" included in GraphPad Prism 5 (San Diego, Calif.))₅₀The value is obtained. Ki values were further calculated using the following equation, where [ L]Is the radioligand concentration (1nM) used in this study. The Kd value is 0.332 nM. The results are shown in Table 3.

Ki＝IC₅₀/(1+[L]/Kd)

Ar transactivation: human AR cloned into CMV vector stem was used for transactivation studies. HEK-293 cells were plated in DME + 5% csFBS at 80,000 cells per well in 24-well plates. Twenty-four hours later, cells were transfected in OPTIMEM medium using lipofectamine (Invitrogen, Carlsbad, CA) with 0.25 μ g GRE-LUC, 0.01 μ g CMV-LUC (renilla luciferase) and 25ng AR. With various ligands (10) ^-12To 10^-5M final concentration) cells were treated 24 hours after transfection and luciferase assays were performed 48 hours after transfection. Firefly values were normalized to renilla luciferase values and expressed as Relative Light Units (RLU). Assays for agonists and antagonists of the test article were performed in the absence and in combination with 0.1nM R1881, respectively. Data are expressed as EC obtained from four parametric logistic curves₅₀(for agonists) and IC₅₀(for antagonists). Each experiment was performed with R1881 as an agonist. The results of AR antagonism are shown in table 2.

d. Cell culture and proliferation assays: LNCaP, PC-3, 22RV1, MCF-7, HT-29 and HCT-116 cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured in the media recommended by ATCC. Cell culture media was obtained from Fisher scientific (Waltham, MA), and serum was obtained from sea clone (Hyclone) (San Angelo, TX), santa arrele, texas).

Cells were plated at different densities in corresponding growth media in 96-well plates. After 24 hours, cells were treated in triplicate or quadruplicate with a concentration of test article by serial dilution of DMSO stock solution in growth medium and incubated for three to seven days. Three days after treatment, the number of viable 22RV1 and HT-29 cells was measured using the CellTiter Glo assay (CTG, Promega, Madison, Wis.). Other types of cancer cells were treated after three or four days, and the number of surviving cells was measured using the CTG assay after a total treatment time of six or seven days. Cell viability data was plotted using GraphPad Prism (glaxpander software, san diego, ca). In addition, the IC of individual test articles was calculated using a non-linear regression model with sigmoidal dose response and variable slope within GraphPad Prism ₅₀The value is obtained. The assay results are shown in table 4.

e. Other cell proliferation assays: other cancer cell lines were tested in cell proliferation assays. For example, SK-OV-3 or OVCAR3 ovarian cancer cells were tested. Cells were grown in supplier's recommended medium (e.g., ATCC or JCRB gene bank) at 37 ℃ in 5% CO₂Culturing in the environment. For proliferation assays, cells were plated in growth medium in 96-well plates. The seeding density was adjusted according to the cell type. After 24 hours, cells are treated in triplicate or quadruplicate with test article prepared at a concentration by serial dilution of DMSO stock solution in growth medium, and the medium containing the test article is replaced after typically three to seven days of incubation, three or four days. The number of viable cells was measured using the CellTiter Glo assay (CTG, promega, madison, wisconsin) or similar methods. GraphPad Prism (Graaffade Praded, san Diego, Calif.) was usedSoftware company) to plot cell viability data. In addition, IC of individual test articles was calculated using a nonlinear regression model with sigmoidal dose response and variable slope within GraphPad Prism₅₀The value is obtained. Similarly, HEK-293 and HeLa cells (HeLa cells) can also be tested by methods known in the art.

f. Nuclear translocation: LNCaP cells were plated on coverslips in 24-well plates in growth medium. Twenty-four hours after plating, the medium was changed to RPMI + 1% csFBS and the cells were kept in this medium for two days. The medium was replaced again and the cells were processed. Cells were fixed 4 hours after treatment and AR was immunostained using AR N20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Nuclei were stained with DAPI. Cells were imaged with confocal microscopy.

Er binding assay: by the Saimer Feishel (Thermo Fisher)The TR-FRET era competitive binding assay assesses era binding. In this assay, a terbium-labeled anti-GST antibody was used to indirectly label the GST-tagged era-Ligand Binding Domain (LBD) by binding to its GST tag. Competitive binding to ER α -LBD (GST) by replacement of fluorescent ligand (Fluormone) from ER α -LBD (GST) by test compound^TMES2 green tracer) which results in a loss of FRET signal between the Tb-anti-GST antibody and the tracer. When the operation is detected, Fluormone is added^TMThe ES2 green tracer was added to the ligand test compound or solvent control, followed by a mixture of ER α -LBD (GST) and terbium anti-GST antibody. After an incubation period at room temperature, the emission at the TR-FRET ratio of 520: 495 was calculated and used to determine the IC from the dose response curve of the compound ₅₀. The assay results are shown in table 3.

Er and PR functional assays: COS cells were transfected with 25ng of rat Progesterone Receptor (PR) and 250ng of GRE-LUC or 50ng of human estrogen receptor alpha (ER) and 250ng of ERE-LUC. Cells were also transfected with 10ng CMV-Renilla LUC in OptiMEM medium using lipofectamine transfection reagents. Twenty-four hours after transfection of the medium became DME + 5% csFBS free and treated with compounds in the presence of 0.1nM progesterone against PR and estradiol against ER. After twenty-four hours of treatment, cells were collected and subjected to luciferase assay using a dual luciferase assay kit. Firefly values were normalized to renilla luciferase values and expressed as a ratio.

i. Test compounds were evaluated in a mouse xenograft model: to examine the in vivo anti-tumor activity of the test compounds, tumor growth experiments were performed in a cell line xenograft model. Male NOD SCID γ (NSG) mice were housed in five animals per cage and allowed free access to water and commercial rodent chow. 22RV1 cells (grown in RPMI + 10% FBS) mixed with 50% matrix gum base membrane were implanted subcutaneously into castrated mice. Alternatively, an antiandrogen-resistant cell line other than 22RV1, such as MR49F or VCaP, is used. Once the tumor reaches 200-500mm ³Animals were randomized and treated intraperitoneally with vehicle (DMSO: PEG-300: corn oil ratio 10: 30: 60) or test compound. Tumors were measured three times a week and volume was calculated using the formula length x width x 0.5. Animals were sacrificed at the end of 28 days of treatment, and tumors were weighed and stored for further processing. Tumor Growth Inhibition (TGI) was calculated by comparing tumor measurements from the control group with the other study groups. TGI was calculated for each group using the following formula:

TGI(％)＝[1-(TV_{treatment _ day N}-TV_{Treatment _ day 0})/(TV_{Vehicle _ Nth day}-TV_{Medium _ day 0})]×100％

TV_{Treatment _ day N}Mean tumor volume on a given day for treatment groups, TV_{Treatment _ day 0}Mean tumor volume for treatment group on day one of treatment, TV_{Vehicle _ Nth day}Mean tumor volume on a given day for vehicle control group, and TV_{Medium _ day 0}Mean tumor volume on the first day of treatment for vehicle group.

DNA-PK inhibition: inhibition of DNA-PK Activity by test Compounds in a radioisotope-based Filter binding assay (BioProcesses Co., Ltd.)Evaluation was performed in a HotSpot Kinase Assay (Reaction Biology Corporation HotSpot Kinase Assay)). Test compounds were dissolved in 100% DMSO until the specified concentration. Serial dilutions of test compounds were performed by Integra Viaflo Assist in DMSO. Substrate DNA-PKtide (Anas Pai, Anaspec, Fremont, Calif.; No. 60210-5) in reaction buffer (20mM Hepes, pH 7.5, 10mM MgCl) ₂，1mM EGTA，0.02％Brij35，0.02mg/ml BSA，0.1mM Na₃VO₄2mM DTT, 1% DMSO) so that its final concentration in the reaction will be 20 μ M. The dsDNA-containing DNA-PK activator was delivered into solution (10. mu.g/mL in the final reaction). DNA-PK (Invitrogen, Calsbad, Calif.; No. PR 9107A) was delivered to the substrate solution (5 nM in the final reaction) and the solution was gently mixed. Compounds in 100% DMSO were delivered to the kinase reaction mixture by Acoustic technology (Echo 550; nanoliter range) and the reaction was incubated for 20 minutes at room temperature. Will be provided with³³P-ATP (specific activity 10. mu. Ci/. mu.L) was delivered to the reaction mixture to initiate the reaction, which was incubated at room temperature for 2 hours. Radioactivity was detected by filter binding. Kinase activity data is expressed as a percentage of the kinase activity remaining in the test sample compared to the vehicle (dimethyl sulfoxide) response. IC acquisition Using GraphPad Prism₅₀Values and curve fitting. The assay results are shown in table 5.

TABLE 2 in vitro results

Compound numbering	PARP1 IC50(μM)	PARP2 IC50(μM)	AR Ant.IC50(μM)
				1.1	0.002	0.0002	0.033
1.2	0.003	0.0002	0.481
				1.3	0.002	0.0005
Enantiomer-1.3	0.079	0.016
				1.4	0.002	0.0004
1.5	0.023	0.002	0.75
				1.68	0.0003	0.0002	3.3
1.83	0.0007	0.0003	0.57
				1.96	0.006	0.0005
1.97	0.002	0.0004
				1.98	0.0002	0.0001
1.99	0.0008	0.0002
				1.104	0.0012	0.0003
1.107	0.0003	0.0001
				1.110	0.0007	0.0002
1.111	0.0016	0.0002
				1.114	0.0279	0.0019
1.115	0.0257	0.0015
				1.116	0.0005	0.0002
1.117	0.0003	0.0001
				1.118	0.0136	0.0010
1.119	0.0016	0.0003
				2.4	0.004	0.0003
2.13	0.0033	0.0005
				2.14	0.274	0.0124

TABLE 3 NHR binding in vitro

Compound numbering	AR Ki(nM)	ERαKi(nM)
			1.2	1.31
1.5	291
			1.68	907
1.83	139
			1.97	504
1.98	181
			1.99	197
1.104	167
			1.107	＞7469
1.110	60
			1.111	36
1.114	19
			1.115	30
1.116	4969
			1.117	60
1.118	＞7469
			1.119	20
2.4		5
			2.13		3.3
2.14		3.5
			3.1	21
3.2	19
			3.3	14

TABLE 4 in vitro cell growth inhibition

Table 5: in vitro enzyme inhibition

Compound numbering	DNA-PK IC50(μM)
		3.1	＞10
3.2	＞10
		3.3	0.0018