Tri-functional constructs with tunable pharmacokinetics useful for imaging and anti-tumor therapy

文档序号：1651746 发布日期：2019-12-24 浏览：16次中文

阅读说明：本技术 可用于成像和抗肿瘤治疗的具有可调的药代动力学的三功能构建体 (Tri-functional constructs with tunable pharmacokinetics useful for imaging and anti-tumor therapy ) 是由约翰·W·百祺詹姆斯·M·凯利亚历杭德罗·埃莫-科尔拉萨沙希坎特·庞纳拉于 2018-04-05 设计创作，主要内容包括：本技术提供了可用于神经胶质瘤、乳腺癌、肾上腺皮质癌、宫颈癌、外阴癌、子宫内膜癌、原发性卵巢癌、转移性卵巢癌、非小细胞肺癌、小细胞肺癌、膀胱癌、结肠癌、原发性胃腺癌、原发性结直肠腺癌、肾细胞癌、和/或前列腺癌的成像和/或治疗的化合物以及包括此类化合物的组合物。所述化合物由以下式(I)表示、或其药学上可接受的盐。<Image he="394" wi="700" file="DDA0002265297870000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The present technology provides compositions useful for glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and/or prostate cancerAnd compositions comprising such compounds. The compound is represented by the following formula (I), or a pharmaceutically acceptable salt thereof.)

1. A compound of formula I

Or a pharmaceutically acceptable salt and/or solvate thereof, wherein

ABD is an antigen binding domain;

W¹is-C (O) -, - (CH)₂)_n-, or- (CH)₂)_o–NH₂-C(O)–；

R¹、R²And R³Is one of

And R is¹、R²And R³The other two are both H;

X¹is absent, O, S or NH;

L¹is absent, -C (O) -NR⁴-、-C(O)-NR⁵-C₁-C₁₂Alkylene-, -C₁-C₁₂alkylene-C (O) -, -C (O) -NR⁶-C₁-C₁₂alkylene-C (O) -, -arylene-, -O (CH)₂CH₂O)_r–CH₂CH₂C (o) -, an amino acid, a peptide of 2,3, 4,5, 6,7,8, 9, or 10 amino acids, or a combination of any two or more thereof, wherein R is 0,1, 2,3, 4,5, 6,7,8, or 9, and wherein R is⁴、R⁵And R⁶Each independently is H, alkyl or aryl;

tox is a cytotoxic-and/or imaging agent-containing domain;

L²is absent, -C (O) -, - (CH)₂CH₂O)_s–CH₂CH₂C (o) -, a peptide of 2,3, 4,5, 6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 amino acids, or a combination of any two or more thereof, wherein s is 0,1, 2,3, 4,5, 6,7,8, 910, 11, 12, 13, 14, 15, 16, 17, 18, or 19;

alb is an albumin binding moiety;

m is 0 or 1;

n is 1 or 2;

o is 1 or 2;

p is 0,1, 2 or 3, with the proviso that X is when p is 0¹Is absent; and is

q is 1 or 2.

2. The compound of claim 1, wherein the compound of formula I has formula II

Or a pharmaceutically acceptable salt and/or solvate thereof, wherein

P¹、P²And P³Each independently is H, methyl, benzyl, 4-methoxybenzyl, or tert-butyl;

R¹、R²and R³Is one of

And R is¹、R²And R³The other two are both H;

rad is a moiety capable of comprising a metal ion, optionally further comprising a metal ion.

3. The compound of claim 2, wherein P¹、P²And P³Each independently is H or t-butyl.

4. The compound of claim 2, wherein P¹、P²And P³Each independently is H.

5. The compound of claim 2, wherein Rad comprises a chelating agent that chelates metal ions.

6. The compound of claim 5, wherein the metal ion is a radionuclide that:¹⁷⁷Lu³⁺、¹⁷⁵Lu³⁺、⁴⁵Sc³⁺、⁶⁶Ga³⁺、⁶⁷Ga³⁺、⁶⁸Ga³⁺、⁶⁹Ga³⁺、⁷¹Ga³⁺、⁸⁹Y³⁺、⁸⁶Y³⁺、⁸⁹Zr⁴⁺、⁹⁰Y³⁺、^99mTc⁺¹、¹¹¹In³⁺、¹¹³In³ ⁺、¹¹⁵In³⁺、¹³⁹La³⁺、¹³⁶Ce³⁺、¹³⁸Ce³⁺、¹⁴⁰Ce³⁺、¹⁴²Ce³⁺、¹⁵¹Eu³⁺、¹⁵³Eu³⁺、¹⁵²Dy³⁺、¹⁴⁹Tb³⁺、¹⁵⁹Tb³⁺、¹⁵⁴Gd³ ⁺、¹⁵⁵Gd³⁺、¹⁵⁶Gd³⁺、¹⁵⁷Gd³⁺、¹⁵⁸Gd³⁺、¹⁶⁰Gd³⁺、¹⁸⁸Re⁺¹、¹⁸⁶Re⁺¹、²¹³Bi³⁺、²¹¹At⁺、²¹⁷At⁺、²²⁷Th⁴⁺、²²⁶Th⁴⁺、²²⁵Ac³⁺、²³³Ra²⁺、¹⁵²Dy³⁺、²¹³Bi³⁺、²¹²Bi³⁺、²¹¹Bi³⁺、²¹²Pb²⁺、²¹²Pb⁴⁺、²⁵⁵Fm³⁺or uranium 230.

7. The compound of claim 5, wherein the metal ion is an alpha-emitting radionuclide selected from the group consisting of:²¹³Bi³⁺、²¹¹At⁺、²²⁵Ac³⁺、¹⁵²Dy³⁺、²¹²Bi³⁺、²¹¹Bi³⁺、²¹⁷At⁺、²²⁷Th⁴⁺、²²⁶Th⁴⁺、²³³Ra²⁺、²¹²Pb²⁺and²¹²Pb⁴⁺。

8. a pharmaceutical composition comprising an effective amount of a compound of any one of claims 1-7 for detecting mammalian tissue that overexpresses prostate specific membrane antigen ("PSMA"), and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the mammalian tissue comprises one or more of glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

10. A method comprising

Administering to a subject an effective amount of a compound of any one of claims 1-7 for imaging cancer; and

after the administration, one or more of positron emission, gamma rays from the positron emission and annihilation, and cerenkov radiation due to the positron emission are detected.

11. The method of claim 10, wherein the cancer comprises one or more of glioma, breast cancer, adrenocortical cancer, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

12. The method of claim 10, wherein the subject is suspected of having mammalian tissue overexpressing prostate specific membrane antigen ("PSMA").

13. The method of claim 12, wherein the mammalian tissue comprises one or more of glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

14. The method of claim 10, wherein administering the compound comprises parenteral administration.

15. A pharmaceutical composition comprising an effective amount of a compound of any one of claims 1-7 for treating mammalian tissue that overexpresses prostate specific membrane antigen ("PSMA"), and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, wherein the mammalian tissue comprises one or more of glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

17. A method comprising

Administering to a subject an effective amount of a compound of any one of claims 1-7 for treating cancer.

18. The method of claim 17, wherein the cancer comprises one or more of glioma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

19. The method of claim 17, wherein administering the compound comprises parenteral administration.

20. A method of achieving in vivo tissue distribution of a radiotherapeutic agent in a mammalian subject, wherein a ratio of tumor activity to renal activity of 1 or greater is observed within about 4 hours to about 24 hours of administration of the radiotherapeutic agent to the mammalian subject, wherein

The method comprises administering the radiotherapeutic agent to the mammalian subject; and is

The radiotherapeutic agent includes a first moiety that targets prostate specific membrane antigen ("PSMA"), a second moiety with a radionuclide, and a third moiety having an affinity for serum albumin, the first moiety being separated from the second moiety by a first covalent linker and the third moiety being separated from the second moiety by a second covalent linker,

wherein the spacing between the first and second moieties (based on the number of consecutive atoms associated with the first covalent linker) is from about 8 atoms to about 40 atoms, and the spacing between the third moieties and the first and second moieties (based on the number of consecutive atoms associated with the second covalent linker) is from about 10 atoms to about 100 atoms.

21. The method of claim 20, wherein the method further comprises: an image of the mammalian subject is obtained from about 4 hours to about 24 hours after administration of the radiotherapeutic agent.

22. The method of claim 20, wherein a ratio of tumor activity to renal activity of 1 or greater persists for up to about 24 hours after administration of the radiotherapeutic agent.

23. The method of claim 20, wherein substantially no radionuclide activity is observed in the salivary glands of the mammalian subject from about 24 hours to about 48 hours after administration of the radiotherapeutic agent.

24. The method of claim 20, wherein the consecutive number of atoms associated with the first covalent linker is from about 10 atoms to about 30 atoms.

25. The method of claim 20, wherein the number of consecutive atoms associated with the second covalent linker is from about 15 atoms to about 40 atoms.

26. The method of claim 20, wherein the administering comprises intravenous administration.

Technical Field

The present technology relates generally to a trifunctional construct comprising: (1) an antigen binding domain, (2) a cytotoxin-containing and/or imaging agent-containing domain; and (3) an albumin binding moiety. The technology also provides compositions comprising such compounds and methods of use in imaging and/or anti-tumor therapy. For example, the compounds and compositions of the present technology are useful therapeutic compounds.

Summary of The Invention

In one aspect, there is provided a compound of formula I:

or a pharmaceutically acceptable salt and/or solvate thereof, wherein

ABD is an antigen binding domain;

W¹is-C (O) -, - (CH)₂)_n-, or- (CH)₂)_o–NH₂-C(O)–；

R¹、R²And R³Is one of

And R is¹、R²And R³The other two are both H;

X¹is absent, O, S or NH;

tox is a cytotoxic-and/or imaging agent-containing domain;

alb is an albumin binding moiety;

m is 0 or 1; n is 1 or 2; o is 1 or 2; p is 0,1, 2 or 3, with the proviso that X is when p is 0¹Is absent; and q is 1 or 2.

In any of the embodiments herein, the compound of formula I may be a compound of formula II

Or a pharmaceutically acceptable salt and/or solvate thereof, wherein

P¹、P²And P³Each independently is H, methyl, benzyl, 4-methoxybenzyl, or tert-butyl;

R¹、R²and R³Is one of

And R is¹、R²And R³The other two are both H;

rad is a moiety capable of comprising a metal ion, optionally further comprising a metal ion; p is 0,1, 2 or 3, with the proviso that X is when p is 0¹Is absent; and q is 1 or 2.

In another aspect, the present technology also provides compositions (e.g., pharmaceutical compositions) and medicaments comprising any of the embodiments of the compounds of formula I (or pharmaceutically acceptable salts thereof) disclosed herein and a pharmaceutically acceptable carrier or one or more excipients or fillers.

In another aspect, the present technology provides methods of treatment and/or imaging by administering to a subject in need of treatment and/or imaging an effective amount of any one of the embodiments of a compound of formula I (or a pharmaceutically acceptable salt thereof) disclosed herein.

In another aspect, the present technology provides a method of achieving in vivo tissue distribution of a radiotherapeutic agent in a mammalian subject, wherein a ratio of tumor activity to renal activity of 1 or greater is observed within about 4 hours to about 24 hours of administration of the radiotherapeutic agent to the mammalian subject, wherein the method comprises administering the radiotherapeutic agent to the mammalian subject; and the radiotherapeutic agent includes a first moiety that targets prostate specific membrane antigen ("PSMA"), a second moiety with a radionuclide, and a third moiety having an affinity for serum albumin, the first moiety being separated from the second moiety by a first covalent linker and the third moiety being separated from the second moiety by a second covalent linker, wherein the spacing between the first and second moieties (based on the number of consecutive atoms associated with the first covalent linker) is from about 8 atoms to about 40 atoms, and the spacing between the third moiety and the first and second moieties (based on the number of consecutive atoms associated with the second covalent linker) is from about 10 atoms to about 100 atoms.

Drawings

FIG. 1 provides injections 1 and 3 hours after injection⁶⁸Ga-RPS-055 (Top) or⁶⁸PET images of Ga-DKFZ-617 (bottom) mice. All images are of the same mouse with a small LNCaP tumor (arrow) on the right shoulder. Images were taken over consecutive days using a Siemens Inveon μ PET/μ CT system (Siemens Corp., Munich, Germany). All images were windowed to a maximum injection dose of 4% per cc of tissue and attenuation and dose corrected.

FIG. 2 provides injection at 1 hour (top) and 3 hours (bottom) post-injection⁶⁸Ga-RPS-055 (left panel) or⁶⁸PET images of Ga-RPS-056 (right panel) mice. All images are of the same mouse with a small LNCaP tumor on the right shoulder. Images were taken over consecutive days using a Siemens Inveon μ PET/μ CT system (Siemens Corp., Munich, Germany). All images were windowed to a maximum injection dose of 22% per cc of tissue and attenuation and dose corrected.

FIG. 3 provides 4 hours (top) and 3 hours (bottom) post-injection¹⁷⁷SPECT images of mice Lu-RPS-055. All images are of the same mouse with a small LNCaP tumor on the right shoulder (left and right panels). A Siemens Inveon μ PET/μ CT system (Siemens Corp., Munic) was usedh, Germany) were taken on the same day.

FIG. 4 shows LNCaP tumor xenografts carried and injected intravenously⁶⁸Ga-RPS-061、⁶⁸Ga-PSMA-617 or⁶⁸PET images of BALB/C nu/nu mice of Ga-RPS-030. Mice were imaged using a Siemens Inveon μ PET/μ CT system (Siemens corp., Munich, Germany) for 1 hour (left) and 3 hours (right) consecutive days post-injection.

FIGS. 5A-C provide histograms illustrating selected organs after intravenous injection in mice²²⁵Ac(NO₃)₃(FIG. 5A), and²²⁵Ac(macropa)]⁺(FIG. 5B) and [, [2 ]²²⁵Ac(DOTA)]^–(FIG. 5C) biodistribution. Adult C57BL/6 mice were sacrificed 15 minutes, 1 hour, or 5 hours after injection. The values for each time point are given as the average% ID/g. + -. 1 SD.

FIG. 6 shows the results after intravenous injection in LNCaP tumor xenograft mice²²⁵Biodistribution of Ac-macropa-RPS-070. Mice were sacrificed 4, 24 or 96 hours after injection. The values for each time point are given as the mean% ID/g. + -. 1 SEM.

FIG. 7 provides 1h, 3h, 6h and 24h post-injection with⁶⁶PET images of LNCaP xenografted mice with Ga-labeled tracer. Mice were injected intravenously with 0.56-5.4MBq (15-145. mu. Ci) of tracer. The total mass of injected ligand was 4. mu.g. Mice were anesthetized with isoflurane prior to imaging, and then imaged for 30 minutes. The attenuation of the image and the activity of the injection are corrected.

FIG. 8 provides¹⁷⁷Lu-RPS-068、¹⁷⁷Lu-RPS-063、¹⁷⁷Lu-RPS-061、¹⁷⁷Lu-RPS-069、¹⁷⁷Lu-RPS-066、¹⁷⁷Lu-RPS-067 and¹⁷⁷the biodistribution of Lu-PSMA-617. Male athymic nude mice bearing LNCaP xenograft tumors (n ═ 5 at each time point) were injected intravenously with 348-. The total mass of injected ligand was 37-50ng (23-25 pmol).

FIG. 9 provides the differences at 4 hours, 24 hours and 96 hours post injection¹⁷⁷Blood pool activity of Lu-labeled ligandsAnd (6) comparing. The error is expressed as SEM. RPS ligands are shown in order of increasing size.

FIG. 10 provides results in male athymic nude mice bearing LNCaP xenograft tumors¹⁷⁷Lu-PSMA-617、¹⁷⁷Lu-RPS-061、¹⁷⁷Lu-RPS-063、¹⁷⁷Lu-RPS-066、¹⁷⁷Lu-RPS-067、¹⁷⁷Lu-RPS-068 and¹⁷⁷time-activity curve (TAC) of tumor and kidney uptake of Lu-RPS-069. The intake is expressed as% ID/g.

FIG. 11 provides a graph of the results after injection¹⁷⁷Lu-labeled compounds and comparison of relative dose scores in tumors of male LNCaP xenograft tumor-bearing mice studied over 96 hours. Relative to the value¹⁷⁷Lu-PSMA-617 for standardization.

FIG. 12 shows the active uptake in blood, normal tissues and tumors in male BALB/C nu/nu mice bearing LNCaP xenograft tumors. Mice (n ═ 4/time point) were injected intravenously with 105kBq²²⁵Ac-RPS-074 and sacrificed at 4 hours, 24 hours, 7 days, 14 days and 21 days post-injection.

FIG. 13 depicts LNCaP bearing xenograft tumors and treated with a)138kBq²²⁵Ac-RPS-074、b)74kBq²²⁵Ac-RPS-074、c)37kBq²²⁵Ac-RPS-074、d)133kBq ²²⁵Ac-DOTA-Lys-IPBA and e) Change in mean tumor volume of vector treated male BALB/C nu/nu mice.

FIG. 14 provides a graph using 138kBq²²⁵Ac-RPS-074 (left) or 74kBq²²⁵Ac-RPS-074 (Right) treated mice⁶⁸Ga-PSMA-11 mu PET/CT images. Images were collected 60 minutes after injection and corrected for attenuation and injection activity.

FIG. 15 depicts a Kaplan-Meier curve illustrating the overall survival of mice. Administered²²⁵Ac-DOTA-Lys-IPBA activity. When the tumor volume exceeds 2000mm³Mice were sacrificed at time.

Detailed Description

The following terms are used throughout and are defined as follows.

As used herein and in the appended claims, the singular articles such as "a," "an," and "the" and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as/for example") provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If there is a term usage that is not clear to one of ordinary skill in the art, "about" means up to plus or minus 10% of the particular term, given the context of the usage.

In general, reference to a particular element, such as hydrogen or H, is intended to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Thus, containing radioactive isotopes such as tritium, C¹⁴、P³²And S³⁵Are within the scope of the present technology. Based on the disclosure herein, procedures for inserting such labels into the compounds of the present technology will be apparent to those skilled in the art.

Typically, "(substituted)" refers to an organic group (e.g., alkyl group) as defined below, wherein one or more bonds to a hydrogen atom contained therein are replaced with bonds to a non-hydrogen or non-carbon atom. Substituted radicals also include those in which one or more bonds to one or more carbon or hydrogen atoms are replaced by one or more bonds to a heteroatomSub-bonded bonds (including double or triple bonds) are substituted. Thus, unless otherwise specified, a substituted group is substituted with one or more substituents. In some embodiments, substituted groups are substituted with 1,2,3, 4,5, or 6 substituents. Examples of the substituent include: halogen (i.e., F, Cl, Br, and I); a hydroxyl group; alkoxy, alkenyloxy, aryloxy, aralkoxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy; carbonyl (oxo); a carboxylate; an ester; a carbamate; an oxime; a hydroxylamine; an alkoxyamine; an arylalkoxyamine; a thiol; a sulfide; a sulfoxide; a sulfone; a sulfonyl group; pentafluorothio (i.e. SF)₅) A sulfonamide; an amine; an N-oxide; hydrazine; a hydrazide; hydrazone; an azide; an amide; urea; amidines; guanidine; an enamine; an imide; an isocyanate; an isothiocyanate; a cyanate; a thiocyanate salt; an imine; a nitro group; nitriles (i.e., CN); and so on.

Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and ring systems in which the bond to a hydrogen atom is replaced with a bond to a carbon atom. Thus, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl and alkynyl groups as defined below.

As used herein, C when used before a group_m-C_nE.g. C₁-C₁₂、C₁-C₈Or C₁-C₆And means a group containing m to n carbon atoms.

Alkyl groups include straight and branched chain alkyl groups having from 1 to 12 carbon atoms, typically from 1 to 10 carbons, or in some embodiments, from 1 to 8,1 to 6, or 1 to 4 carbon atoms. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isoamyl, and 2, 2-dimethylpropyl. The alkyl group may be substituted or unsubstituted. Representative substituted alkyl groups can be substituted one or more times with substituents such as those listed above and include, but are not limited to, haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

Cycloalkyl groups include mono-, di-, or tricycloalkyl groups having from 3 to 12 carbon atoms, or in some embodiments, from 3 to 10, 3 to 8, or 3 to 4,5, or 6 carbon atoms in the ring. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, cycloalkyl groups have 3 to 8 ring members, while in other embodiments the number of ring carbon atoms ranges from 3 to 5,3 to 6, or 3 to 7. Bicyclic and tricyclic systems include bridged cycloalkyl groups and fused rings such as, but not limited to, bicyclo [2.1.1] hexane, adamantyl, decahydronaphthyl, and the like. Cycloalkyl groups may be substituted or unsubstituted. Substituted cycloalkyl groups may be substituted one or more times by non-hydrogen and non-carbon groups as defined above. However, substituted cycloalkyl also includes rings substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, 2-, 2,3-, 2,4-, 2,5-, or 2, 6-disubstituted cyclohexyl, which may be substituted with substituents such as those listed above.

Cycloalkylalkyl is an alkyl group as defined above in which a hydrogen or carbon bond of the alkyl group is replaced by a bond to the cycloalkyl group as defined above. In some embodiments, cycloalkylalkyl groups have from 4 to 16 carbon atoms, from 4 to 12 carbon atoms, and typically from 4 to 10 carbon atoms. Cycloalkylalkyl groups may be substituted or unsubstituted. Substituted cycloalkylalkyl groups may be substituted on the alkyl, cycloalkyl, or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups may be mono-substituted or substituted more than once, for example, but not limited to, mono-, di-, or tri-substituted with substituents such as those listed above.

Alkenyl includes straight and branched chain alkyl groups as defined above, except that at least one double bond is present between two carbon atoms. Alkenyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons, or in some embodimentsHaving 2 to 8,2 to 6, or 2 to 4 carbon atoms. In some embodiments, an alkenyl group has one, two, or three carbon-carbon double bonds. Examples include, but are not limited to, vinyl, allyl, -CH ═ CH (CH)₃)、-CH＝C(CH₃)₂、-C(CH₃)＝CH₂、-C(CH₃)＝CH(CH₃)、-C(CH₂CH₃)＝CH₂And so on. The alkenyl group may be substituted or unsubstituted. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, for example, but not limited to, mono-, di-, or tri-substituted with substituents such as those listed above.

Cycloalkenyl includes cycloalkyl groups as defined above having at least one double bond between two carbon atoms. The cycloalkenyl group may be substituted or unsubstituted. In some embodiments, cycloalkenyl groups can have one, two, or three double bonds, but do not include aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbon atoms, or in some embodiments, from 5 to 14 carbon atoms, from 5 to 10 carbon atoms, or even 5,6,7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl.

Cycloalkenylalkyl is an alkyl group as defined above wherein a hydrogen or carbon bond of the alkyl group is replaced by a bond to the cycloalkenyl group as defined above. Cycloalkenylalkyl groups may be substituted or unsubstituted. Substituted cycloalkenylalkyl groups may be substituted on the alkyl, cycloalkenyl or both the alkyl and cycloalkenyl portions of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above.

Alkynyl includes straight and branched chain alkyl groups as defined above except that at least one triple bond exists between two carbon atoms. Alkynyl groups have 2 to 12 carbon atoms, and typically 2 to 10 carbons, or in some embodiments, 2 to 8,2 to 6, or 2 to 4 carbon atoms. In some embodiments, alkynyl groups have one, two, or three carbon-carbon triple bonds. Examples include, but are not limited to, -C ≡ CH, -C ≡ CCH₃、-CH₂C≡CCH₃、-C≡CCH₂CH(CH₂CH₃)₂And the like. The alkynyl group may be substituted or unsubstituted. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, for example but not limited to mono-, di-or tri-substituted with substituents such as those listed above.

Aryl is a cyclic aromatic hydrocarbon that does not contain heteroatoms. Aryl groups herein include monocyclic, bicyclic and tricyclic ring systems. Thus, aryl groups include, but are not limited to, phenyl, azaenyl, heptenyl, biphenyl, fluorenyl, phenanthryl, anthracenyl, indenyl, indanyl, pentenyl, and naphthyl. In some embodiments, the aryl group contains 6 to 14 carbons in the ring portion of the group, and in others 6 to 12 or even 6 to 10 carbon atoms. In some embodiments, aryl is phenyl or naphthyl. The aryl group may be substituted or unsubstituted. The phrase "aryl" includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like). Representative substituted aryl groups may be mono-substituted or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.

An aralkyl group is an alkyl group as defined above, wherein a hydrogen or carbon bond of the alkyl group is replaced by a bond to an aryl group as defined above. In some embodiments, an aralkyl group contains 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. The aralkyl group may be substituted or unsubstituted. Substituted aralkyl groups may be substituted on the alkyl, aryl, or both the alkyl and aryl portions of the group. Representative aralkyl groups include, but are not limited to, benzyl and phenethyl and fused (cycloalkylaryl) alkyl groups, such as 4-indanylethyl. Representative substituted aralkyl groups may be substituted one or more times with substituents such as those listed above.

Heterocyclyl includes aromatic (also referred to as heteroaryl) and non-aromatic ring compounds containing 3 or more ring members, one or more of which is a heteroatom such as, but not limited to N, O and S. In some embodiments, heterocyclyl contains 1,2,3, or 4 heteroatoms. In some embodiments, heterocyclyl includes monocyclic, bicyclic, and tricyclic rings having 3 to 16 ring members, while other such groups have 3 to 6,3 to 10, 3 to 12, or 3 to 14 ring members. Heterocyclyl includes aromatic, partially unsaturated and saturated ring systems, such as imidazolyl, imidazolinyl and imidazolidinyl. The phrase "heterocyclyl group" includes fused ring materials, including those containing fused aromatic and non-aromatic groups, such as benzotriazolyl, 2, 3-dihydrobenzo [1,4] dioctyl and benzo [1,3] dioxolyl. The phrase also includes bridged polycyclic ring systems containing heteroatoms such as, but not limited to, quinolinyl. The heterocyclic group may be substituted or unsubstituted. Heterocyclyl groups include, but are not limited to, azido, azetidinyl, pyrrolidinyl, imidazolinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothienyl, tetrahydrofuranyl, dioxolanyl, furyl, thienyl, pyrrolyl, pyrrolidinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, xanthenyl, dioxy, dithienyl, pyranyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridinyl, dihydrodithienyl, dihydrodisulfinyl, homopiperazinyl, quinuclidinyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), Indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothienyl, benzothiazolyl, benzooxadiazole, benzoxazinyl, benzodithienyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo [1,3] dioxazolyl, pyrazolopyridyl, imidazopyridyl (azabenzoimidazolyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthyl, adenine, guanidinyl, quinolyl, isoquinolyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thienyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, indolinyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, benzoxazolyl, pyrazolopyridyl, pyrazol, Tetrahydropyrrolopyridinyl, tetrahydropyrazolopyridinyl, tetrahydroimidazopyridinyl, tetrahydrotriazolopyridinyl and tetrahydroquinolinyl. Representative substituted heterocyclyl groups may be mono-or substituted more than once, such as, but not limited to, pyridyl or morpholinyl, substituted with 2-, 3-, 4-, 5-, or 6-, or with various substituents such as those listed above.

Heteroaryl is an aromatic ring compound containing 5 or more ring members, one or more of which is a heteroatom, such as, but not limited to N, O and S. Heteroaryl groups include, but are not limited to, groups such as: pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, benzothienyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thienyl, purinyl, xanthine, adenine, guanidino, quinolyl, isoquinolyl, tetrahydroquinolyl, quinoxalyl and quinazolinyl. Heteroaryl includes fused ring compounds in which all rings are aromatic, such as indolyl, and includes fused ring compounds in which only one ring is aromatic, such as 2, 3-indolinyl. Heteroaryl groups may be substituted or unsubstituted. Thus, the phrase "heteroaryl" includes fused ring compounds as well as heteroaryl groups having other groups, such as alkyl, bonded to one ring member. Representative substituted heteroaryl groups may be substituted one or more times with various substituents such as those listed above.

Heterocyclylalkyl is an alkyl group as defined above in which a hydrogen or carbon bond of the alkyl group is replaced by a bond to a heterocyclyl group as defined above. The heterocycloalkyl group may be substituted or unsubstituted. Substituted heterocyclylalkyl groups may be substituted on the alkyl, heterocyclyl, or both the alkyl and heterocyclyl portions of the group. Representative heterocyclylalkyl groups include, but are not limited to, morpholin-4-ylethyl, furan-2-ylmethyl, imidazol-4-ylmethyl, pyridin-3-ylmethyl, tetrahydrofuran-2-yl-ethyl, and indol-2-ylpropyl. Representative substituted heterocyclylalkyl groups may be substituted one or more times with substituents such as those listed above.

Heteroaralkyl is an alkyl group as defined above in which a hydrogen or carbon bond of the alkyl group is replaced by a bond to a heteroaryl group as defined above. Heteroaralkyl groups may be substituted or unsubstituted. Substituted heteroaralkyl groups may be substituted on the alkyl, heteroaryl, or both the alkyl and heteroaryl portions of the group. Representative substituted heteroaralkyl groups may be substituted one or more times with substituents such as those listed above.

Groups described herein that have two or more points of attachment (i.e., divalent, trivalent, or multivalent) within the compounds of the present technology are designated by the use of the prefix "sub". For example, a divalent alkyl group is an alkylene group, a divalent aryl group is an arylene group, a divalent heteroaryl group is a divalent heteroarylene group, and the like. Substituents having a single point of attachment in compounds of the present technology are not referred to using the "sub" name. Thus, for example, chloroethyl is not referred to herein as chloroethylene. Such groups may be further substituted or unsubstituted.

An alkoxy group is a hydroxyl group (-OH) in which the bond to a hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy, and the like. Alkoxy groups may be substituted or unsubstituted. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.

As used herein, the terms "alkanoyl" and "alkanoyloxy" may refer to-c (O) -alkyl and-O-c (O) -alkyl, respectively, wherein, in some embodiments, alkanoyl or alkanoyloxy each contains 2-5 carbon atoms. Similarly, the terms "aroyl" and "aroyloxy" refer to the groups-C (O) -aryl and-O-C (O) -aryl, respectively.

The terms "aryloxy" and "arylalkoxy" refer to a substituted or unsubstituted aryl group bonded to an oxygen atom and a substituted or unsubstituted aralkyl group bonded to an oxygen atom at an alkyl group, respectively. Examples include, but are not limited to, phenoxy, naphthoxy, and benzyloxy. Representative substituted aryloxy and arylalkoxy groups may be substituted one or more times with substituents such as those listed above.

As used herein, the term "carboxylic acid" refers to a compound having a-C (O) OH group. As used herein, the term "carboxylate" refers to-C (O) O^–A group. "protected carboxylate" means-C (O) O-G, wherein G is a carboxylate protecting group. Carboxylate protecting groups are well known to those of ordinary skill in the art. Can be found in Protective Groups in organic synthesis, Greene, t.w.; wuts, p.g.m., John Wiley&A broad list of protecting groups for carboxylate functionality is found in Sons, New York, NY, (3rd Edition,1999), which can be added or deleted using the procedures set forth therein, incorporated herein by reference in its entirety for any and all purposes, as if fully set forth herein.

As used herein, the term "ester" refers to-COOR⁷⁰A group. R⁷⁰Is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein.

The term "amide" (or "amide group") includes C-and N-amide groups, i.e., -C (O) NR, respectively⁷¹R⁷²and-NR⁷¹C(O)R⁷²A group. R⁷¹And R⁷²Independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Thus, amide groups include, but are not limited to, carbamoyl (-C (O) NH)₂) And a carboxamide group (-NHC (O) H). In some casesIn embodiments, the amide group is-NR⁷¹C(O)-(C_1-5Alkyl), and the group is referred to as "carbonylamino", and in other embodiments, the amide group is-nhc (o) -alkyl, and the group is referred to as "alkanoylamino".

As used herein, the term "nitrile" or "cyano" refers to a-CN group.

The carbamate groups include N-and O-carbamate groups, i.e., -NR, respectively⁷³C(O)OR⁷⁴and-OC (O) NR⁷³R⁷⁴A group. R⁷³And R⁷⁴Independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group, as defined herein. R⁷³Or may be H.

As used herein, the term "amine group" (or "amino group") refers to-NR⁷⁵R⁷⁶Group, wherein R⁷⁵And R⁷⁶Independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. In some embodiments, the amino group is an alkylamino, dialkylamino, arylamino, or alkylarylamino group. In other embodiments, amino is NH₂Methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino or benzylamino.

The term "sulfonamide" includes S-and N-sulfonamide groups, i.e., -SO, respectively₂NR⁷⁸R⁷⁹and-NR⁷⁸SO₂R⁷⁹A group. R⁷⁸And R⁷⁹Independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Thus, sulfonamide groups include, but are not limited to, sulfamoyl (-SO)₂NH₂). In some embodiments herein, the sulfonamide group is-NHSO₂-alkyl, known as "alkylsulfonylamino" group.

The term "thiol" refers to the-SH group, while sulfides include-SR⁸⁰The group, sulfoxide, including-S (O) R⁸¹The group, sulfone, includes-SO₂R⁸²The sulfonyl group includes-SO₂OR⁸³。R⁸⁰、R⁸¹、R⁸²And R⁸³Each independently is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylaralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein. In some embodiments, the sulfide is alkylthio, -S-alkyl.

The term "urea" refers to-NR⁸⁴-C(O)-NR⁸⁵R⁸⁶。R⁸⁴、R⁸⁵And R⁸⁶The groups are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

The term "amidine" refers to-C (NR)⁸⁷)NR⁸⁸R⁸⁹and-NR⁸⁷C(NR⁸⁸)R⁸⁹Wherein R is⁸⁷、R⁸⁸And R⁸⁹Each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylalkyl, heterocyclyl or heterocycloalkyl group, as defined herein.

The term "guanidine" refers to-NR⁹⁰C(NR⁹¹)NR⁹²R⁹³Wherein R is⁹⁰、R⁹¹、R⁹²And R⁹³Each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylalkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

The term "enamine" refers to-C (R)⁹⁴)＝C(R⁹⁵)NR⁹⁶R⁹⁷and-NR⁹⁴C(R⁹⁵)＝C(R⁹⁶)R⁹⁷Wherein R is⁹⁴、R⁹⁵、R⁹⁶And R⁹⁷Each independently is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, arylaralkyl, heterocyclyl or heterocyclylalkyl as defined herein.

As used herein, the term "halogen" refers to bromine, chlorine, fluorine or iodine. In some embodiments, the halogen is fluorine. In other embodiments, halogen is chlorine or bromine.

As used herein, the term "hydroxy" may refer to-OH or its ionized form-O-.

The term "imide" refers to-C (O) NR⁹⁸C(O)R⁹⁹Wherein R is⁹⁸And R⁹⁹Each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylalkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

The term "imino" refers to the group-CR¹⁰⁰(NR¹⁰¹) and-N (CR)¹⁰⁰R¹⁰¹) Group, wherein R¹⁰⁰And R¹⁰¹Each independently hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylalkyl, heterocyclyl or heterocyclylalkyl group as defined herein, with the proviso that R is¹⁰⁰And R¹⁰¹Not hydrogen at the same time.

As used herein, the term "nitro" refers to-NO₂A group.

As used herein, the term "trifluoromethyl" refers to-CF₃。

As used herein, the term "trifluoromethoxy" refers to-OCF₃。

The term "azido" refers to-N₃。

The term "trialkylammonium" refers to-N (alkyl)₃A group. Trialkylammonium groups are positively charged and therefore typically have associated anions, such as halide anions.

The term "trifluoromethyl diazo" refers to

The term "isocyano" refers to — NC.

The term "isothiocyanato" refers to-NCS.

The term "pentafluorosulfanyl" refers to-SF₅。

As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily identified as fully descriptive and the same range can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein may be readily broken down into a lower third, a middle third, and an upper third, and so on. As will also be understood by those of skill in the art, all language such as "at most," "at least," "greater than," "less than," and the like includes the recited number and refers to ranges that can subsequently be broken down into the aforementioned sub-ranges. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to a group having 1,2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1,2,3, 4, or 5 atoms, and so forth.

Pharmaceutically acceptable salts of the compounds described herein are within the scope of the present technology and include acid or base addition salts that retain the desired pharmacological activity and are not biologically undesirable (e.g., the salts are not unduly toxic, allergenic, or irritating, and are bioavailable). When the compounds of the present technology have a basic group (e.g., an amino group), a pharmaceutically acceptable salt can be formed with an inorganic acid (e.g., hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), an organic acid (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid), or an acidic amino acid (e.g., aspartic acid and glutamic acid). When a compound of the present technology has an acidic group (e.g., a carboxylic acid group), it can be reacted with metals such as alkali metals and alkaline earth metals (e.g., Na)⁺、Li⁺、K⁺、Ca²⁺、Mg²⁺、Zn²⁺) Salts with ammonia or organic amines (e.g. dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or with basic amino acids (e.g. arginine, lysine and ornithine). Such salts may be prepared in situ during the isolation and purification of the compound or by reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.

One skilled in the art will appreciate that compounds of the present technology may exhibit tautomerism, conformational isomerism, geometric isomerism and/or stereoisomerism. Since the structural diagrams in the specification and claims represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it is to be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and/or geometric isomeric form of the compounds having one or more of the uses described herein, as well as mixtures of these various different forms.

"tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The presence and concentration of the isomeric forms will depend on the environment in which the compound is found and may vary depending on, for example, whether the compound is a solid or an organic or aqueous solution. For example, in aqueous solution, quinazolinones may exhibit the following isomeric forms, referred to as tautomers for each other:

as another example, guanidine can exhibit the following isomeric forms, also referred to as tautomers of each other, in protic organic solutions:

as a result of the limitations of the structural formulae representing compounds, it is understood that all chemical formulae of the compounds described herein represent all tautomeric forms of the compounds and are within the scope of the technology.

Stereoisomers (also referred to as optical isomers) of compounds include all chiral, diastereomeric and racemic forms of the structure, unless the specific stereochemistry is explicitly indicated. Thus, as is apparent from the figures, compounds useful in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms. Both racemic and diastereomeric mixtures and individual optical isomers can be separated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and such stereoisomers are within the scope of the technology.

The compounds of the present technology may exist in the form of solvates, in particular hydrates. Hydrates can form during the manufacture of the compound or composition containing the compound, or hydrates form over time due to the hygroscopic nature of the compound. The compounds of the present technology may also exist in the form of organic solvates, including DMF, ethers, and alcohol solvates. The identification and preparation of any particular solvate is within the skill of one of ordinary skill in synthetic organic or pharmaceutical chemistry.

Throughout this disclosure, various publications, patents, and published patent specifications are cited by identifying citations. Also within this disclosure are arabic numerals that cite references, all bibliographic details of which are provided prior to the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into this disclosure to more fully describe the present technology.

The present technology

In general, it is desirable that the radiotherapeutic compound accumulate to a greater extent in the tumor without being taken up unacceptably in normal organs, since the absorbed dose is a function of the cumulative integral of activity. Furthermore, although targeted radiotherapy has been carried out for some time using macrocyclic complexes of radionuclides, the complexes formed with radionuclides by currently used macrocyclic compounds (e.g. DOTA) are often insufficiently stable, especially for radionuclides of larger size (e.g. actinium, radium, bismuth and lead isotopes). These larger radionuclides are typically alpha-emitting radionuclides, i.e., radionuclides that have much higher energy, and thus are substantially more efficient, than beta-emitting radionuclides. The instability of currently known macrocyclic-containing compounds results in dissociation of the radionuclide from the macrocycle, which results in a lack of selectivity for the target tissue, which also results in toxicity to non-target tissues.

The present technology provides novel trifunctional compounds that overcome these problems, which accumulate to a greater extent, particularly in tumors, without unacceptable uptake in normal organs. The present technology also includes the use of macrocyclic complexes that are substantially more stable than conventional technologies for emitting alpha radionuclides rather than beta radionuclides. Thus, the compounds of the present technology advantageously target cancer cells more efficiently, with much lower toxicity to non-targeted tissues than the complexes of the prior art. Furthermore, in contrast to DOTA-type complexes, which typically require elevated temperatures (e.g. at least 80 ℃) to complex with radionuclides, new complexes can be advantageously produced at room temperature.

Accordingly, in one aspect of the present technology, there is provided a compound of formula I:

or a pharmaceutically acceptable salt and/or solvate thereof, wherein

ABD is an antigen binding domain;

W¹is-C (O) -, - (CH)₂)_n-, or- (CH)₂)_o–NH₂-C(O)–；

R¹、R²And R³Is one of

And R is¹、R²And R³The other two are both H;

X¹is absent, O, S or NH;

tox is a cytotoxic-and/or imaging agent-containing domain;

alb is an albumin binding moiety;

m is 0 or 1; n is 1 or 2; o is 1 or 2; p is 0,1, 2 or 3, with the proviso that X is when p is 0¹Is absent; and q is 1 or 2.

For clarity, in the compounds of the present technology, reference is made to a compound such as X¹、L¹And L²The term "absent" of a divalent group of (a) means that instead of the divalent group is a bond. For example, when L is¹When "is absent", R¹、R²And R³One of them is

The antigen binding domain includes a portion capable of recognizing or interacting with a molecular target on the surface of a cell. These molecular targets include cell surface proteins, such as receptors, enzymes, and antigens. For example, the molecular target may be a receptor, enzyme and/or antigen expressed on the surface of a tumor cell (e.g., a tumor-specific cell surface protein) that is capable of interacting with the antigen binding domain. An example of such a tumor targeting moiety is the glutamate-urea-lysine motif, which is recognized by the Prostate Specific Membrane Antigen (PSMA), expressed on the surface of most prostate cancer cells. Another example is the eletripeptide, which is recognized by somatostatin receptors expressed on the surface of many neuroendocrine cancers. Thus, the antigen binding domain of any of the embodiments herein may comprise a moiety capable of binding to one or more of PSMA, somatostatin peptide receptor 2(SSTR2), somatostatin peptide receptor 5(SSTR5), integrins (e.g., alphavbeta6, alphavbeta3 and/or alphavbeta5), gastrin-releasing peptide receptor, seprase, fibroblast activation protein alpha (FAP-alpha), incretin receptor, glucose-dependent insulinotropic polypeptide receptor, VIP-1, NPY, folate receptor, LHRH, neuronal transporters (e.g., noradrenaline transporter (NET)), EGFR, HER-2, VGFR, MUC-1, CEA, c-4, ED2, TF antigen, endothelial-specific markers, neuropeptide Y, uPAR, TAG-72, CCK analogs, VIP, VEGFR, GLP-1, CXCR4, hepsin, tmed 2, pace, and cMET.

The albumin binding moiety plays a role in modulating the rate of plasma clearance of the compound in a subject, thereby increasing circulation time and compartmentalizing the cytotoxic effect of the cytotoxic-containing domain and/or the imaging capacity of the imaging agent-containing domain in the plasma space rather than in normal organs and tissues that may express the antigen. Without being bound by theory, it is believed that this component of the structure reversibly interacts with serum proteins (e.g., albumin and/or cellular elements). The affinity of the albumin binding moiety for the plasma or cellular components of blood may be configured to affect the residence time of the compound in the blood pool of the subject. In any of the embodiments herein, the albumin binding moiety may be configured such that it binds to albumin reversibly or irreversibly when in plasma. In any of the embodiments herein, the albumin binding moiety can be selected such that the compound has a binding affinity to human serum albumin of about 5 μ Μ to about 15 μ Μ.

For example, the albumin binding moiety of any embodiment herein may comprise a short chain fatty acid, a medium chain fatty acid, a long chain fatty acid, myristic acid, a substituted or unsubstituted indole-2-carboxylic acid, a substituted or unsubstituted 4-oxo-4- (5,6,7, 8-tetrahydronaphthalen-2-yl) butanoic acid, a substituted or unsubstituted naphthylsulfonamide, a substituted or unsubstituted diphenylcyclohexanol phosphate, a substituted or unsubstituted 2- (4-iodophenyl) acetic acid, a substituted or unsubstituted 3- (4-iodophenyl) propanoic acid, or a substituted or unsubstituted 4- (4-iodophenyl) butanoic acid. Some representative examples of albumin binding moieties that may be included in any embodiment herein include one or more of the following:

and

in any of the embodiments herein, the compound may comprise an albumin binding moiety as

Wherein Y is¹、Y²、Y³、Y⁴And Y⁵Independently is H, halogen or alkyl, X³、X⁴、X⁵And X⁶Each independently is O or S, a is independently at each occurrence 0,1 or 2, b is independently at each occurrence 0 or 1, c is independently at each occurrence 0 or 1, and d is independently at each occurrence 0,1, 2,3 or 4. In any embodiment herein, it may be that b and c cannot be the same value. In any embodiment herein, may be Y³Is I, and Y¹、Y²、Y⁴And Y⁵Each of which is independently H.

As noted above, the Tox group of formula I is a cytotoxic-and/or imaging agent-containing domain, such as a cytotoxic chemical moiety, a moiety capable of containing a metal ion, a chelator, a moiety bearing a metal ion, or a combination of any two or more thereof. By way of example of a compound comprising a moiety capable of comprising a metal ion (e.g., by chelation of the metal ion and/or by covalent bond with the metal ion), in any embodiment herein, the compound of formula I may be a compound of formula II:

or a pharmaceutically acceptable salt and/or solvate thereof, wherein

W¹、X¹、L¹、L²R, s, m, n and o (and any other variables) are as provided herein for any embodiment of formula I;

P¹、P²and P³Each independently is H, methyl, benzyl, 4-methoxybenzyl, or tert-butyl;

R¹、R²and R³Is one of

And R is¹、R²And R³The other two are both H;

rad is a moiety capable of comprising a metal ion, optionally further comprising a metal ion; p is 0,1, 2 or 3, with the proviso that X is when p is 0¹Is absent; and q is 1 or 2.

In any embodiment herein, P¹、P²And P³May each independently be H.

Rad in the compound of formula II may or may not contain a metal ion, wherein the compound does not contain a metal ion. In any of the embodiments herein, Tox and/or Rad may comprise a chelating agent; in any of the embodiments herein, Tox and/or Rad may include a chelating agent that chelates metal ions. Such chelated metal ions may make the compounds of the present technology useful, for example, for magnetic resonance imaging, luminescence imaging, radiotherapy, or any two or more thereofA combination of a plurality. The metal ion for any embodiment of Tox and/or Rad herein can be a radionuclide, for example¹⁷⁷Lu³⁺、¹⁷⁵Lu³⁺、⁴⁵Sc³⁺、⁶⁶Ga³⁺、⁶⁷Ga³⁺、⁶⁸Ga³⁺、⁶⁹Ga³⁺、⁷¹Ga³⁺、⁸⁹Y³⁺、⁸⁶Y³⁺、⁸⁹Zr⁴⁺、⁹⁰Y³⁺、^99mTc⁺¹、¹¹¹In³⁺、¹¹³In³⁺、¹¹⁵In³⁺、¹³⁹La³⁺、¹³⁶Ce³⁺、¹³⁸Ce³⁺、¹⁴⁰Ce³⁺、¹⁴²Ce³⁺、¹⁵¹Eu³⁺、¹⁵³Eu³⁺、¹⁵²Dy³⁺、¹⁴⁹Tb³⁺、¹⁵⁹Tb³⁺、¹⁵⁴Gd³⁺、¹⁵⁵Gd³⁺、¹⁵⁶Gd³⁺、¹⁵⁷Gd³⁺、¹⁵⁸Gd³⁺、¹⁶⁰Gd³⁺、¹⁸⁸Re⁺¹、¹⁸⁶Re⁺¹、²¹³Bi³⁺、²¹¹At⁺、²¹⁷At⁺、²²⁷Th⁴⁺、²²⁶Th⁴⁺、²²⁵Ac³⁺、²³³Ra²⁺、¹⁵²Dy³⁺、²¹³Bi³⁺、²¹²Bi³⁺、²¹¹Bi³⁺、²¹²Pb²⁺、²¹²Pb⁴⁺、²⁵⁵Fm³⁺Or uranium 230. For example, the metal ion may be selected from²¹³Bi³⁺、²¹¹At⁺、²²⁵Ac³⁺、¹⁵²Dy³⁺、²¹²Bi³⁺、²¹¹Bi³⁺、²¹⁷At⁺、²²⁷Th⁴⁺、²²⁶Th⁴⁺、²³³Ra²⁺、²¹²Pb²⁺And²¹²Pb⁴⁺alpha emitting radionuclide.

Chelating agents that may be used in any embodiment of the present technology include, but are not limited to, the following covalently conjugated substituted or unsubstituted chelating agents:

1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA),

1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA),

p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS),

PIP-DOTA，

diethylenetriaminepentaacetic acid (DTPA),

PIP-DTPA，

AZEP-DTPA，

ethylene Diamine Tetraacetic Acid (EDTA),

triethylenetetramine-N, N, N ', N ", N'" -hexane-acetic acid (TTHA),

7- [2- (biscarboxymethylamino) -ethyl ] -4, 10-biscarboxymethyl-1, 4,7, 10-tetraaza-cyclododecan-1-yl-acetic acid (DEPA),

2,2',2 "- (10- (2- (bis (carboxymethyl) amino) -5- (4-isothiocyanatophenyl) pentyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid (3p-C-DEPA-NCS),

NETA，

{ 4-carboxymethyl-7- [2- (carboxymethylamino) -ethyl ] -perhydro-1, 4, 7-triazol-1-yl } -acetic acid (NPTA),

diacetylpyridine bis (benzoylhydrazone),

1,4,7,10,13, 16-hexaazacyclooctadecane-N, N ', N' '' '', N '' '' -hexaacetic acid (HEHA),

an octadentate terephthalic acid amide ligand is prepared,

an iron carrier, wherein the iron carrier is a ferrite,

2,2' - (4- (2- (bis (carboxymethyl) amino) -5- (4-isothiocyanatophenyl) pentyl) -10- (2- (bis (carboxymethyl) amino) ethyl) -1,4,7, 10-tetraazacyclododecane-1, 7-diyl) diacetic acid,

n, N' -bis [ (6-carboxy-2-pyridyl) methyl group]-4, 13-diaza-18-crown-6 (H)₂macropa)，

6- ((16- ((6-carboxypyridin-2-yl) methyl) -1,4,10, 13-tetraoxa-7, 16-diazooctadecan-7-yl) methyl) -4-isothiocyanato-phthalic acid (macropa-NCS), and

3, 9-carboxymethyl-6- (2-methoxy-5-isothiocyanatophenyl) carboxymethyl-3, 6,9, 15-tetraazabicyclo- [9.3.1] pentadecan-1- (15),11, 13-triene.

Some members of this exemplary group are shown below.

It is to be understood that "covalently conjugated" chelating agents refer to chelating agents (such as those listed above) wherein one or more bonds to a hydrogen atom contained therein are bonded to an atom of the remainder of the TOX and/or RAD moiety, to L¹Bonded to and/or with L²Bonded bonds, or a pi bond between two atoms is bonded by one of the two atoms to an atom of the remainder of the TOX and/or RAD moiety, to L¹Bonded to and/or with L²The bonded bonds are substituted and the other of the two atoms includes a new bond, such as a hydrogen bond (e.g., reaction of a-NCS group in a chelator provides a covalently conjugated chelator).

In any of the embodiments herein, the Tox and/or Rad groups comprising the covalently conjugated chelating agent may be represented by the formula:

wherein L is³Is absent, -C (O) -, -C₁-C₁₂Alkylene-, -C₁-C₁₂alkylene-C (O) -, -C₁-C₁₂alkylene-NR¹⁰-, or-arylene-; r¹⁰Is H, alkyl or aryl, and CHEL is a covalently conjugated chelator that may or may not include the chelated metal of any of the embodiments described hereinIons. For example, a compound of formula I having such a Tox group or a compound of formula II having such a Rad group may be a compound wherein R is¹、R²And R³Is one of

And R is¹、R²And R³The other two are both H; l is³Is absent, -C (O) -, -C₁-C₁₂Alkylene-, -C₁-C₁₂alkylene-C (O) -, -C₁-C₁₂alkylene-NR¹⁰-, or-arylene-; r¹⁰Is H, alkyl or aryl; and CHEL is a covalently conjugated chelator, optionally including chelated metal ions.

As another example, a compound of formula II having such a Rad group may be a compound of formula III

Or a pharmaceutically acceptable salt and/or solvate thereof, wherein

W¹、X¹、L¹、L²、P¹、P²、P³R, s, m, n, o, p, q (and any other variables) are as provided herein for any embodiment of formulas I and II;

R¹、R²and R³Is one of

In any of the embodiments herein, L is¹May be-O (CH)₂CH₂O)_r–CH₂CH₂C (o) -, an amino acid, a peptide of 2,3, 4,5, 6,7,8, 9, or 10 amino acids, or a combination of any two or more thereof. In any of the embodiments herein, L is¹May be-O (CH)₂CH₂O)_r–CH₂CH₂C (o) -, glycine, polyglycine consisting of 2,3, 4,5, 6,7,8, 9, or 10 glycine residues, or a combination of any two or more thereof.

In any of the embodiments herein, L is²May be-C (O) -, - (CH)₂CH₂O)_s–CH₂CH₂C (o) -, 2,3, 4,5, 6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 amino acid peptide, or a combination of any two or more thereof. In any of the embodiments herein, L is²May be-C (O) -, - (CH)₂CH₂O)_s–CH₂CH₂C (o) -, polyglycine consisting of 2,3, 4,5, 6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 amino acids, or a combination of any two or more thereof.

The present technology also provides compositions and medicaments comprising any of the embodiments of the compounds of formulae I, II and III (or pharmaceutically acceptable salts thereof) disclosed herein and a pharmaceutically acceptable carrier or one or more excipients or fillers (collectively referred to as "pharmaceutically acceptable carriers" unless otherwise specified). The compositions may be used in the methods and treatments described herein. The present technology also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a compound of any one of the aspects and embodiments of the compounds of formulae I-III for imaging and/or treating a disorder; and the disorder can include glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and/or prostate cancer. For example, such disorders can include PSMA-overexpressing mammalian tissue, such as PSMA-expressing cancer (including cancer tissue, neovasculature associated with cancer, or a combination thereof), crohn's disease or IBD.

In another related aspect, there is provided a method of imaging comprising administering to a subject a compound of any of the aspects and embodiments of compounds of formulae I-III (e.g., administering an effective amount) or a pharmaceutical composition comprising an effective amount of a compound of any of the aspects and embodiments of compounds of formulae I-III, and detecting positron emission, detecting gamma rays from the positron emission and annihilation (e.g., by positron emission tomography), and/or detecting cerenkov radiation due to the positron emission (e.g., by cerenkov luminescence imaging). In any embodiment of the imaging method, the subject may be suspected of having a disorder comprising: glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer, PSMA-overexpressing mammalian tissue (e.g., PSMA-expressing cancer (including cancer tissue, neovasculature associated with cancer, or a combination thereof)), crohn's disease, or IBD. This detection step may be performed during surgery of the subject, for example, to remove mammalian tissue that overexpresses PSMA. The detecting step may include performing the detecting step using a handheld device. For example, the cerenkov luminescence image may be acquired by detecting cerenkov light using an ultra-high sensitivity optical camera such as an Electron Multiplying Charge Coupled Device (EMCCD) camera.

In any of the above embodiments, the effective amount may be determined relative to the subject. An "effective amount" refers to the amount of a compound or composition required to produce the desired effect. One non-limiting example of an effective amount includes an amount or dose that results in an acceptable level of toxicity and bioavailability for therapeutic use, including but not limited to treatment of, for example, glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer. Another example of an effective amount includes an amount or dose that is capable of alleviating the symptoms associated with: such as glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer, for example, to reduce proliferation and/or metastasis. An effective amount of a compound of the present technology can include an amount sufficient to detect binding of the compound to a target of interest, including, but not limited to, glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer (e.g., castration-resistant prostate cancer). Another example of an effective amount includes an amount or dose capable of providing a detectable gamma ray emission (above background) from positron emission and annihilation (e.g., a statistically significant emission above background) in a subject with PSMA-overexpressing tissue. Another example of an effective amount includes an amount or dose capable of providing a detectable positron emission-induced cerenkov radiation emission (above background) (e.g., a statistically significant emission above background) in a subject having tissue overexpressing PSMA. An effective amount may be from about 0.01 μ g to about 1mg of the compound per gram of the composition, preferably from about 0.1 μ g to about 500 μ g of the compound per gram of the composition.

As used herein, a "subject" or "patient" is a mammal, e.g., a cat, dog, rodent, or primate. Typically, the subject is a human, and preferably a human having or suspected of having glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, or prostate cancer. The terms "subject" and "patient" are used interchangeably.

In particular, an effective amount of a compound of any of the embodiments herein for treating cancer and/or PSMA-overexpressing mammalian tissue can be about 0.1 μ g to about 50 μ g per kilogram of the subject's mass. Thus, for the treatment of cancer (e.g., glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer, and/or castration-resistant prostate cancer) and/or mammalian tissue overexpressing PSMA, an effective amount of a compound of any of the embodiments described herein can be about 0.1 μ g/kg, about 0.2 μ g/kg, about 0.3 μ g/kg, about 0.4 μ g/kg, about 0.5 μ g/kg, about 0.6 μ g/kg, about 0.7 μ g/kg, about 0.8 μ g/kg, about 0.9 μ g/kg, about 1 μ g/kg, about 2 μ g/kg, about 3. mu.g/kg, about 4. mu.g/kg, about 5. mu.g/kg, about 6. mu.g/kg, about 7. mu.g/kg, about 8. mu.g/kg, about 9. mu.g/kg, about 10. mu.g/kg, about 11. mu.g/kg, about 12. mu.g/kg, about 13. mu.g/kg, about 14. mu.g/kg, about 15. mu.g/kg, about 16. mu.g/kg, about 17. mu.g/kg, about 18. mu.g/kg, about 19. mu.g/kg, about 20. mu.g/kg, about 22. mu.g/kg, about 24. mu.g/kg, about 26. mu.g/kg, about 28. mu.g/kg, about 30. mu.g/kg, about 32. mu.g/kg, about 34. mu.g/kg, about 36. mu.g/kg, about 38. mu.g/kg, about 40. mu.g/kg, about 42, About 44 μ g/kg, about 46 μ g/kg, about 48 μ g/kg, about 50 μ g/kg, or any range encompassing and/or between any two of these values.

In particular, an effective amount of a compound of any of the embodiments herein for imaging cancer and/or PSMA-overexpressing mammalian tissue can be about 0.1 μ g to about 50 μ g per kilogram mass of the subject. Thus, for the treatment/therapy of cancer (e.g., glioma, breast cancer, adrenocortical cancer, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer, and/or castration-resistant prostate cancer) and/or mammalian tissue overexpressing PSMA, an effective amount of a compound of any of the embodiments described herein can be about 0.1 μ g/kg, about 0.2 μ g/kg, about 0.3 μ g/kg, about 0.4 μ g/kg, about 0.5 μ g/kg, about 0.6 μ g/kg, about 0.7 μ g/kg, about 0.8 μ g/kg, about 0.9 μ g/kg, about 1 μ g/kg, about 2 μ g/kg, about 0.6 μ g/kg, about 0.7 μ g/kg, about 0.8 μ g/kg, about 0.9 μ g/kg, about 1 μ g/, About 3. mu.g/kg, about 4. mu.g/kg, about 5. mu.g/kg, about 6. mu.g/kg, about 7. mu.g/kg, about 8. mu.g/kg, about 9. mu.g/kg, about 10. mu.g/kg, about 11. mu.g/kg, about 12. mu.g/kg, about 13. mu.g/kg, about 14. mu.g/kg, about 15. mu.g/kg, about 16. mu.g/kg, about 17. mu.g/kg, about 18. mu.g/kg, about 19. mu.g/kg, about 20. mu.g/kg, about 22. mu.g/kg, about 24. mu.g/kg, about 26. mu.g/kg, about 28. mu.g/kg, about 30. mu.g/kg, about 32. mu.g/kg, about 34. mu.g/kg, about 36. mu.g/kg, about 38. mu.g/kg, about 40. mu.g/kg, about 42, About 44 μ g/kg, about 46 μ g/kg, about 48 μ g/kg, about 50 μ g/kg, or any range encompassing and/or between any two of these values.

The compounds of the technology can also be administered to a patient with other conventional imaging agents that can be used for imaging and/or treatment of glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer, or mammalian tissue that overexpresses PSMA. Such mammalian tissues include, but are not limited to PSMA-expressing cancers (including cancer tissue, neovasculature associated with cancer, or combinations thereof), crohn's disease or IBD. Accordingly, the pharmaceutical compositions and/or methods of the present technology can further include an imaging agent other than a compound of formulas I-III; the pharmaceutical compositions and/or methods of the present technology can include therapeutic agents other than compounds of formulas I-III; the pharmaceutical compositions and/or methods of the present technology may further comprise an imaging agent according to any embodiment of the compounds of formulas I-III and a therapeutic agent also according to any embodiment of the compounds of formulas I-III. The compound, which may be according to any embodiment of the compounds of formulae I, II and/or III, is both a therapeutic agent and an imaging agent. Administration may include oral, parenteral or nasal administration. In any of these embodiments, administration may include subcutaneous injection, intravenous injection, intraperitoneal injection, or intramuscular injection. In any of these embodiments, administering may comprise oral administration. The methods of the present technology may further comprise administering, sequentially or in combination with one or more compounds of the present technology, a conventional imaging agent in an amount that is potentially or synergistically effective for imaging PSMA-overexpressing mammalian tissue.

In any of the embodiments of the present technology described herein, the pharmaceutical composition can be packaged as a unit dosage form. The unit dosage form is effective in treating glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and/or prostate cancer. In general, the unit dosage of a compound comprising the present technology will vary depending upon patient considerations. Such considerations include, for example, age, regimen, condition, sex, extent of disease, contraindications, concomitant therapy, and the like. Exemplary unit dosages based on these considerations may also be adjusted or modified by those skilled in the art. For example, a unit dose for a patient comprising a compound of the present technology may be in the range of 1X 10^–4Varying between g/kg and 1g/kg, preferably between 1X 10^–3Varying between g/kg and 1.0 g/kg. The dosage of the compounds of the technology can also vary from 0.01mg/kg to 100mg/kg, or preferably from 0.1mg/kg to 10 mg/kg. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injections, implantable sustained release formulations, mucosal membranes (rnucoadherentfilm), topical varnishes, lipid complexes, and the like.

Pharmaceutical compositions can be prepared by mixing one or more compounds of formulae I, II and III, pharmaceutically acceptable salts thereof, stereoisomers thereof, tautomers thereof or solvates thereof, with pharmaceutically acceptable carriers, excipients, binders, diluents, and the like, to prepare pharmaceutical compositions for the prevention and treatment of cancer-related diseases (e.g., glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer). The compounds and compositions described herein are useful for the preparation of formulations and medicaments for the treatment of, for example, glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer. Such compositions may be in the form of, for example, granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions or solutions. The compositions of the present invention may be formulated for various routes of administration, for example, by oral, parenteral, topical, rectal, nasal, vaginal administration, or by implanted depot. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular injection. The following formulations are given as examples and should not be construed as limiting the technology of the present invention.

For oral, buccal and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, soft capsules and caplets can be accepted as solid dosage forms. These may be prepared, for example, by mixing one or more compounds of the present technology, or pharmaceutically acceptable salts or tautomers thereof, with at least one additive (e.g., starch or other additive). Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, acacia gum, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, the oral dosage form may contain other ingredients to aid in administration, such as inert diluents, or lubricating agents (e.g., magnesium stearate), or preserving agents (e.g., parabens or sorbic acid), or antioxidants (e.g., ascorbic acid, tocopherol or cysteine), disintegrating agents, binding agents, thickening agents, buffering agents, sweetening agents, flavoring agents, or perfuming agents. The tablets and pills may be further treated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions and solutions, which may contain an inactive diluent, for example water. The pharmaceutical formulations and medicaments may be prepared as liquid suspensions or solutions using sterile liquids such as, but not limited to, oils, water, alcohols, and mixtures thereof. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents may be added for oral or parenteral administration.

As mentioned above, the suspension may comprise an oil. These oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. Suspension formulations may also contain esters of fatty acids, such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols such as, but not limited to, ethanol, isopropanol, cetyl alcohol, glycerol, and propylene glycol. Ethers (such as, but not limited to, polyethylene glycol), petroleum hydrocarbons (such as mineral oil and petrolatum), and water may also be used in the suspension formulation.

Injectable dosage forms typically comprise aqueous or oily suspensions, which may be prepared using suitable dispersing or wetting agents and suspending agents. Injectable forms may be in the form of a solution phase or a suspension prepared in a solvent or diluent. Acceptable solvents or excipients include sterile water, ringer's solution or isotonic saline solution. Alternatively, sterile oils may be employed as a solvent or suspending agent. Typically, the oil or fatty acid is non-volatile and includes natural or synthetic oils, fatty acids, mono-, di-or triglycerides.

For injection, the pharmaceutical formulation and/or medicament may be a powder suitable for reconstitution with a suitable solution as described above. Examples of these include, but are not limited to, freeze-dried, spin-dried or spray-dried powders, amorphous powders, granules, precipitates or microparticles. For injections, the formulations may optionally include stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations thereof.

The compounds of the present technology can be administered to the lung by nasal or oral inhalation. Pharmaceutical formulations suitable for inhalation include solutions, sprays, dry powders or aerosols containing any suitable solvent and optionally other compounds such as, but not limited to, stabilizers, antimicrobials, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations thereof. The carrier and stabilizer will vary with the requirements of a particular compound, but will typically include a non-ionic surfactant (tween, pluronic or polyethylene glycol), a non-toxic protein (such as serum albumin), a sorbitol ester, oleic acid, lecithin, an amino acid (such as glycine), a buffer, a salt, a sugar or a sugar alcohol. Aqueous and non-aqueous (e.g. in fluorocarbon propellants) aerosols are commonly used to deliver the compounds of the present technology by inhalation.

Pharmaceutically acceptable excipients and carriers, in addition to those representative dosage forms described above, are generally known to those skilled in the art and are therefore included in the present technology. Such excipients and carriers are described, for example, in "remington pharmaceutical Sciences" Mack pub. co., New Jersey (1991), which is incorporated herein by reference. The compositions of the invention may also include, for example, micelles or liposomes, or some other encapsulated form.

The specific dose can be adjusted according to disease conditions, age, body weight, general health, sex, and diet, dosage interval, administration route, excretion rate, and drug combination of the subject. It is within the skill of the art to include an effective amount of any of the above dosage forms in a range that is routine experimentation.

In accordance with the present techniques, various assay and model systems can be readily used to determine the therapeutic effect of a treatment.

For a given condition, the test subject will reduce the reduction of one or more symptoms caused by or associated with the subject's disease by 10%, 20%, 30%, 50% or more, up to 75-90% or 95% or more, as compared to placebo treatment or other suitable control subjects.

The present technology further provides a method of achieving in vivo tissue distribution of a radiotherapeutic agent in a mammalian subject, wherein a ratio of tumor activity to renal activity of 1 or greater is observed within about 4 hours to about 24 hours of administration of the radiotherapeutic agent to the mammalian subject. Such methods comprise administering a radiotherapeutic agent to a mammalian subject, wherein the radiotherapeutic agent comprises a first moiety that targets prostate specific membrane antigen ("PSMA"), a second moiety with a radionuclide, and a third moiety that has affinity for serum albumin, the first moiety being separated from the second moiety by a first covalent linker and the third moiety being separated from the second moiety by a second covalent linker. The spacing between the first and second moieties (based on the number of consecutive atoms associated with the first covalent linker) is from about 8 atoms to about 40 atoms, and the spacing between the third moieties and the first and second moieties (based on the number of consecutive atoms associated with the second covalent linker) is from about 10 atoms to about 100 atoms.

The method may include obtaining an image of the mammalian subject from about 4 hours to about 24 hours after administration of the radiotherapeutic agent; thus, obtaining an image after administration of the radiotherapeutic agent may occur after about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 4 hours later, 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, or any range including and/or between these two values. A ratio of tumor activity to renal activity equal to or greater than 1 may last for about 24 hours after radiation therapy. In any of the embodiments of the methods herein, there may be no substantial observed radionuclide activity in the salivary glands of the mammalian subject from about 24 hours to about 48 hours after administration of the radiotherapeutic agent. In any of the embodiments of the methods herein, there may be from about 10 atoms to about 30 atoms in consecutive atoms associated with the first covalent linker. In any of the embodiments herein of the method, there may be from about 15 atoms to about 40 atoms in consecutive atoms associated with the second covalent linker. In any of the embodiments of the methods herein, it may be that said administering comprises intravenous administration.

The examples herein are provided to illustrate the advantages of the present technology and to further assist those of ordinary skill in the art in making and using the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs or tautomeric forms thereof. In order to more fully illustrate preferred aspects of the present technology, examples herein are also presented. These examples should in no way be construed as limiting the scope of the present technology as defined by the appended claims. These examples may include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may further each include or incorporate variations of any or all of the other variations, aspects, or embodiments of the present technology.

Examples

Section 1.1

Materials and instruments. Unless otherwise indicated, all solvents and reagents were purchased from commercial sources and used directly without further purification. Is stored inAfter sieving, a "dry" solvent is obtained. The reaction was monitored by thin layer chromatography (TLC, Whatman UV254 aluminium supported silica gel). An HPLC system for analysis and purification of compounds consists of a CBM-20A communication bus module, LC-20AP (preparative) pump, and SPD-20AV UV/Vis detector (Shimadzu, Japan) monitored at 270 nm. Unless otherwise stated, usePurification was carried out at a flow rate of 14mL/min on a 10 μm,25cm by 20mm Epic Polar preparative column (ES Industries, West Berlin, NJ). Using a catalyst comprising H₂Gradient H of binary mobile phase of O (A) and MeOH (B) or ACN (C)A PLC method. HPLC method A comprises 10% B (0-5 min), 10-100% B (5-25 min). The method B comprises 10% C (0-5 min), 10-100% C (5-25 min). Method C comprises 10% C (0-5 min), 10-100% C (5-40 min). Method D comprises 10% C (0-5 min), 10-100% C (5-20 min). The solvent system contained 0.2% trifluoroacetic acid (TFA). NMR spectra were recorded at ambient temperature on a Varian Inova 300MHz, 400MHz, 500MHz or 600MHz spectrometer or on a Bruker AV III HD 500MHz spectrometer equipped with a broadband Prodigy cryoprobe. Chemical shifts are reported in ppm.¹H and¹³c NMR spectra were referenced to TMS internal standard (0ppm), residual solvent peak, or acetonitrile internal standard (D)₂2.06ppm in the O spectrum).¹⁹The F NMR spectrum was referenced to a fluorobenzene internal standard (-113.15 ppm). Of reports¹The split of the proton resonance in the H spectrum is defined as: s is singlet, d is doublet, t is triplet, q is quartet, m is multiplet, dt is doublet of triplets, td is triplet of doublets, br is broad. IR spectroscopy of KBr particles of samples was performed using Nicolet Avatar 370DTGS (ThermoFisher Scientific, Waltham, Mass.). High Resolution Mass Spectra (HRMS) were recorded in positive ESI mode (ThermoFisher Scientific, Waltham, MA) on an exact Orbitrap mass spectrometer. Unless otherwise stated, UV/visible spectra were recorded on Cary 8454UV-Vis (Agilent Technologies, Santa Clara, Calif.) using a 1cm quartz cuvette. Elemental Analysis (EA) was performed by Atlantic Microlab, Inc (Norcross, GA).

((1- (tert-butoxy) -6- (3- (3-ethynylphenyl) ureido) -1-oxohex-2-yl) carbamoyl) glutam Preparation of Di-tert-butyl ester (5) acid

(S) -di-tert-butyl 2- [ (imidazole-1-carbonyl) amino ] glutarate (2): to a suspension of glutamic acid L-di-tert-butyl ester hydrochloride (15.0g, 51mmol) in DCM (150mL) cooled to 0 deg.C was added TEA (18mL) and DMAP (250 mg). After the mixture was stirred for 5 minutes, CDI (9.0g, 56mmol) was added, and the mixture was stirred overnight while being heated to room temperature. The mixture was diluted with DCM (150mL) and washed with saturated sodium bicarbonate (60mL), water (2X 100mL) and brine (100 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product as a semi-solid, which slowly solidified upon standing. The crude material was triturated with hexane/ethyl acetate to give a white solid, which was filtered, washed with hexane (100mL) and dried to give 2 as a white solid (15.9g, 45mmol, 88%).

(S) -2- [3((S) - (5-benzyloxycarbonylamino) -1-tert-butoxycarbonylpentylureido)]Di-tert-butyl glutarate (3): to a solution of 2(1g, 2.82mmol) in DCE (10mL) at 0 deg.C was added MeOTf (0.47g, 2.85mmol) and TEA (0.57g, 5.65 mmol). After stirring the solution for 30 min, Cbz-L-Lys-Ot-Bu (1.06g, 2.82mmol) was added in one portion and stirred at 40 ℃ for 1 h. The mixture was concentrated to dryness and purified by column chromatography (SiO)₂) Purification yielded 3 as a white solid (1.37g, 79%).

Di-tert-butyl 2- [3- (5-amino-1-tert-butoxycarbonylpentyl) ureido ] glutarate (4): to a solution of 3(630mg, 1.0mmol) in ethanol (20mL) was added ammonium formate (630mg, 10eq) under hydrogen atmosphere followed by 10% Pd-C. The suspension was stirred overnight from time to time until complete. The mixture was filtered through celite and concentrated to give the desired product (479mg, 98%) as a waxy solid.

Di-tert-butyl ((1- (tert-butoxy) -6- (3- (3-ethynylphenyl) ureido) -1-oxohex-2-yl) carbamoyl) glutamate (5): in N₂To a solution of 4(0.488, 1mmol) in DCM (10mL) was added 1-ethynyl-3-isocyanatobenzene (185mg, 1.3mmol) in DCM (5mL) at room temperature. The resulting reaction mixture was stirred at the same temperature for 12 hours and transferred to a separatory funnel and washed with water (2X 50mL) and brine (30 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product as a semi-solid which was purified by column chromatography (SiO)₂) Purification yielded 5 as a white foam (84%).

N2- (N2- (((9H-fluoren-9-yl) methoxy) carbonyl) glycylglycinyl glycyl-N6- (tert-butoxycarbonyl) Preparation of lysyl) -N6- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (10)

N2- (((9H-fluoren-9-yl) methoxy) carbonyl) -N6- (tert-butoxycarbonyl) -L-lysine 2, 5-dioxapyrrolidin-1-ester (7): n2- (((9H-fluoren-9-yl) methoxy) carbonyl) -N6- (tert-butoxycarbonyl) -L-lysine 6(4.68g, 10mmol) was dissolved in dry DCM (20mL) and DIPEA (1.74mL, 10mmol) was added. The reaction mixture was stirred at room temperature for 10 minutes and solid bis (N-succinimidyl) carbonate (3.84g, 15mmol) was added in one portion. The resulting reaction mixture was stirred for 3-4 hours and diluted with DCM, transferred to a separatory funnel and washed with excess water. Collecting the organic layer, using MgSO₄Drying and evaporating to dryness to obtain semi-solid. The crude product was recrystallized from ethanol and diethyl ether to give 7 as a milky white solid (3.44g, 61%).

N2- (N2- (((9H-fluoren-9-yl) oxy) carbonyl) -N6- (tert-butoxycarbonyl) -L-lysyl) -N6- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (8): to a suspension of H-Lys (Z) -OtBu HCl (3.72g, 10mmol) in DCM (25mL) at 0 deg.C was added DIPEA (1.74mL, 10mmol) and then a solution of compound 7(5.65g, 10mmol) in DCM (20mL) was added dropwise. The resulting clear solution was stirred at room temperature overnight. The solvent was evaporated and the crude compound was purified by column chromatography (SiO)₂) Purification yielded 8 as a white solid (76%).

N6- ((benzyloxy) carbonyl) -N2- (N6- (tert-butoxycarbonyl) -L-lysyl) -L-lysine tert-butyl ester (9) to a solution of compound 8(1.156g, 2mmol) in DCM was added diethylamine (6mL) dropwise and the resulting reaction mixture was stirred at room temperature for 4-5 hours. The solvent was evaporated under reduced pressure and the crude product was redissolved in DCM and washed with water (2 × 100mL) and brine (100 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product as a semi-solid, which was used as such without any further purification.

N2- (N2- (((9H-fluoren-9-yl) methoxy) carbonyl) glycylglycylglycinyl glycyl-N6- (tert-butoxycarbonyl) -L-lysyl) -N6- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (10): in N₂Next (((9H-fluoren-9-yl) methoxy) carbonyl) glycylglycine (246mg, 0)6mmol) and HATU (230mg, 0.6mmol) was added to dry DMF and stirred at room temperature for 5 min. DIPEA (0.12mL, 0.7mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 min. A solution of compound 9(282mg, 0.5mmol) in DMF was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (10mL), transferred to a separatory funnel and washed with water (2X 20mL) and brine (15 mL). Collecting the organic layer, using MgSO₄Drying and evaporating to dryness to obtain semi-solid. By column chromatography (SiO)₂) The crude compound was purified to afford the desired product 10 as a brown solid (41%).

N2- (N2- (2-azidoacetyl) glycylglycinyl glycyl-N6- (tert-butoxycarbonyl) lysyl) -N6- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (12)

N6- ((benzyloxy) carbonyl) -N2- (N6- (tert-butoxycarbonyl) -N2-glycylglycosyl-L-lysyl) -L-lysine tert-butyl ester (11): to a solution of compound 10(0.478g, 0.5mmol) in DCM (10mL) was added diethylamine (2mL) dropwise and the resulting reaction mixture was stirred at rt for 3 h. The solvent was evaporated under reduced pressure and redissolved in DCM and washed with water (2 × 20mL) and brine (20 mL). The organic layer was dried over sodium sulfate and concentrated to give crude product 11 as a semi-solid, which was used without further purification.

N2- (N2- (2-azidoacetyl) glycylglycylglycinyl-N6- (tert-butoxycarbonyl) -L-lysyl) -N6- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (12): in N₂To a solid mixture of azidoacetic acid (101mg, 1mmol) and HATU (383mg, 1mmol) was added dry DMF (5mL) and the mixture was stirred at room temperature for 5 min. DIPEA (0.17mL, 1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 min. A solution of compound 11(367mg, 0.5mmol) in DMF (5mL) was added dropwise at room temperature and stirred at the same temperature for 12 hours. Evaporating DMF under reduced pressure to obtain a suspension, andit was dissolved in DCM (10mL) and washed with water (2X 20mL) and brine (15 mL). The crude compound was used without further purification.

10- (6-Alkanamido-1- (tert-butoxy) -1-oxohex-2-yl) -24,28, 30-tri-tert-butyl-2, 2-dimethyl Yl-4, 12,21, 26-tetraoxy-3-oxa-5, 11,20,25, 27-pentaazatriacontane-10, 24,28, 30-tetracarboxylate (14) Preparation of

((S) -1- (tert-butoxy) -6- (3- (3- (1- ((9S,12S) -9- (tert-butoxycarbonyl) -12- (4- ((tert-butoxycarbonyl) amino) butyl) -3,11,14,17,20, 23-hexyloxy-1-phenyl-2-oxa-4, 10,13,16,19, 22-hexaazalign-24-yl) -1H-1,2, 3-triazol-5-yl) phenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamic acid di-tert-butyl ester (13): compound 12(140mg, 0.1mmol) and compound 5(63mg, 0.1mmol) were dissolved in DMF (2mL) followed by the addition of 0.5M CuSO₄And 0.5M sodium ascorbate in water. The resulting reaction was stirred at room temperature for 3 hours. DMF was evaporated and used without further purification crude compound 13.

10- (6-alkanoylamino-1- (tert-butoxy) -1-oxohex-2-yl) -24,28, 30-tri-tert-butyl-2, 2-dimethyl-4, 12,21, 26-tetraoxy-3-oxa-5, 11,20,25, 27-pentaazatriacontane-10, 24,28, 30-tetracarboxylate (14): compound 13(144mg, 0.1mmol) was dissolved in a mixture of methanol: THF (1:1, 10mL) and 10% Pd-C was added. The resulting suspension is taken up in H₂Stirring was carried out under an atmosphere (balloon pressure) for 3 hours. The mixture was filtered through celite and concentrated to give the corresponding amine (not shown) as a semi-solid, which was used immediately in the next step. In N₂Next, dry DMF (3mL) was added to a solid mixture of the acid RCOOH (0.1mmol) and HATU (38mg, 0.1mmol), and the mixture was stirred at room temperature for 5 minutes. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. A solution of amine (0.1mmol) in DMF (2mL) was added dropwise at room temperature and stirred at the same temperature for 12 h. DMF was evaporated under reduced pressure to give a suspension which was dissolved in DCM (5mL) and washed with water (2X 10mL) andbrine (10mL) wash. The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purify and isolate product 14 as a semi-solid.

15 preparation of

To compound 14(1 eq; R ═ 4-iodophenyl) CH in dioxane (2mL) was added₂-) to a solution in dioxane (2mL) was added 4M HCl. The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The amine hydrochloride formed was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly subjected to LCMS purification using 0.1% formic acid in ACN and water. The product was collected and lyophilized.¹H NMR(500MHz,DMSO-d₆):δ12.25(bs,7H),9.40(bs,1H),8.64-8.62(m,1H,N-H),8.54-8.52(m,1H,N-H),8.42(s,1H),8.34-8.31(m,1H,N-H),8.15-8.11(m,3H),8.04-8.02(m,1H,N-H),7.94-7.91(m,2H,N-H),7.64-7.63(m,3H),7.37-7.31(m,5H),7.28-7.25(m,1H),7.17-7.16(m,2H),7.05-7.04(m,3H),6.34-6.30(m,2H),6.17(bs,1H),5.21(s,2H),4.34-4.30(m,1H),4.13-4.04(m,4H),3.84-3.83(m,2H),3.75-3.74(m,4H),3.63-3.60(m,3H),3.16-3.13(m,4H),3.12-3.05(m,4H),3.02-2.98(m,6H),2.30-2.18(m,3H),1.95-1.88(m,1H),1.71-1.65(m,5H),1.58-1.49(m,6H),1.45-1.22(m,12H).¹³C NMR(500MHz,DMSO-d₆) Delta 174.3,174.0,173.5,173.2,171.4,169.3,168.9,168.7,168.2,165.6,157.1,154.9,146.1,140.9,136.7,136.1,131.2,130.8,129.0,122.6,118.4,117.8,116.9,116.0,113.9,53.4,52.1,51.9,51.6,51.4,41.9,41.8,41.6,41.5,38.2,31.8,31.7,30.3,29.7,29.3,28.5,28.0,27.3,22.7,22.5,22.4,17.9,16.5,12.3 pairs C₇₃H₁₀₂IN₁₉O₂₄S([M+2H]⁺) Calculated HRMS,1787.6110, found 1787.6048.

2- [3- (5-amino-1-tert-butoxycarbonylpentyl) ureido]Preparation of di-tert-butyl glutarate (17)

1- (tert-butyl) (((S) -1, 5-di-tert-butoxy-1, 5-dioxolan-2-yl) carbamoyl) -L-glutamic acid 5-benzyl ester (16): to a solution of 2(1g, 2.82mmol) in DCE (10mL) at 0 deg.C was added MeOTf (0.47g, 2.85mmol) and TEA (0.57g, 5.65 mmol). After stirring the solution for 30 minutes, H-L-Glu (Bzl) -OtBu hydrochloride (0.927g, 2.82mmol) was added in one portion and stirred at 40 ℃ for 1H. The mixture was concentrated to dryness and purified by column chromatography (SiO)₂) Purification gave the desired product as a white solid (79%).

(S) -5- (tert-butoxy) -4- (3- ((S) -1, 5-di-tert-butoxy-1, 5-dioxolan-2-yl) ureido) -5-oxopentanoic acid (17): ammonium formate (630mg, 10eqv) was added to a solution of 16 in ethanol (20mL) under a hydrogen atmosphere, followed by 10% Pd-C, and the suspension was allowed to stand stirring overnight from time to time until complete. The mixture was filtered through celite and concentrated to give 17 as the desired product as a waxy solid (479mg, 98%).

Preparation of 2, 5-dioxopyrrolidin-1-yl 8- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) octanoic acid (19)

8- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) octanoic acid 18(1.43g, 3mmol) was dissolved in anhydrous DCM (20mL) and DIPEA (0.522mL, 3mmol) was added. The reaction mixture was stirred at room temperature for 10 minutes and solid bis (N-succinimidyl) carbonate (1.152g, 4.5mmol) was added in one portion. The resulting reaction mixture was stirred for 3 hours, diluted with DCM, and transferred to a separatory funnel and washed with excess water. Collecting the organic layer, using MgSO₄Drying and evaporation to dryness gave a semi-solid which was recrystallized from ethanol and ether to give the desired product as an off-white solid (0.932g, 65.08%).

(3S,7S,21S24S) -28-amino-21- (4- ((tert-butoxycarbonyl) amino) butyl) -5,10,19, 22-tetrakis Preparation of oxy-4, 6,11,20, 23-pentaazaoctacosane-1, 3,7, 24-tetracarboxylic acid tetra-tert-butyl ester (23)

N2- (N2- (8- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) octanoyl) -N6- (tert-butoxycarbonyl) -L-lysyl) -N6- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (20): compound 9(0.551g, 1mmol) and compound 19(0.573, 1.2mmol) were dissolved in DCM (10mL) and stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. The mixture was concentrated to dryness and purified by column chromatography (SiO)₂) Purification gave the desired product 20 as a brown solid (41%).

N2- (N2- (8-aminocaproyl) -N6- (tert-butoxycarbonyl) -L-lysyl) -N6- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (21): to a solution of compound 20(0.457g, 0.5mmol) in DCM (10mL) was added diethylamine (3mL) dropwise and the resulting reaction mixture was stirred at rt for 3 h. The solvent was evaporated under reduced pressure and redissolved in DCM and washed with water (2 × 20mL) and brine (20 mL). The organic layer was dried over sodium sulfate and concentrated to give crude product 21 as a semi-solid, which was used without further purification.

(9S,12S,26S,30S) -12- (4- ((tert-butoxycarbonyl) amino) butyl) -3,11,14,23, 28-pentaoxo-1-phenyl-2-oxa-4, 10,13,22,27, 29-hexaazadotriacontane-9, 26,30, 32-tetracarboxylic acid tetra-tert-butyl ester (22): in N₂To a solid mixture of compound 17(0.114, 0.24mmol) and HATU (0.092g, 0.24mmol) was added dry DMF next and the mixture was stirred at room temperature for 5 min. DIPEA (0.041mL, 0.24mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 min. A solution of compound 21(0.141, 0.2mmol) in DMF was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (10mL) and washed with water (2X 20mL) and brine (15 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purifying the mixture to obtain a purified mixture,product 22 was obtained in semi-solid form (28%).

(3S,7S,21S,24S) -28-amino-21- (4- ((tert-butoxycarbonyl) amino) butyl) -5,10,19, 22-tetraoxy-4, 6,11,20, 23-pentaazaoctacosane-1, 3,7, 24-tetracarboxylic acid tetra-tert-butyl ester (23): compound 22(0.1g, 0.085mmol) was dissolved in a mixture of methanol THF (1:1, 10mL) and 10% Pd-C was added. The resulting suspension is taken up in H₂Stirring was carried out under an atmosphere (balloon pressure) for 3 hours. The mixture was filtered through celite and concentrated to give the desired product 23 (91%) as a waxy solid.

Preparation of 24(a-g) and 25(a-h)

(2S) -5- (4- ((tert-butoxycarbonyl) amino) butyl) -2- (4- (4- (4-iodophenyl) butanamido) butyl) -1,4,7,16, 21-pentaoxo-3, 6,15,20, 22-pentaazapentacosan-1, 19,23, 25-tetracarboxylic acid tetra-tert-butyl ester (24 a): in N₂To a solid mixture of 4- (4-iodophenyl) butanoic acid (0.029g, 0.1mmol) and HATU (0.038g, 0.1mmol) was added dry DMF (2mL) and the mixture was stirred at room temperature for 5 min. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. A solution of compound 23(0.052, 0.05mmol) in DMF (1mL) was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (5mL) and washed with water (2X 10mL) and brine (10 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purification, product 24a (18%) was isolated in semi-solid form.

33- (4-iodophenyl) -5,10,19,22, 30-pentaoxo-21- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododecan-2-yl) methyl) phenyl) thioureido) butyl) -4,6,11,20,23, 29-hexaazadotriacontane-1, 3,7, 24-tetracarboxylic acid (25 a): to a solution of compound 24a (0.025mmol, 1eq) in dioxane (2mL) was added 4M HCl in dioxane (2 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The amine HCl salt was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.050mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly subjected to LCMS purification using 0.1% formic acid in ACN and water.

(2S) -2- (4- (4- (4-bromophenyl) butanamido) butyl) -5- (4- ((tert-butoxycarbonyl) amino) butyl) -1,4,7,16, 21-pentaoxo-3, 6,15,20, 22-pentaazapentacosan-1, 19,23, 25-tetracarboxylic acid tetra-tert-butyl ester (24 b): in N₂To a solid mixture of 4- (4-bromophenyl) butanoic acid (0.024g, 0.1mmol) and HATU (0.038g, 0.1mmol) was added dry DMF (2mL) and the mixture was stirred at room temperature for 5 minutes. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. A solution of compound 23(0.052, 0.05mmol) in DMF (1mL) was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (5mL) and washed with water (2X 10mL) and brine (10 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purify and isolate product 24b in semi-solid form (6%).

33- (4-bromophenyl) -5,10,19,22, 30-pentaoxo-21- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododecan-2-yl) methyl) phenyl) thioureido) butyl) -4,6,11,20,23, 29-hexaazadotriacontane-1, 3,7, 24-tetracarboxylic acid (25 b): to a solution of compound 24b (0.020mmol, 1eq) in dioxane (2mL) was added 4M HCl in dioxane (2 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The amine HCl salt was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.050mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly LCMS purified using 0.1% formic acid in ACN and water to give 25 b.

(2S,5S,19S,23S) -5- (4- ((tert-butoxycarbonyl) amino) butyl) -2- (4- (4- (4-iodophenyl) butanamido) butyl) -1,4,7,16, 21-pentaoxo-3, 6,15,20, 22-pentaazapentacosan-1, 19,23, 25-tetracarboxylic acid tetra-tert-butyl ester (24 a): in N₂To a solid mixture of 4- (4-iodophenyl) butanoic acid (0.029g, 0.1mmol) and HATU (0.038g, 0.1mmol) was added dry DMF (2mL) and the mixture was stirred at room temperature for 5 min. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. A solution of compound 23(0.052, 0.05mmol) in DMF (1mL) was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (5mL) and washed with water (2X 10mL) and brine (10 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purification, product 24a (18%) was isolated in semi-solid form.

(3S,7S,21S,24S) -33- (4-iodophenyl) -5,10,19,22, 30-pentaoxo-21- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -4,6,11,20,23, 29-hexaazadotriacontane-1, 3,7, 24-tetracarboxylic acid (25 a): to a solution of compound 24a (0.025mmol, 1eq) in dioxane (2mL) was added 4M HCl in dioxane (2 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The amine HCl salt was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.050mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly purified by LCMS using 0.1% formic acid in ACN and water.

(2S,5S,19S,23S) -2- (4- (4- (4-bromophenyl) butanamido) butyl) -5- (4- ((tert-butoxycarbonyl) amino) butyl) -1,4,7,16, 21-pentaoxo-3, 6,15,20, 22-pentaazapentacosan-1, 19,23, 25-tetracarboxylic acid tetra-tert-butyl ester (24 b): in N₂To a solid mixture of 4- (4-bromophenyl) butanoic acid (0.024g, 0.1mmol) and HATU (0.038g, 0.1mmol) was added dry DMF (2mL) and the mixture was stirred at room temperature for 5 minutes. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. A solution of compound 23(0.052, 0.05mmol) in DMF (1mL) was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (5mL) and washed with water (2X 10mL) and brine (10 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purify and isolate product 24b in semi-solid form (6%).

(3S,7S,21S,24S) -33- (4-bromophenyl) -5,10,19,22, 30-pentaoxo-21- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -4,6,11,20,23, 29-hexaazadotriacontane-1, 3,7, 24-tetracarboxylic acid (25 b): to a solution of compound 24b (0.020mmol, 1eq) in dioxane (2mL) was added 4M HCl in dioxane (2 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The amine HCl salt was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.050mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly LCMS purified using 0.1% formic acid in ACN and water to give 25 b.

(2S,5S,19S,23S) -5- (4- ((tert-butoxycarbonyl) amino) butyl) -1,4,7,16, 21-pentaoxo-2- (4- (4- (p-tolyl) butanamido) butyl) -3,6,15,20, 22-pentaazapentacosan-1, 19,23, 25-tetracarboxylic acid tetra-tert-butyl ester (24 c): in N₂Next, dry DMF (2mL) was added to a solid mixture of 4- (p-tolyl) butanoic acid (0.0178g, 0.1mmol) and HATU (0.038g, 0.1mmol), and the mixture was stirred at room temperature for 5 minutes. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. A solution of compound 23(0.052, 0.05mmol) in DMF (1mL) was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (5mL) and washed with water (2X 10mL) and brine (10 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purification, product 24c (18%) was isolated in semi-solid form.

(3S,7S,21S,24S) -5,10,19,22, 30-pentaoxo-21- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -33- (p-tolyl) -4,6,11,20,23, 29-hexaazadotriacontane-1, 3,7, 24-tetracarboxylic acid (25 c): to a solution of compound 24c (0.03mmol, 1eq) in dioxane (2mL) was added 4M HCl in dioxane (2 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The amine HCl salt was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.050mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly subjected to LCMS purification using 0.1% formic acid in ACN and water.

(2S,5S,19S,23S) -5- (4- ((tert-butoxycarbonyl) amino) butyl) -1,4,7,16, 21-pentaoxo-2- (4- (4-phenylbutylamido) butyl) -3,6,15,20, 22-pentaazapentacosan-1, 19,23, 25-tetracarboxylic acid tetra-tert-butyl ester (24 d): in N₂To a solid mixture of 4-phenylbutyric acid (0.0164g, 0.1mmol) and HATU (0.038g, 0.1mmol) was added dry DMF (2mL) and the mixture was stirred at room temperature for 5 min. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. A solution of compound 23(0.052, 0.05mmol) in DMF (1mL) was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (5mL) and washed with water (2X 10mL) and brine (10 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purification, product 24d (21%) was isolated in semi-solid form.

(3S,7S,21S,24S) -5,10,19,22, 30-pentaoxo-33-phenyl-21- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -4,6,11,20,23, 29-hexaazadotriacontane-1, 3,7, 24-tetracarboxylic acid (25 d): to a solution of compound 24d (0.04mmol, 1eq) in dioxane (3mL) was added 4M HCl in dioxane (3 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The amine HCl salt was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.080mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly LCMS purified using 0.1% formic acid in ACN and water to give 25 d.

(2S,5S,19S,23S) -5- (4- ((tert-butoxycarbonyl) amino) butyl) -1,4,7,16, 21-pentaoxo-2- (4- (4-phenylbutylamido) butyl) -3,6,15,20, 22-pentaazapentacosan-1, 19,23, 25-tetracarboxylic acid tetra-tert-butyl ester (24 e): in N₂Next, to a solid mixture of 4-oxo-4- (5,6,7, 8-tetrahydronaphthalen-2-yl) butanoic acid (0.023g, 0.1mmol) and HATU (0.038g, 0.1mmol) was added dry DMF (2mL) and the mixture was stirred at room temperature for 5 minutes. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. Compound 23(0.052, 0.05mmol) in DMF (1mL) was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (5mL) and washed with water (2X 10mL) and brine (10 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purification, product 24e (17%) was isolated in semi-solid form.

(3S,7S,21S,24S) -5,10,19,22,30, 33-hexyloxy-33- (5,6,7, 8-tetrahydronaphthalen-2-yl) -21- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -4,6,11,20,23, 29-hexaazadotriacontane-1, 3,7, 24-tetracarboxylic acid (25 e): to a solution of compound 24e (0.02mmol, 1eq) in dioxane (3mL) was added 4M HCl in dioxane (3 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The HCl salt of the amine was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.080mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly LCMS purified using 0.1% formic acid in ACN and water to give 25 e.

(2S,5S,19S,23S) -5- (4- ((tert-butoxycarbonyl) amino) butyl) -2- (4- (2- (4-iodophenyl) acetylamino) butyl) -1,4,7,16, 21-pentaoxo-3, 6,15,20, 22-pentaazapentacosan-1, 19,23, 25-tetracarboxylic acid tetra-tert-butyl ester (24 f): in N₂To a solid mixture of 2- (4-isophenylphenyl) acetic acid (0.026g, 0.1mmol) and HATU (0.038g, 0.1mmol) was added dry DMF (2mL) and the mixture was stirred at room temperature for 5 minutes. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. A solution of compound 23(0.052, 0.05mmol) in DMF (1mL) was added dropwise at room temperature and stirred at the same temperature for 12 hours. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (5mL) and washed with water (2X 10mL) and brine (10 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purification, product 24f (10%) was isolated in semi-solid form.

(8S,11S,25S,29S) -1- (4-iodophenyl) -2,10,13,22, 27-pentaoxo-11- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -3,9,12,21,26, 28-hexaazatriundecane-8, 25,29, 31-tetracarboxylic acid (25 f): to a solution of compound 24f (0.02mmol, 1eq) in dioxane (3mL) was added 4M HCl in dioxane (3 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The HCl salt of the amine was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.080mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly LCMS purified using 0.1% formic acid in ACN and water to give 25 f.

(2S,5S,19S,23S) -2- (4- (1H-indole-2-carboxamide) butyl) -5- (4- ((tert-butoxycarbonyl) amino) butyl) -1,4,7,16, 21-pentaoxo-3, 6,15,20, 22-pentaazapentacosane-1, 19,23, 25-tetracarboxylic acid tetra-tert-butyl ester (24 g): in N₂Next, dry DMF (2mL) was added to a solid mixture of indole-2-acetic acid (0.016g, 0.1mmol) and HATU (0.038g, 0.1mmol), and the mixture was stirred at room temperature for 5 minutes. DIPEA (0.017mL, 0.1mmol) was added to the reaction mixture and stirring was continued at room temperature for 10 minutes. A solution of compound 23(0.052, 0.05mmol) in DMF (1mL) is added dropwise at room temperature and stirred at the same temperature for 12 h. DMF was evaporated under reduced pressure to give a suspension, which was dissolved in DCM (5mL) and washed with water (2X 10mL) and brine (10 mL). The organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (SiO)₂) Purification 24g (15%) of the product were isolated in semi-solid form.

(7S,10S,24S,28S) -1- (1H-indol-2-yl) -1,9,12,21, 26-pentaoxo-10- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -2,8,11,20,25, 27-hexaazatriacontane-7, 24,28, 30-tetracarboxylic acid (25 g): to a solution of 24g (0.02mmol, 1eq) of the compound in dioxane (3mL) was added 4M HCl in dioxane (3 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The HCl salt of the amine was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.080mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was directly subjected to LCMS purification using 0.1% formic acid in CAN and water to give 25 g.

(9S,12S,26S,30S) -3,11,14,23, 28-pentaoxo-1-phenyl-12- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -2-oxa-4, 10,13,22,27, 29-hexaazadotrialkyl-9, 26,30, 32-tetracarboxylic acid (25 h): to a solution of compound 22 (produced as defined herein) (0.02mmol, 1eq) in dioxane (3mL) was added 4M HCl in dioxane (3 mL). The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was monitored by TLC. The solvent was removed under reduced pressure and co-distilled with toluene (2X 5 mL). The HCl salt of the amine was dissolved in DMF (1.5mL) and DIPEA (0.5mmol, 20eq) was added. The resulting reaction mixture was stirred for 10 minutes, then p-NCS-Bn-DOTA (0.080mmol, 2eq) and distilled water (0.5mL) were added. Stirring was continued at room temperature for 3 hours. The reaction mixture was purified directly by LCMS using 0.1% formic acid in ACN and water to give 25 h.

Section 1.2

Preparation of dimethyl 4-aminopyridine-2, 6-dicarboxylate (204).

Combining dimethyl 4-azidopyridine-2, 6-2, 6-dicarboxylate in a round-bottomed flask^[245](0.9445g, 4.0mmol), 10% Pd/C (0.1419g) and DCM: MeOH (1:1, 18 mL). By H₂After balloon purging the flask, the reaction was allowed to proceed at room temperature under H₂Stir vigorously under atmosphere for 46 hours. The gray mixture was diluted with DMF (450mL) and filtered through a celite bed. At the following stageAfter filtration through a 0.22 μm nylon membrane, the filtrate was concentrated at 60 ℃ under reduced pressure and further dried in vacuo to afford 204 as a light tan solid (0.824g, 98% yield).¹H NMR(500MHz,DMSO-d₆):δ＝7.36(s,2H),6.72(s,2H),3.84(s,6H).¹³C{¹H}APT NMR(126MHz,DMSO-d6):δ＝165.51,156.24,148.05,111.99,52.29.IR(cm^–1):3409,3339,3230,1726,1639,1591,1443,1265,996,939,787,630,543.HPLC t_R9.369min (method B), HRMS (M/z):211.07213[ M + H: (M/z):211.07213]⁺；Calc:211.07133。

Preparation of ethyl 4-amino-6- (hydroxymethyl) picolinate (205).

To a refluxing suspension of 204(0.677g, 3.22mmol) in anhydrous EtOH (27mL) was added NaBH in portions over 1 hour₄(0.1745g, 4.61mmol) to give a pale yellow suspension. The reaction was then quenched with acetone (32mL) and concentrated to a tan solid at 60 ℃ under reduced pressure. The crude product is dissolved in H₂O (60mL) and washed with ethyl acetate (4X 150 mL). The combined organics were dried over sodium sulfate and concentrated at 40 ℃ under reduced pressure. Further drying in vacuo gave 205 as a pale yellow solid (0.310g, 49% yield).¹H NMR(300MHz,DMSO-d₆):δ＝7.07(d,J＝2.1Hz,1H),6.78(m,1H),6.32(s,2H),5.30(t,J＝5.8Hz,1H),4.39(d,J＝5.6Hz,2H),4.26(q,J＝7.1Hz,2H),1.28(t,J＝7.1Hz,3H).¹³C APT NMR(126MHz,DMSO-d₆)δ＝165.57,162.38,155.68,147.25,108.50,107.01,63.95,60.61,14.24.IR(cm^-1):3439,3217,2974,2917,1717,1643,1600,1465,1396,1378,1239,1135,1022,974,865,783.HPLC t_R8.461min (method B), HRMS (M/z):197.09288[ M + H: (M/z):197.09288]⁺；Calc:197.09207。

Preparation of ethyl 4-amino-6- (chloromethyl) picolinate (206).

A mixture of thionyl chloride (2.5mL) and 205(0.301g, 1.53mmol) was stirred in an ice bath for 1 hour, then at room temperature for 30 minutes. The orange-yellow emulsion was concentrated at 40 ℃ under reduced pressure to an oily residue. The residue was taken up with saturated NaHCO₃The aqueous solution (12mL) was neutralized and then extracted with ethyl acetate (75 mL). H for organic extracts₂O (2mL), dried over sodium sulfate, and concentrated at 40 ℃ under reduced pressure. Further drying in vacuo afforded 206 as an amber wax (0.287g, 80% yield, corrected for residual ethyl acetate).¹H NMR(500MHz,DMSO-d₆)δ＝7.18(d,J＝2.1Hz,1H),6.78(d,J＝2.1Hz,1H),6.62(br s,2H),4.62(s,2H),4.29(q,J＝7.1Hz,2H),1.30(t,J＝7.1Hz,3H).¹³C{¹H}APT NMR(126MHz,DMSO-d₆)δ＝164.75,156.42,156.19,147.17,109.79,109.50,60.97,46.47,14.15.IR(cm^–1):3452,3322,3209,2978,2922,1726,1639,1604,1513,1465,1378,1248,1126,1026,983,861,783,752,700.HPLC t_R12.364min (method B), HRMS (M/z):215.05903[ M + H: (M/z):215.05903]⁺；Calc:215.05818。

Methyl 6- ((1,4,10, 13-tetraoxa-7, 16-diazacyclooctadecan-7-yl) methyl) picolinate (209.2 TFA.1H)₂O) preparation.

To a clear colorless solution of 1,7,10, 16-tetraoxa-4, 13-diazacyclooctadecane (7, 1.9688g, 7.5mmol) and diisopropylethylamine (0.8354g, 6.5mmol) in dry ACN (1.075L) at 75 deg.C, a solution of 206(0.9255g, 5.0mmol) in dry ACN (125mL) was added dropwise over 2 hours and 40 minutes. The flask was then equipped with a condenser and drying tube, and the slightly yellowish solution was heated under reflux for 42 hours. Subsequently, the dark gold solution containing the fine white precipitate was concentrated at 60 ℃ under reduced pressure to an amber oil. To the crude oil was added 10% MeOH/H with 0.1% TFA₂O (10 mL). The slight suspension was filtered and the filtrate was purified by preparative HPLC (method a). The pure fractions were combined, concentrated at 60 ℃ under reduced pressure, then lyophilized,209(1.6350g, 50% yield) was obtained as a light orange solid.¹H NMR(500MHz,DMSO-d₆)δ＝8.75(br s,2H),8.17–8.06(m,2H),7.83(dd,J＝7.4,1.5Hz,1H),4.68(br s,2H),3.91(s,3H),3.85(br t,J＝5.1Hz,4H),3.69(t,J＝5.1Hz,4H),3.59(br s,8H),3.50(brs,4H),3.23(br t,J＝5.1Hz,4H).¹³C{¹H}APT NMR(126MHz,DMSO-d₆)δ164.68,158.78-157.98(q,TFA),151.44,147.13,139.01,128.63,124.87,120.08-113.01(q,TFA),69.33,69.00,65.31,64.60,56.43,53.29,52.67,46.32.¹⁹F NMR(470MHz,DMSO-d₆) Delta-73.84. EA found C, 43.88; h, 5.29; n,6.28.C₂₀H₃₃N₃O₆·2CF₃COOH·1H₂C, 43.84; h, 5.67; n,6.39.HPLC t_R12.372min (method B), HRMS (M/z):412.24568[ M + H: (M/z):412.24568]⁺；Calc:412.24421。

Preparation of ethyl 4-amino-6- ((16- ((6- (methoxycarbonyl) pyridin-2-yl) methyl) -1,4,10, 13-tetraoxa-7, 16-diazacyclooctadecan-7-yl) methyl) picolinate (210).

To a round bottom flask equipped with a condenser and drying tube was added 209(0.4210g, 0.64mmol), Na₂CO₃(0.3400g, 3.2mmol) and dried ACN (10 mL). The light yellow suspension was heated to reflux for 15 min, then 206(0.1508g, 0.70mmol, corrected for residual ethyl acetate) was added as a slight suspension in dry ACN (3.5 mL). The mixture was heated to reflux for 44 hours and then filtered. The orange filtrate was concentrated at 60 ℃ under reduced pressure to an orange brown oil (0.612g), which was used in the next step without further purification. HRMS (M/z) 590.32021[ M + H]⁺；Calc:590.31844。

Preparation of 4-amino-6- ((16- ((6-carboxypyridin-2-yl) methyl) -1,4,10, 13-tetraoxa-7, 16-diazacyclooctadecan-7-yl) methyl) picolinic acid (211. 4 TFA).

Compound 210(0.612g) was dissolved in 6M HCl (7mL) and heated at 90 ℃ for 17 h. The orange-brown solution containing a small amount of precipitate was concentrated at 60 ℃ under reduced pressure to a light tan solid. To the solid was added 10% MeOH/H containing 0.1% TFA₂O (3 mL). The slight suspension was filtered and the filtrate was purified by preparative HPLC using method a. The pure fractions were combined, concentrated under reduced pressure at 60 ℃ and then lyophilized to give 211 as an off-white solid (0.2974g, 2 steps 46% yield).¹H NMR(500MHz,DMSO-d₆)δ＝8.13–8.08(m,2H),7.80(dd,J＝7.3,1.6Hz,1H),7.64(br s),7.24(d,J＝2.3Hz,1H),6.76(d,J＝2.3Hz,1H),4.74(s,2H),4.15(s,2H),3.85(t,J＝5.0Hz,4H),3.63(t,J＝5.1Hz,4H),3.57–3.50(m,12H),3.09(br t,J＝5.2Hz,4H).¹³C{¹H}NMR(126MHz,DMSO-d₆)δ165.96,163.37,159.47,158.78–157.98(q,TFA),151.93,151.64,148.25,144.68,139.59,128.43,124.96,120.79–113.68(q,TFA),109.40,108.96,70.03,69.89,67.09,65.16,57.28,55.85,54.47,53.81.¹⁹F NMR(470MHz,DMSO-d₆) EA found to be C, 40.60; h, 4.29; n,7.04.C₂₆H₃₇N₅O₈·4CF₃Calculation of COOH C, 40.69; h, 4.12; n,6.98.IR (cm)^-1):3387,3161,1735,1670,1204,1130,791,722.HPLC t_R11.974min (method B); 11.546min (method D), HRMS (M/z):548.26883[ M + H]⁺；Calc:548.27149。

Preparation of 6- ((16- ((6-carboxypyridin-2-yl) methyl) -1,4,10, 13-tetraoxa-7, 16-diazacyclooctadecan-7-yl) methyl) -4-isothiocyanatopyridinecarboxylic acid (212, macropa-NCS).

White 211(0.1598g, 0.16mmol) and Na were mixed₂CO₃(0.2540g, 2.4mmol) of the suspension was heated at reflux in acetone (10mL) for 30 min, then CSCl was slowly added₂(305μL CSCl₂85%, Acros Organics). The resulting orange suspension was heated to reflux for 3 hours and then reduced at 30 deg.CThe concentrate was pressed to a pale orange solid. The solid was dissolved in portions in 10% ACN/H containing 0.2% TFA₂O (total 8mL), filtered, and used immediately as method C^[246]Purification was performed by preparative HPLC. The pure fractions were combined, concentrated at room temperature under reduced pressure to remove the organic solvent, and then lyophilized. The fraction which could not be concentrated immediately was frozen at-80 ℃. Isothiocyanate 212(0.0547g) was obtained as a mixture of white and light yellow solids and stored in a Drierite tank at-80 ℃. From 212 samples spiked with known concentrations of fluorobenzene¹H NMR and¹⁹f NMR spectroscopy calculated that isolated 212 was estimated to be the tetra TFA salt.¹H NMR(400MHz,DMSO-d₆)δ＝8.17–8.06(m,2H),8.00(s w/fine splitting,1H),7.84(d,J＝1.5Hz,1H),7.81–7.75(d w/fine splitting,J＝7.16Hz,1H),4.71(s,2H),4.64(s,2H),3.89–3.79(m,8H),3.62–3.46(m,16H).¹⁹F NMR(470MHz,DMSO-d₆)δ＝–74.17.IR(cm^-1):～3500–2800,2083,2026,1735,1670,1591,1448,1183,1130,796,717.HPLC t_R15.053min (method B); 13.885min (method D), HRMS (M/z):590.22600[ M + H]⁺；Calc:590.22791。

Preparation of di-tert-butyl ((S) -1- (tert-butoxy) -6- (3- (3-ethynylphenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamate (214).

According to the disclosed method^[247]Alkyne 214 was prepared and isolated as an off-white powder.¹H NMR(500MHz,CDCl₃)δ＝7.90(s,1H),7.58(t,1H,J＝1.7Hz),7.51(dd,1H,J₁＝8.2Hz,J₂＝1.3Hz),7.18(t,1H,J＝7.9Hz),7.05(d,1H,J＝7.7Hz),6.38(d,1H,J＝7.9Hz),6.28(br s,1H),5.77(d,1H,J＝6.9Hz),4.32(m,1H),4.02(m,1H),3.53(m,1H),3.05(m,1H),3.00(s,1H),2.39(m,2H),2.07(m,1H),1.88(m,1H),1.74(m,1H),1.62(m,1H),1.49–1.37(m,4H),1.41(s,18H),1.37(s,9H)。

N²- (((9H-fluoren-9-yl) methoxy) carbonyl) -N⁶- (tert-butoxycarbonyl) -L-lysine 2, 5-dioxopyrrolidin-1-yl ester (2)15) And (4) preparing.

Will be in CH₂Cl₂A suspension of Fmoc-L-Lys (Boc) -OH (5.0g, 10.7mmol) and N, N' -disuccinimidyl carbonate (2.74g, 10.7mmol) in (50mL) was stirred at room temperature under argon. DIPEA (1.86mL, 10.7mmol) was then added and the suspension was stirred overnight. The solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography (0-100% EtOAc in hexanes). Lysine 215 was isolated as a white powder (2.5g, 41%).¹H NMR(500MHz,CDCl₃)δ＝7.76(d,2H,J＝7.6Hz),7.59(d,2H,J＝7.3Hz),7.40(t,2H,J＝7.4Hz),7.32(t,2H,J＝7.3Hz),5.46(br s,1H),4.71(m,2H),4.45(m,2H),4.23(t,1H,J＝6.6Hz),3.14(brs,2H),2.85(s,4H),2.02(m,1H),1.92(m,1H),1.58(m,4H),1.44(s,9H)。

N²-(N²- (((9H-fluoren-9-yl) methoxy) carbonyl) -N⁶- (tert-Butoxycarbonyl) -L-lysyl) -N⁶Preparation of t-butyl (- (benzyloxy) carbonyl) -L-lysine (216).

Treatment with DIPEA (0.87mL, 5.0mmol) in CH₂Cl₂(15mL) of L-Lys (Z) -OtBu HCl (1.49g, 4.0 mmol). To the resulting mixture was added₂Cl₂(10mL) of lysine 215(2.2g, 3.9mmol), and the reaction was stirred at room temperature under argon overnight. It was then washed with saturated NaCl solution and the organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (0-100% EtOAc in hexanes) and isolated as a white powder of dilysine 216(2.2g, 72%).¹H NMR(500MHz,CDCl₃)δ＝7.76(d,2H,J＝7.5Hz),7.59(d,2H,J＝7.3Hz),7.40(t,2H,J＝7.5Hz),7.32(m,8H),6.69(br s,1H),5.60(br s,1H),5.06(m,4H),4.72(br s,1H),4.43(m,1H),4.38(m,1H),4.21(m,1H),3.14(m,4H),1.85(m,2H),1.73(m,2H),1.50(m,4H),1.46(s,9H),1.44(s,9H),1.39(m,4H)。

Preparation of 2- (4-iodophenyl) acetic acid 2, 5-dioxopyrrolidin-1-yl ester (217).

Will be in CH₂Cl₂A solution of 2- (4-iodophenyl) acetic acid (786mg, 3.0mmol) and EDC & HCl (671mg, 3.5mmol) in (20mL) was stirred at room temperature under argon for 15 min. N-hydroxysuccinimide (368mg, 3.2mmol) and NEt were then added₃(0.56mL, 4.0mmol) and the reaction stirred for 7 h. It was then washed with saturated NaCl solution and the organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography (0-100% EtOAc in hexanes) to isolate the NHS ester 217 as a white solid (760mg, 70%).¹H NMR(500MHz,CDCl₃)δ＝7.69(d,2H,J＝7.9Hz),7.09(d,2H,J＝7.9Hz),3.88(s,2H),2.83(s,4H)。

N²-(N²- (1-azido-3, 6,9,12,15, 18-hexaoxaheneicosane-21-acyl) -N⁶- (tert-Butoxycarbonyl) -L-lysyl) -N⁶Preparation of t-butyl (- (benzyloxy) carbonyl) -L-lysine (218).

To in CH₂Cl₂To a solution of Fmoc-protected dilysine 216(768mg, 0.97mmol) in (4mL) was added NHEt₂(2.07mL, 20 mmol). The solution was stirred at room temperature overnight. The solvent was removed under reduced pressure and the crude yellow oil was used without further purification. To in CH₂Cl₂To a solution of this oil (183mg, 0.32mmol) in (3mL) was added CH in order₂Cl₂NEt in (1mL)₃(57. mu.L, 0.41mmol) in CH₂Cl₂azido-PEG in (1mL)₆-NHS ester (100mg, 0.21 mmol; Broadpharmm, USA), the reaction was stirred at room temperature overnight. Then using it with CH₂Cl₂Diluting with H₂And washing with saturated NaCl solution. The organic layer was MgSO₄Drying, filtration and concentration under reduced pressure gave azide 218 as a colorless oil without further purification (184 mg; 95%). Mass (ESI +: 926.4[ M + H ]]⁺.Calc.Mass＝925.54。

Preparation of di-tert-butyl ((S) -1- (tert-butoxy) -6- (3- (3- (1- ((9S,12S) -9- (tert-butoxycarbonyl) -12- (4- ((tert-butoxycarbonyl) amino) butyl) -3,11, 14-trioxo-1-phenyl-2, 17,20,23,26,29, 32-heptaoxa-4, 10, 13-triaza-tridecan-34-yl) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamate (219).

mu.L of 0.5M CuSO in DMF (0.5mL)₄And 100. mu.L of a 1.5M solution of sodium ascorbate were mixed for 5 minutes and then added to a solution of 218(184mg, 0.20mmol) and 214(132mg, 0.21mmol) in DMF (2.5 mL). The resulting mixture was stirred at room temperature for 45 minutes. It was then concentrated under reduced pressure and the crude residue was purified by flash chromatography (0-30% MeOH in EtOAc) to give triazole 219 as an orange oil (285 mg; 87%). Mass (ESI +: 1557.2[ M + H ]]⁺.Calc.Mass＝1555.90。

Preparation of di-tert-butyl ((S) -1- (tert-butoxy) -6- (3- (3- (1- ((23S,26S) -26- (tert-butoxycarbonyl) -23- (4- ((tert-butoxycarbonyl) amino) butyl) -33- (4-iodophenyl) -21,24, 32-trioxo-3, 6,9,12,15, 18-hexa-oxa-22, 25, 31-triazatritrialky) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamate (220).

In a two-necked flask, Cbz-protected triazole 219(285mg, 0.18mmol) was dissolved in MeOH (15 mL). To the solution was added 10% Pd/C (20mg), and the suspension was shaken and the flask evacuated. The suspension is then placed in H₂Stirred under atmosphere overnight. Will be provided withIt was filtered through celite and the filter cake was washed 3 times with MeOH. The combined filtrates were concentrated under reduced pressure to give the free amine as a colorless oil (117 mg; 45%), which was used without further purification. Mass (ESI +: 1423.8[ M + H ]]⁺Mass 1422.77. To in CH₂Cl₂To a solution of amine (117mg, 82. mu. mol) in CH (4mL)₂Cl₂A solution of DIPEA (23 μ L, 131mmol) in (1mL) and the mixture was stirred at room temperature under argon. Then added to CH₂Cl₂(2mL) of 217(37mg, 103. mu. mol), and the reaction was stirred at room temperature for 2 h. It is then poured into H₂O (10mL) and the layers were separated. The organic layer was MgSO₄Drying, filtration and concentration under reduced pressure gave the crude product as a colorless semisolid. The crude product was purified by preparative TLC (10% MeOH in EtOAc) to give phenyl iodide 220(34 mg; 25%) as a colorless oil. Mass (ESI +: 1666.6[ M + H ]]⁺.Calc.Mass＝1665.80。

((S) -1-carboxy-5- (3- (3- (1- ((23S,26S) -26-carboxy-23- (4- (3- (2-carboxy-6- ((16- ((6-carboxypyridin-2-yl) methyl) -1,4,10, 13-tetraoxa-7, 16-diazacyclooctadecan-7-yl) methyl) pyridin-4-yl) thioureido) butyl) -33- (4-iodophenyl) -21,24, 32-trioxo-3, 6,9,12,15, 18-hexaoxa-22, 25, 31-triazatririacontanyl) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) pentyl) carbamoyl) Preparation of L-glutamic acid (221, macropa-RPS-070).

To in CH₂Cl₂To a solution of 220(34mg, 20. mu. mol) in (2mL) was added TFA (0.5mL), and the reaction was stirred at room temperature for 5 h. It is then concentrated under reduced pressure and the crude product is taken up in H₂Dilution with O and lyophilization gave the free amine as a TFA salt. Mass (ESI +: 1342.5[ M + H ]]⁺.Mass(ESI-):1340.6[M-H]^-Mass 1341.50. To a solution of amine (9mg, 6.7. mu. mol) in DMF (0.5mL) was added a solution of Macropa-NCS 212(15mg, 25.4. mu. mol) in DMF (0.5 mL). DIPEA (300. mu.L, 1.72mmol) was then added and the reaction stirred at room temperatureFor 2 hours. The volatiles were removed under reduced pressure and the crude product was purified by preparative HPLC to give Macropa-RPS-070(221) (5.4 mg; 42%) as a white powder. Mass (ESI +: 1932.76[ M + H ]]⁺.1931.09[M+H]^-.Calc.Mass＝1931.91。

²²⁵Preparation of Ac-macropa-RPS-070 by radiosynthesis.

Overview. All reagents were purchased from Sigma Aldrich and were reagent grade unless otherwise indicated. Hydrochloric acid (HCl) is(>99.999%) with trace analytical mass. Aluminum-supported silica Thin Layer Chromatography (TLC) plates were purchased from Sigma Aldrich. By being atDilution in Water to prepare 0.05M HCl and 1M NH₄Stock solutions of OAc.

And (4) performing a radioactive labeling procedure. To a solution in 0.05M HCl²²⁵Ac(NO₃)₃To a solution of (Oak Ridge National Laboratory, USA) (17.9 MBq in 970. mu.L) was added 20. mu.L of a 1mg/mL solution of macropa-RPS-070 in DMSO. By adding 90. mu.L of 1M NH₄OAc raises the pH to 5-5.5. The reaction was allowed to stand at room temperature for 20 minutes with regular shaking. Then, 200. mu.L of the reaction solution was removed and diluted with 3.8mL of physiological saline (0.9% NaCl in deionized water; VWR) to give a solution with a concentration of 910 kBq/mL. An aliquot was taken from the final solution and spotted onto an aluminum-supported silica TLC plate to determine the radiochemical yield. Will be in 0.05M HCl²²⁵Ac(NO₃)₃An aliquot of the solution was spotted in parallel lanes as a control. Immediately, the plate was run in 10% v/v MeOH/10mM EDTA mobile phase and then left for 8 hours to reach radiochemical equilibrium. After 3 minutes exposure on the screen, the plate was observed on a Cyclone Plus storage fluorescence system (PerkinElmer). The radiochemical yield is expressed as²²⁵The ratio of Ac-macropa-RPS-070 to total activity was determined to be 98.1%.

²²⁵Organisms of Ac-macropa-RPS-070And (5) distribution research.

And (5) culturing the cells. The human prostate cancer cell line LNCaP expressing PSMA was obtained from the american type culture collection. Unless otherwise indicated, cell cultures were from Invitrogen. At 37 deg.C/5% CO₂In a humidified incubator of (1), LNCaP cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone), 4mM L-glutamine, 1mM sodium pyruvate, 10mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 2.5mg/mL D-glucose, and 50. mu.g/mL gentamicin. Cells were removed from the flask by incubating them with 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) to pass or transfer to a 12-well assay plate.

Mice were inoculated with xenografts. All animal studies have been approved by the animal protection and use committee of the wil kanel institute of medicine and conducted according to guidelines set forth in the united states pharmacopeia for policy on human care and use of experimental animals. Animals were housed in approved facilities under standard conditions for 12 hour light/dark cycles. Food and water were provided ad libitum throughout the study. Hairless nu/nu mice were purchased from Jackson laboratories. For inoculation in mice, LNCaP cells were plated at 4x 10⁷The ratio of individual cells/mL was suspended in a 1:1 mixture of PBS: Matrigel (BD Biosciences). The left flank of each mouse was injected with 0.25mL of cell suspension. When the tumor is 100-400mm³Within range, biodistribution is performed.

²²⁵Biodistribution of Ac-macropa-RPS-070 in LNCaP xenograft mice. Fifteen tumor-bearing mice (5 per time point) were xenografted with LNCaP intravenously injected with bolus injections of 85-95kBq and 100ng (50pmol) of each ligand. Mice were sacrificed by cervical dislocation at 4, 24 and 96 hours post injection. Blood samples were taken and a complete biodistribution study was performed on the following organs (including contents): heart, lung, liver, small intestine, large intestine, stomach, spleen, pancreas, kidney, muscle, bone, and tumor. Tissues were weighed and counted on a 2470Wizard automated gamma counter (Perkin Elmer). Samples at 1% ID/mL were counted before and after each group of tissue samples for attenuation correction. Correcting counts to reduce fadingThe injection activity was varied and tissue uptake was expressed as a percentage of injected dose per gram (% ID/g). The standard error for each data point was calculated.

TABLE 1T 4h, 24h and 96h post intravenous injection in LNCaP xenograft mice²²⁵Organ distribution of Ac-macropa-RPS-070 (n ═ 5 at each time point). The values are expressed as% ID/g.

The above [2 ]²²⁵Ac(macropa)]⁺Discussion of the results of the complexes.

By comparison²²⁵Ac(macropa)]⁺And²²⁵Ac(NO₃)₃and 2²²⁵Ac(DOTA)]^–To assess its stability in vivo. C57BL/6 mice were injected 10-50 kBq of each radiometal complex via the tail vein and sacrificed after 15 minutes, 1 hour or 5 hours. Quantification of the amount retained in each organ by gamma counting²²⁵The amount of Ac is reported as a percentage of injected dose per gram of tissue (% ID/g).²²⁵The Ac complex has insufficient stability, resulting in a loss of radioisotope in vivo as evidenced by loss of radioisotope²²⁵Accumulation of Ac in mouse liver, spleen and bone^[11,12,31]. Not compounded²²⁵Ac(NO₃)₃The biodistribution profile of (fig. 5A) showed slow blood clearance and excretion, associated with massive accumulation in the liver and spleen. [²²⁵Ac(macropa)]⁺Biodistribution characteristics (FIG. 5B) and²²⁵Ac(NO₃)₃are significantly different. [²²⁵Ac(macropa)]⁺Cleared rapidly from the mice and had little activity measured in the blood 1h after injection. The majority of the injected dose is excreted through the kidney and subsequently detected in the urine, which explains the moderate degree of [2 ] observed in mice at 15 minutes and 1 hour post-injection²²⁵Ac(macropa)]⁺Kidney and bladder uptake. Importantly, the term²²⁵Ac(macropa)]⁺No accumulation in any organ during the study indicated that the complex was presentNot released free in vivo²²⁵Ac³⁺. Its biodistribution characteristics are similar to those of [ alpha ], [ beta ]²²⁵Ac(DOTA)]^–Has previously been shown to be retained in vivo (FIG. 5C)²²⁵Ac^3+[7]. Of note are²²⁵Ac(DOTA)]^–It appears that it is cleared more rapidly by urine and is absorbed to a lesser extent in the thyroid gland. These differences may be due in part to the opposite charge of the complex. In general, the results of these biodistribution studies indicate that [ alpha ], [ alpha ]²²⁵Ac(macropa)]⁺Is highly stable in vivo.

RPS-070 is conjugated to macropha-NCS, where the construct bears a glutamate-urea-lysine moiety that inhibits Prostate Specific Membrane Antigen (PSMA)^[237–241]This is a membrane-bound glycoprotein that is overexpressed in prostate cancer cells^[242]. The albumin binding function (in this case a group comprising iodophenyl) is also a key component of these compounds to extend their circulating half-life^[243,244]. By using²²⁵The process of Ac radiolabeling macropa-RPS-070 was carried out at room temperature and pH 5-5.5 for 20 minutes to achieve 98% RCY. Then will be²²⁵Ac-macropa-RPS-070 (85-95 kBq) was injected into mice bearing LNCaP (prostate cancer) tumor xenografts and the biodistribution of the complexes was determined at 4, 24 and 96h post-injection (table 1, figure 6 above).²²⁵Ac-macropa-RPS-070 cleared rapidly from the blood and was mainly distributed in the kidneys and tumors (52. + -. 16% ID/g and 13. + -. 3% ID/g, respectively, 4h after injection). After 4 hours, most of the activity was cleared from the kidney and a gradual tumor clearance was observed. Importantly, the uptake of the complex by other organs was negligible (96 hours after injection,<1% ID/g) and will not accumulate in any organ over time. Activity cleared from tumors at 4-96 hours was still sequestered by macropa-RPS-070, which was deficient in liver, spleen and bone in mice at this time²²⁵This is evidenced by the accumulation of Ac. These results are important because they demonstrate that macropa-RPS-070 can be stably retained in vivo for several days²²⁵Ac, and the construct may be selectivelyThe tumor is targeted.

Reference is made to section 1.2

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Section 1.3

General procedure. All solvents were purchased from Sigma Aldrich and of reagent grade quality unless otherwise stated. The solvent was dried by distillation on an activated stainless steel column (Pure Process Technology, LLC) or by drying on an activated molecular sieve. Reagents were purchased from Sigma Aldrich, 2-azidoacetic acid-NHS ester and azido-PEG_nExcept for the-NHS ester compound, which was purchased from BroadPharm. The reagents are all reagent grade and can be used without further purification.

All reactions described below were carried out in dry glassware. Is used inHigh Purity Silica GelSilica gel chromatography on silica, preparative TLC on silica-coated glass plates (Analtech) and by subjectingPurification was performed by flash chromatography using the CombiFlash Rf + (Teledyne Isco) system. Using Xbridge^TMPrep C18 5μm OBD^TMPreparative HPLC was performed on a 19X 100mm column (Waters) on a two-pump Agilent ProStar HPLC equipped with an Agilent ProStar 325Dual wavelet UV-Vis detector. UV absorption was monitored at 220nm and 280 nm. Using a binary solvent system, wherein solvent A comprises H₂O + 0.01% TFA, solvent B contained 90% v/v MeCN/H₂O + 0.01% TFA. Purification was performed using the following gradient HPLC method: 0-1 minute of 0% B, 0-100 minutes of B1-28 minutes, 100-0 minutes of B28-30 minutes.

The final product was identified and characterized using thin layer chromatography, analytical HPLC and mass spectrometry. NMR spectroscopy was used to confirm the structures of compounds 7a, 8a, 26 and 28. Using XSelect^TMCSH^TMAnalytical HPLC was performed on C185 μm 4.6X 50mm columns (Waters). Use of Waters ACQUITY connected to Waters SQ Detector 2Mass determination was performed by LCMS analysis. NMR analysis was performed using a Bruker Avance III 500MHz spectrometer. Spectra are expressed in ppm and referenced to solvent resonance in chloroform-d (Sigma Aldrich). All compounds evaluated in the biological assay were greater than 95% pure as judged by analytical HPLC.

((S) -1- (tert-butoxy) -6- (3- (3-ethynylphenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamic acid di-tert-butyl ester (26):

according to Kelly J, Amor-Coarasa A, Nikolopoulou A, Kim D, Williams C, Jr, Ponnala S, Babich JW¹⁸Eur J Nucl Med Mol Imaging 2017; the protocol described in 44:647-61 produced alkyne 26 and was isolated as an off-white powder.¹H NMR(500MHz,CDCl₃)δ7.90(s,1H),7.58(t,1H,J＝1.7Hz),7.51(dd,1H,J₁＝8.2Hz,J₂＝1.3Hz),7.18(t,1H,J＝7.9Hz),7.05(d,1H,J＝7.7Hz),6.38(d,1H,J＝7.9Hz),6.28(br s,1H),5.77(d,1H,J＝6.9Hz),4.32(m,1H),4.02(m,1H),3.53(m,1H),3.05(m,1H),3.00(s,1H),2.39(m,2H),2.07(m,1H),1.88(m,1H),1.74(m,1H),1.62(m,1H),1.49-1.37(m,4H),1.41(s,18H),1.37(s,9H)。

For N²-(N²- (((9H-fluoren-9-yl) methoxy) carbonyl) -N⁶- (tert-Butoxycarbonyl) -L-lysyl) -N⁶- Synthesis procedure in section 1.1 of t-butyl ((benzyloxy) carbonyl) -L-lysine (8a)

N²- (((9H-fluoren-9-yl) methoxy) carbonyl) -N⁶- (tert-Butoxycarbonyl) -L-lysine 2, 5-dioxopyrrolidin-1-yl ester (7a) to be in CH₂Cl₂A suspension of Fmoc-L-Lys (Boc) -OH 6a (5.0g, 10.7mmol) and N, N' -disuccinimidyl carbonate (2.74g, 10.7mmol) in (50mL) was stirred at room temperature under argon. DIPEA (1.86mL, 10.7mmol) was then added and the suspension was stirred overnight. The solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography (0-100% EtOAc in hexanes). The NHS ester 7a was isolated as a white powder (2.5g, 41%).¹H NMR(500MHz,CDCl₃)δ7.76(d,2H,J＝7.6Hz),7.59(d,2H,J＝7.3Hz),7.40(t,2H,J＝7.4Hz),7.32(t,2H,J＝7.3Hz),5.46(br s,1H),4.71(m,2H),4.45(m,2H),4.23(t,1H,J＝6.6Hz),3.14(br s,2H),2.85(s,4H),2.02(m,1H),1.92(m,1H),1.58(m,4H),1.44(s,9H)。

N²-(N²- (((9H-fluoren-9-yl) methoxy) carbonyl) -N⁶- (tert-Butoxycarbonyl) -L-lysyl) -N⁶- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (8a) treatment with DIPEA (0.87mL, 5.0mmol) in CH₂Cl₂Suspension of L-Lys (Z) -OtBu. HCl (1.49g, 4.0mmol) in (15 mL). To the resulting mixture was added₂Cl₂(10mL) of a solution of compound 7a (2.2g, 3.9mmol), and the reaction was stirred at room temperature under argon overnight. It is then washed with a saturated NaCl solution and will haveThe organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (0-100% EtOAc in hexanes) and the white powder of dilysine 8a (2.2g, 72%) was isolated.¹H NMR(500MHz,CDCl₃)δ7.76(d,2H,J＝7.5Hz),7.59(d,2H,J＝7.3Hz),7.40(t,2H,J＝7.5Hz),7.32(m,8H),6.69(br s,1H),5.60(br s,1H),5.06(m,4H),4.72(br s,1H),4.43(m,1H),4.38(m,1H),4.21(m,1H),3.14(m,4H),1.85(m,2H),1.73(m,2H),1.50(m,4H),1.46(s,9H),1.44(s,9H),1.39(m,4H)。

2- (4-iodophenyl) acetic acid 2, 5-dioxopyrrolidin-1-ester (28):

will be in CH₂Cl₂A solution of 2- (4-iodophenyl) acetic acid 27(786mg, 3.0mmol) and EDC.HCl (671mg, 3.5mmol) in (20mL) was stirred at room temperature under argon for 15 min. N-hydroxysuccinimide (368mg, 3.2mmol) and TEA (0.56mL, 4.0mmol) were then added and the reaction stirred for 7 hours. It was then washed with saturated NaCl solution and the organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography (0-100% EtOAc in hexanes) and the NHS ester 28 was isolated as a white solid (760mg, 70%).¹H NMR(500MHz,CDCl₃)δ7.69(d,2H,J＝7.9Hz),7.09(d,2H,J＝7.9Hz),3.88(s,2H),2.83(s,4H)。

Synthesis of trifunctional ligands (RPS-061, RPS-063, RPS-066, RPS-067, RPS-068, RPS-069) and substitution Synthesis of RPS-069 by the exemplary procedure

N²-(N²- (1-azido-3, 6,9,12,15, 18-hexaoxaheneicosane-21-acyl) -N⁶- (tert-butoxycarbonyl)Yl) -L-lysyl) -N⁶- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (29e) in CH₂Cl₂To a solution of Fmoc-protected compound 8a (768mg, 0.97mmol) in (4mL) was added diethylamine (2.07mL, 20 mmol). The solution was stirred at room temperature overnight. The solvent was removed under reduced pressure and the crude product was used as a yellow oil without further purification. To in CH₂Cl₂To a solution of this yellow oil (183mg, 0.32mmol) in (3mL) was added CH₂Cl₂(1mL) of a solution of TEA (57. mu.L, 0.41mmol) in CH₂Cl₂azido-PEG in (1mL)₆-NHS ester (100mg, 0.21mmol) and the reaction was stirred at room temperature overnight. Then using it with CH₂Cl₂Diluting with H₂And washing with saturated NaCl solution. The organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure to give azide 29e as a colorless oil (184 mg; 95%) without further purification. Mass (ESI +: 926.4[ M + H ]]⁺.Calc.Mass＝925.54。

((S) -1- (tert-butoxy) -6- (3- (3- (1- ((9S,12S) -9- (tert-butoxycarbonyl) -12- (4- ((tert-butoxycarbonyl) amino) butyl) -3,11, 14-trioxo-1-phenyl-2, 17,20,23,26,29, 32-heptaoxa-4, 10, 13-triaza-tridecan-34-yl) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamic acid di-tert-butyl ester (30e) 100. mu.L of 0.5M CuSO in DMF (0.5mL)₄And 100. mu.L of a 1.5M solution of sodium ascorbate were mixed for 5 minutes and then added to a solution of 29e (184mg, 0.20mmol) and 26(132mg, 0.21mmol) in DMF (2.5 mL). The resulting mixture was stirred at room temperature for 45 minutes. It was then concentrated under reduced pressure and the crude residue was purified by flash chromatography (0-30% MeOH in EtOAc) to give triazole 30e as an orange oil (285 mg; 87%). Mass (ESI +: 1557.2[ M + H ]]⁺.Calc.Mass＝1555.90。

((S) -1- (tert-butoxy) -6- (3- (3- (1- ((23S,26S) -26- (tert-butoxycarbonyl) -23- (4- ((tert-butoxycarbonyl) amino) butyl) -33- (4-iodophenyl) -21,24, 32-trioxo-3, 6,9,12,15, 18-hexaoxa-22, 25, 31-triazatritrialky) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamic acid di (tert-butoxy) -1- (3- (4- ((tert-butoxycarbonyl) amino) butyl) -3- (4-iodophenyl) -21,24, 32-trioxo-3, 6Tert-butyl ester (31e) Cbz protected triazole 30e (285mg, 0.18mmol) was dissolved in MeOH (15mL) in a two-necked flask. To the solution was added 10% Pd/C (20mg), and the suspension was shaken and the flask evacuated. The suspension is then placed in H₂Stirred under atmosphere overnight. It was filtered through celite and the filter cake was washed 3 times with MeOH. The combined filtrates were concentrated under reduced pressure to give the free amine as a colorless oil (117 mg; 45%), which was used without further purification. Mass (ESI +: 1423.8[ M + H ]]⁺Mass 1422.77. To in CH₂Cl₂To a solution of free amine (117mg, 82. mu. mol) in CH (4mL) was added₂Cl₂A solution of DIPEA (23 μ L, 131mmol) in (1mL) and the mixture was stirred at room temperature under argon. . Then added to CH₂Cl₂(2mL) of 28(37mg, 103. mu. mol), and the reaction was stirred at room temperature for 2 h. It is then poured into H₂O (10mL) and the layers were separated. The organic layer was MgSO₄Drying, filtration and concentration under reduced pressure gave the crude product as a colorless semisolid. The crude product was purified by preparative TLC (10% MeOH in EtOAc) to give phenyl iodide 31e (34 mg; 25%) as a colorless oil. Mass (ESI +: 1666.6[ M + H ]]⁺.Calc.Mass＝1665.80。

((1S) -1-carboxy-5- (3- (3- (1- ((23S,26S) -26-carboxy-33- (4-iodophenyl) -21,24, 32-trioxo-23- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -3,6,9,12,15, 18-hexaoxa-22, 25, 31-triazatririacontanyl) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) pentyl) carbamoyl) -L-glutamic acid (RPS-069): is directed to be in CH.₂Cl₂To a solution of 31e (34mg, 20. mu. mol) in (2mL) was added TFA (0.5mL), and the reaction was stirred at room temperature for 5 h. It is then concentrated under reduced pressure and the crude product is taken up in H₂Dilution in O and lyophilization gave the free amine as a TFA salt. Mass (ESI +: 1342.5[ M + H ]]⁺.Mass(ESI-):1340.6[M-H]^-Mass 1341.50. To at H₂p-SCN-Bn-DOTA.25HCl.25H in O (0.5mL)₂To a solution of O (Macrocyclics, Inc.) (13mg,19 μmol) was added a solution of free amine (18mg, 13 μmol) in DMF (1 mL). DIPEA was added until the reactionpH of ≈ 9. The reaction was stirred at room temperature for 3 hours, then the reaction mixture was purified by preparative HPLC. The peak corresponding to the desired product was collected and lyophilized to give RPS-069 as a white powder (8 mg; 32%). Mass (ESI +: 1893.3[ M + H ]]⁺,947.6[(M+2H)/2]⁺.Mass(ESI-):1891.4[M-H]^-,945.5[(M-2H)/2]^-.Calc.Mass＝1892.70。

((1S) -1-carboxy-5- (3- (3- (1- ((17S,20S) -20-carboxy-27- (4-iodophenyl) -15,18, 26-trioxo-17- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -3,6,9, 12-tetraoxa-16, 19, 25-triaza-octacosyl) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) pentyl) carbamoyl) -L-glutamic acid (RPS-061) RPS-061 is according to the procedure described for RPS-069, composed of common structural units 26, 8a and 28 and azido-PEG₄-NHS ester synthesis. Mass (ESI +: 1805.6664[ M + H ]]⁺.Calc.Mass＝1804.6594。

((1S) -1-carboxy-5- (3- (3- (1- ((14S,17S) -17-carboxy-24- (4-iodophenyl) -12,15, 23-trioxo-14- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) butyl) -3,6, 9-trioxa-13, 16, 22-triazacyclotetracosyl) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) pentyl) carbamoyl) -L-glutamic acid (RPS-063) RPS-063 is according to the procedure described for RPS-069, composed of common structural units 26, 8a and 28 and azido-PEG₃-NHS ester synthesis. Mass (ESI +: 1762.4[ M + H ]]⁺.Mass(ESI-):1760.5[M-H]^-.Calc.Mass＝1761.71。

((1S) -1-carboxy-5- (3- (3- (1- ((29S,32S) -32-carboxy-39- (4-iodophenyl) -27,30, 38-trioxo-29- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododecan-2-yl) methyl) phenyl) thioureido) butyl) -3,6,9,12,15,18,21, 24-octaoxa-28, 31, 37-triazahutadecanyl) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) pentyl) carbamoyl) -L-glutamic acid (RPS-066) RPS-066 is according to the procedure described for RPS-069 Composed of the common structural units 26, 8a and 28 and azido-PEG₈-NHS ester synthesis. Mass (ESI +: 1982.3[ M + H ]]⁺,991.5[(M+2H)/2]^-.Calc.Mass＝1980.76。

((1S) -1-carboxy-5- (3- (3- (1- ((41S,44S) -44-carboxy-51- (4-iodophenyl) -39,42, 50-trioxo-41- (4- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododecan-2-yl) methyl) phenyl) thioureido) butyl) -3,6,9,12,15,18,21,24,27,30,33, 36-dodecaoxa-40, 43, 49-triazapentanyl) -1H-1,2, 3-triazol-5-yl) phenyl) ureido) pentyl) carbamoyl) -L-glutamic acid (RPS-067 is based on the comparison of RPS 069, from the common building blocks 26, 8a and 28 and azido-PEG₁₂-NHS ester synthesis. Mass (ESI +) 1079.7[ (M +2H)/2 ]]⁺.Mass(ESI-):2155.6(M-H)^-,1077.7[(M-2H)/2]^-.Calc.Mass＝2156.86。

Synthesis of RPS-068

N²-(N²- (2-azidoacetyl) -N⁶- (tert-Butoxycarbonyl) -L-lysyl) -N⁶- ((benzyloxy) carbonyl) -L-lysine tert-butyl ester (32): to in CH₂Cl₂To a solution of Fmoc-protected 8a (768mg, 0.97mmol) in (4mL) was added diethylamine (2.07mL, 20 mmol). The solution was stirred at room temperature overnight. The solvent was removed under reduced pressure and the crude product was used as the free amine as a yellow oil without further purification. To in CH₂Cl₂To a solution of free amine (356mg, 0.63mmol) in CH (6mL)₂Cl₂(1mL) of TEA (175. mu.L, 1.26mmol) and the resulting mixture was stirred at room temperature. Then added to CH₂Cl₂A solution of NHS ester 2-azidoacetate (138mg, 0.69mmol) in (3mL) was stirred at room temperature. After 3 hours, it is treated with CH₂Cl₂Diluting with H₂And washing with saturated NaCl solution. The organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure to give a pale yellow azide32(374mg, 92%), which was used without further purification. Mass (ESI +: 648.1[ M + H ]]⁺.Calc.Mass＝647.36。

((S) -1- (tert-butoxy) -6- (3- (3- (1- ((9S,12S) -9- (tert-butoxycarbonyl) -12- (4- ((tert-butoxycarbonyl) amino) butyl) -3,11, 14-trioxo-1-phenyl-2-oxa-4, 10, 13-triaza-pentadecan-15-yl) -1H-1,2, 3-triazol-5-yl) phenyl) ureido) -1-oxahex-2-yl) carbamoyl) -L-glutamic acid di-tert-butyl ester (33): 150 μ L of 0.5M CuSO in DMF (0.2mL)₄And 150 μ L of a solution of 1.5M sodium ascorbate were mixed for 5 minutes and then added to a solution of azide 32(374mg, 0.54mmol) and alkyne 26(358mg, 0.54mmol) in DMF (2 mL). The mixture was stirred at room temperature for 2h, then the solvent was removed under reduced pressure. Dissolving the residue in CH₂Cl₂In combination with H₂And O washing. The organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (0-10% MeOH in EtOAc) but small amounts of impurities remained. Thus, a second purification was performed by preparative TLC (100% EtOAc) and the product was isolated as a colorless oil (146 mg; 21%). Mass (ESI +: 1278.6[ M + H ]]⁺.Calc.Mass＝1277.73。

((S) -1- (tert-butoxy) -6- (3- (3- (1- (2- (((10S,13S) -13- (tert-butoxycarbonyl) -20- (4-iodophenyl) -2, 2-dimethyl-4, 11, 19-trioxo-3-oxa-5, 12, 18-triaza-eicosan-10-yl) amino) -2-oxoethyl) -1H-1,2, 3-triazol-5-yl) phenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamic acid di-tert-butyl ester (34): triazole 33(146mg, 0.11mmol) was dissolved in MeOH (10mL) in a two-necked flask. To the solution was added 10% Pd/C (10mg), and the suspension was shaken while evacuating the flask. The suspension is then suspended in H₂Stir under atmosphere for 2 hours, then filter the mixture through celite. The filter cake was washed 3 times with MeOH, the filtrates combined and concentrated under reduced pressure to give the free amine as a black residue (91 mg; 72%) containing traces of minor impurities. The crude product was used without further purification. Mass (ESI +: 1144.6[ M + H ]]⁺Mass 1143.69. To in CH₂Cl₂Free amine (90mg, 79. mu. mol) and NEt in (4mL)₃(14. mu.L, 150. mu. mol) of the solution was addedIn CH₂Cl₂(1mL) of a solution of 28(36mg, 100. mu. mol). The resulting mixture was stirred at room temperature overnight, then it was treated with CH₂Cl₂Diluting with H₂And washing with saturated NaCl solution. The organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure to give a black residue. The residue was dissolved in EtOAc and the black precipitate was removed by filtration. The resulting crude product was purified by preparative TLC (5% MeOH in EtOAc) and isolated as phenyl iodide 34 as a white solid (21 mg; 19%). Mass (ESI +: 1388.4[ M + H ]]⁺.Calc.Mass＝1387.63。

((1S) -1-carboxy-5- (3- (3- (1- (2- (((2S) -1- (((S) -1-carboxy-5- (2- (4-iodophenyl) acetylamino) pentyl) amino) -1-oxo-6- (3- (4- ((1,4,7, 10-tetrakis (carboxymethyl) -1,4,7, 10-tetraazacyclododec-2-yl) methyl) phenyl) thioureido) hexan-2-yl) amino) -2-oxyethyl) -1H-1,2, 3-triazol-5-yl) phenyl) ureido) pentyl) carbamoyl) -L-glutamic acid (RPS-068): to in CH₂Cl₂To a solution of 34(20mg, 15. mu. mol) in (3.5mL) was added TFA (0.5 mL). The reaction was stirred at room temperature for 4h, then concentrated under reduced pressure. The crude residue was dissolved in H₂In O and lyophilized to give the free amine as TFA salt. Mass (ESI +: 1064.1[ M + H ]]⁺Mass 1063.33. To p-SCN-Bn-DOTA.25HCl.25Hg25HgHg1 mL 50% DMF in water₂To a solution of O (Macrocyclics, Inc.) (10mg, 15. mu. mol) was added free amine (16mg, 15. mu. mol) in DMF (0.7 mL). Addition of NEt₃(110. mu.L) until the pH of the reaction was about 9. The reaction was stirred for 1h, then the reaction mixture was purified by preparative HPLC. The peak corresponding to the product was collected and lyophilized to give RPS-068 as a white powder (2.4 mg; 10%). Mass (ESI +: 1615.2[ M + H ]]⁺.Mass(ESI-):1613.3[M-H]^-,806.4[(M-2H)/2]^-.Calc.Mass＝1614.53。

Radiochemistry

The general method comprises the following steps: all reagents were purchased from Sigma Aldrich and were reagent grade unless otherwise indicated. Hydrochloric acid (HCl) and sodium acetate (NaOAc) in mass(>99.999%). All Water used (H)₂O) was high purity water (18 m.OMEGA.). Analytical HPLC was performed on a dual pump Varian Dynamax HPLC (Agilent technologies) equipped with a dual UV-Vis detector and radiochemical purity was determined using a NaI (Tl) flow counter detector (Bioscan). UV absorption was monitored at 220nm and 280 nm. The solvent A is in H₂0.01% trifluoroacetic acid (TFA) in O, solvent B in 90% v/v acetonitrile (MeCN): H₂0.01% TFA in O. In Symmetry 184.6 x 50mm,the analysis was performed on a column (Waters) over 10 minutes at a flow rate of 2mL/min and a gradient from 0% B to 100% B.

Production of Ga-66: gallium 66 (t)_1/29.4h) was determined from a natural zinc target (Alfa Aesar; 0.5g,100 μm thick, 99.999%) of radiation, generated by the (p, n) reaction taking place within 2 hours using a 15MeV beam and a 17.5mA current. Irradiation of native zinc produces Ga-66, Ga-67 and Ga-68. The target was left overnight to allow Ga-68 (t)_1/268min) was degraded before processing. The main radionuclide impurity in the process is Ga-67 (t)_1/278.3h), about 3%. The target was dissolved in HCl solution (5mL) and according to the previously published method [20 ]]By 20mg UTEVA anion exchange (Eichrom)⁶⁶Ga³⁺Ions with Zn²⁺And (5) separating ions. The column was then washed twice with 3ml of 5M HCl solution to eliminate excess Zn²⁺. Finally, purifying the obtained product⁶⁶Ga³⁺H for ions₂O (0.5mL) and the final solution was obtained containing 2.14-2.36GBq/mL (58-64mCi/mL) and about 0.1M HCl.

Radiolabelling of RPS series: prepared according to the following procedure⁶⁶Ga-labelled ligands. 100 μ L of Ga-66 stock solution containing 167-205MBq (4.5-5.5mCi) was diluted with 1mL of 0.05M HCl. To this solution 40-80 μ L of a 1mg/mL solution of the precursor in DMSO was added. The reaction was initiated by the addition of 40. mu.L of 3N NaOAc and the solution was incubated at 95 ℃ in EppendorfC (VWR) for 25 minutes.Then the mixture is taken up with H₂O was diluted and passed through a pre-activated Sep-Pak C18 Plus Light column (Waters). Column H₂O wash and product eluted with 100. mu.L EtOH (300proof, VWR) followed by 900. mu.L saline (0.9% NaCl solution; VWR). The final radioactive concentration is in the range of 7.4-85MBq/mL (0.2-2.3mCi/mL) and the radiochemical purity is greater than 90%.

Labeling with Lu-177: lu-177 without carrierPurchased as a chloride salt from iTG (Garching, Germany) and having an activity of 1.5-3.0GBq (40-80mCi) at the time of calibration. Aliquots of Lu-177 stock containing 0.52-0.93GBq (14-25mCi) were diluted to 1mL with 0.05M HCl. To this solution was added 20. mu.g of precursor, which was a 1mg/mL solution in DMSO. The reaction was initiated by raising the pH to 4-5 using 3N NaOAc (20-30. mu.L). The buffer solution was heated at 95 ℃ for 10 minutes on a simulated heating block (VWR). After the solution was cooled to room temperature, it was washed with H₂O (9mL) was diluted and passed through a pre-activated Sep-Pak C18 Plus Light column (Waters). By H₂The column was washed with O (5mL) and the product was eluted with 500. mu.L EtOH (200proof, VWR) followed by 500. mu.L saline (0.9% NaCl solution; VWR). An aliquot (40-98. mu.L) was removed from the solution and diluted to 4mL with saline. The final concentration of each ligand in the injection solution was 0.23-0.28. mu.M, and the activity ranged from 3.5-8.8MBq/mL (93-240. mu. Ci/mL).¹⁷⁷The specific activity of the Lu-labeled compound ranges from 15.8 to 48.8 GBq/. mu.mol. After purification and reconstitution, the radiochemical yield is 33-80% and the radiochemical purity is greater than 98%.

Cell culture: the human prostate cancer cell line LNCaP expressing PSMA was obtained from the american type culture collection. Cell cultures were obtained from Invitrogen unless otherwise indicated. At 37 deg.C/5% CO₂In a humidified incubator of (1), LNCaP cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone), 4mM L-glutamine, 1mM sodium pyruvate, 10mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 2.5mg/mL D-glucose, and 50. mu.g/mL gentamicin. By incubating it with 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA)Incubations, cells were removed from flasks for passaging or transferred to 12-well assay plates.

IC₅₀The in vitro assay of (1): according to the previously described methods [18,19 ]]After a small change, by aiming at^99mTc- ((7S,12S,16S) -1- (1- (carboxymethyl) -1H-imidazol-2-yl) -2- ((1- (carboxymethyl) -1H-imidazol-2-yl) methyl) -9, 14-dioxo-2, 8,13, 15-tetraazaoctadecane-7, 12,16, 18-tetracarboxylic technetium tricarbonyl complex) ((technetium tricarbonyl complex)^99mTc-MIP-1427) in a multiple concentration competitive binding assay for binding to PSMA on LNCaP cells to determine the IC of unlabeled metal-free ligand₅₀The value is obtained. Briefly, LNCaP cells were plated 48 hours prior to the experiment to achieve approximately 5x 10 in RPMI-1640 medium supplemented with 0.25% bovine serum albumin⁵Density of individual cells/well (in triplicate). Contacting the cells with 1nM in serum-free RPMI-1640 medium in the presence of 0.001-10,000nM test compound^99mTc-MIP-1427 was incubated for 2 hours. The radioactive incubation medium was then removed by pipette and the cells were washed twice using 1mL of ice-cold PBS 1X solution. After treatment with 1mL of 1M NaOH, cells were harvested from the plate and transferred to tubes using 2470Wizard²Radioactivity was counted in an automated gamma counter (Perkin Elmer). A standard solution (10% activity added to each well) was prepared for decay correction. IC was determined by fitting data points to a sigmoidal Hills1 curve in Origin software₅₀The value is obtained.

Inoculation of mice with xenografts: all animal studies have been approved by the animal care and use committee of the wel kannel institute of medicine, and conducted according to the guidelines prescribed by the usps for the policy of humane care and use of experimental animals. Animals were housed in approved facilities under standard conditions for 12 hour light/dark cycles. Food and water were provided ad libitum throughout the study. Hairless nu/nu mice were purchased from Jackson laboratories. For inoculation in mice, LNCaP cells were plated at 4x 10⁷The ratio of individual cells/mL was suspended in a 1:1 mixture of PBS: Matrigel (BDbiosciences). The left flank of each mouse was injected with 0.25mL of cell suspension. When the tumor reaches about 200-400mm³Time to smallImaging of mice, when tumors were at 100-³Within range, biodistribution is performed.

In LNCaP xenograft mice⁶⁶Imaging of Ga-RPS ligands: tumor-bearing mice (2-3 per compound) xenografted with LNCaP were injected intravenously with 0.56-5.4MBq (15-145. mu. Ci)⁶⁶A bolus of Ga-labeled ligand. The specific activity of the tracer was in the range of 14.8-47 MBq/. mu.mol (0.4-1.27 mCi/. mu.mol). Mu PET/CT (Inveon) was used 1,3, 6 and 24 hours after injection following inhalation anesthesia with isoflurane^TM(ii) a Siemens Medical Solutions, Inc.) mice were imaged. The total acquisition time for the 1 hour, 3 hour and 6 hour images was 30 minutes, while the total acquisition time for the 24 hour time point was 60 minutes. A CT scan is performed immediately prior to acquisition for anatomical co-registration and attenuation correction. Using a supplier supplied Inveon^TMThe software reconstructs the image. By comparison with the 10% injected dose per cubic millimeter (% ID/mm) introduced in the imaging field³) Standard comparisons estimated image-derived tumor and kidney uptake. Standards were prepared by diluting 10% of the injection activity to 1mL with saline. The volume of interest (VOI) was rendered by CT and confirmed by PET. The VOI content is integrated and after correction of the injected activity, the calculated counts are converted into% ID/mm by direct comparison with the above-mentioned standards³。

¹⁷⁷Biodistribution studies of Lu-labeled ligands in LNCaP xenograft mice: LNCaP xenograft tumor-bearing mice (5 per compound per time point) were injected intravenously with boluses of each ligand 348-. Mice were sacrificed at 4, 24 and 96 hours post injection. Blood samples were taken and a complete biodistribution study was performed on the following organs (including contents): heart, lung, liver, small intestine, large intestine, stomach, spleen, pancreas, kidney, muscle, bone, and tumor. The tissue is weighed and weighed at 2470Wizard²Count on an automatic gamma counter (Perkin Elmer). Counts were corrected to reduce decay and injection activity and tissue uptake was expressed as percent injected dose per gram (% ID/g). The standard error for each data point was calculated.

And (3) dose determination: dosimetry was calculated by linear interpolation between the three time points. The average injection dose per organ was calculated by using the average of the activity and organ weight of the mice at this time point. Intermediate time points every 4 hours are generated using linear approximation and the time intervals of all time points are corrected. These curves are integrated using trapezoidal approximation and the sum is used to determine residence time.

Statistical analysis: a comprehensive statistical analysis was performed to compare the tissue uptake of each compound over time. The normality hypothesis is visually checked through a quantile (QQ) chart and the data is log transformed to eliminate the skew effect. One-way ANOVA (analysis of variance) and Tukey's true significant difference (HSD) post hoc tests were used to assess the measured differences at the three time points, at each organ and each compound. The overall P-value under F-test and the pair P-value under t-test were determined. In addition, the effect of time, compound and its interaction in each organ was assessed using two-way ANOVA. The P value is reported. The 95% confidence interval was used to determine statistical significance.

Section 1.3 results and discussion

The three moieties were linked by azide-derived polyethylene glycol (PEG) spacers incorporating 0(RPS-068), 3(RPS-063), 4(RPS-061), 6(RPS-069), 8(RPS-066) or 12 PEG subunits (see Table 2). No degradation or decomposition of the ligand was observed during storage at 4 ℃ for three months as determined by analytical HPLC. In contrast, it was found that Gly-Gly-Gly linkers or C were used under the same storage conditions₇H₁₄Similar analogs of the linker instead of the PEG spacer decomposed over a period of several weeks.

In an effort to minimize the use of animals, primary screening of compounds was performed using μ PET/CT imaging to avoid unnecessary testing. For this reason, Ga-66 is preferred over other PET radionuclides (e.g., Ga-68 or Sc-44) due to its longer half-life (t)_1/29.4h) and may yield a greater amount in the cyclotron(s) ((s)>1.85GBq/50 mCi). More than 99% of Ga-66 was recovered during the purge, but the labeling rate remained consistently low (46.4 ± 20.5%, n ═ 7). Variable and low labeling yields are likely due to⁶⁶Ga³⁺The presence of Zn in the labeling reaction due to incomplete separation of ions from dissolved target species²⁺Ions.

After purification and reconstitution, was prepared in a radiochemical yield of 67 ± 17% (n ═ 20)¹⁷⁷Lu-labelled constructs. The variation in final product yield was mainly due to the difference in trapping efficiency of the C18 column, where¹⁷⁷Lu-RPS-067 and¹⁷⁷Lu-RPS-068 showed the lowest capture (about 40%). The labeling yield before purification was typically > 75% for all ligands as determined by radio HPLC. There was no apparent correlation between PEG length and labeling yield. Lu-177 labeled ligand was stable for 24 hours upon storage at 4 ℃. Radiochemical stability was not determined at room temperature.

The total amount of ligand injected per mouse is 22-24pmol to maintain and administer to human subjects¹⁷⁷Clinical Mass dose of Lu-PSMA-617 [ 4]]And (4) in proportion. The specific activity of the preparation is in the range of 15.8-48.8 GBq/. mu.mol, with¹⁷⁷Pre-clinical evaluation of Lu-PSMA-617 values reported [21]And (5) the consistency is achieved. By injection of⁶⁶The mass of Ga-labelled ligand was 4. mu.g per mouse, corresponding to 1.8-2.5 nmol. Greater mass is required to account for the poor labeling yield of this radionuclide.

Cell-based competitive binding assays were used to assess PSMA binding of all compounds in vitro. All compounds have high potency (IC)₅₀<10nM), we validated the 3-ethynylphenyl urea derivative of Glu-urea-Lys we selected as the PSMA targeting pharmacophore. Affinity range was defined by RPS-063 (IC)₅₀1.5 ± 0.3nM) and RPS-067 (IC)₅₀9.5 ± 1.1nM) (table 2). Efficacy generally decreased with increasing PEG linker length, although RPS-068 (PEG)₀；IC₅₀2.1 ± 0.1nM) was slightly less potent than RPS-063. IC of PSMA-617 in the same experiment₅₀Was determined to be 6.6. + -. 0.7nM (Table 2), corresponding to the previously reported value [21 ]]And (5) the consistency is achieved.

Table 2 summary of compound structures and key in vitro and in vivo characteristics. Determination of IC in LNCaP cells by competitive binding assay₅₀The value is obtained. By corresponding to¹⁷⁷Lu-labelled compoundsBiodistribution studies were performed in LNCaP xenograft tumor bearing mice to determine tumor uptake. (a-4 h p.i.; b-24 h p.i.)

By using⁶⁶The μ PET/CT imaging of Ga was used to perform a preliminary screening of compounds in mice (fig. 7) in order to avoid comprehensive biodistribution studies of poorly targeted compounds. The images were analyzed to determine quantitative uptake in tumor and kidney at 1,3, 6 and 24 hours post-injection. In tumors, uptake was higher, but decreased with increasing PEG length.⁶⁶Ga-RPS-068(PEG 0; maximum uptake 9.7. + -. 2.0% ID/cm)³(3h) (ii) a 8.3 +/-2.8% ID/cm at 24h³) And⁶⁶Ga-RPS-063(PEG 3; maximum uptake 9.5. + -. 2.4% ID/cm)³(6h) (ii) a 7.9 +/-3.0% ID/cm at 24h³) Exhibit maximal uptake, and⁶⁶Ga-RPS-061(PEG4；6.1±1.1％ID/cm³) And⁶⁶Ga-RPS-069(PEG6；7.0±3.9％ID/cm³) Indicating comparable uptake at 24h post injection.⁶⁶Ga-RPS-066(PEG 8; maximum uptake 7.8. + -. 0.7% ID/cm)³(3h) (ii) a 5.5. + -. 0.4% ID/cm at 24h³) And⁶⁶Ga-RPS-067(PEG 12; maximum uptake 6.6. + -. 3.2% ID/cm)³(1h) (ii) a 3.1. + -. 1.7% ID/cm at 24h³) Show lower uptake at all time points, but still exceed⁶⁶Ga-PSMA-617 (maximum uptake 3.1 + -0.4% ID/cm)³(1h) (ii) a 1.1. + -. 0.4% ID/cm at 24h³. Renal uptake was generally ranked the same as tumor uptake and was maximal at 1h post-injection. The range of 1 hour post-injection uptake was 10.5. + -. 2.1% ID/cm³(⁶⁶Ga-RPS-061) to 3.7. + -. 0.5% ID/cm³(⁶⁶Ga-RPS-067), 1.9. + -. 0.3% ID/cm 24 hours after injection³(⁶⁶Ga-RPS-068) to 0.2. + -. 0.1% ID/cm³(⁶⁶Ga-RPS-066 and⁶⁶Ga-RPS-067). In contrast to this, the present invention is,⁶⁶the maximal renal uptake of Ga-PSMA-617 was 0.4. + -. 0.1% ID/cm³And the intake at 24 hours was 0.1. + -. 0.1% ID/cm³。

After a promising imaging study, it was possible to,¹⁷⁷biodistribution studies of Lu-labeled ligands confirmed a clear trend in PET images. Although the affinity of the compounds for PSMA was concentrated to within an order of magnitude, the tissue distribution of the ligands showed considerable changes. This was most evident in tissues known to express PSMA, including tumors and kidneys (fig. 8).¹⁷⁷Lu-RPS-068(PEG₀)、¹⁷⁷Lu-RPS-063(PEG₃)、¹⁷⁷Lu-RPS-061(PEG₄)、¹⁷⁷Lu-RPS-069(PEG₆) And¹⁷⁷Lu-RPS-066(PEG₈) The tumor uptake is high and the maintenance is high. For higher affinity compounds¹⁷⁷Lu-RPS-068 and¹⁷⁷Lu-RPS-063, uptake at 4h p.i.was 21.8. + -. 2.8% ID/g and 30.0. + -. 3.1% ID/g, respectively, and at 96h p.i.was maintained at 14.9. + -. 1.5% ID/g and 12.9. + -. 0.5% ID/g. By 24 hours, clearance was statistically insignificant (p)>0.13). At 4h p.i. time¹⁷⁷Lu-RPS-069 and¹⁷⁷the uptake of Lu-RPS-066 was 17.0. + -. 2.1% ID/g and 18.7. + -. 1.1% ID/g, respectively, and dropped to 9.8. + -. 0.8% ID/g and 5.9. + -. 0.7% ID/g at 96h p.i.. However, these uptake values are taken after 24h p.i. and¹⁷⁷observed values of Lu-PSMA-617 (14.4. + -. 1.1% ID/g and 3.5. + -. 0.3% ID/g at 4h and 96h p.i., p.<0.001) is significantly higher. Ligands of lowest affinity¹⁷⁷Lu-RPS-067(PEG₁₂) At 4h p.i. only accumulated at a concentration of 7.6. + -. 1.2% ID/g and had cleared to 3.2. + -. 0.1% ID/g at 96h p.i. Except that¹⁷⁷Beyond Lu-PSMA-617, the uptake was significantly lower than for all other ligands (p < 0.001).

Similar trends in renal uptake were observed within the RPS series, with the lowest affinity ligand¹⁷⁷Lu-RPS-067 differs in that it has a significantly lower uptake at 4h p.i. (54.9. + -. 13.2% ID/g) (p) than the other RPS ligands tested<0.004) (fig. 8). For all other ligands of the RPS series, kidney uptake at 4h p.i. exceeded 100% ID/g, and was also found¹⁷⁷Lu-PSMA-617 fastRapid clearance (14.1. + -. 3.1% ID/g at 4h p.i.), which is consistent with published reports [21 [. ]]。¹⁷⁷Lu-RPS-068 (87.3. + -. 6.7% ID/g at 24h p.i.) and¹⁷⁷long term retention of Lu-RPS-063 (51.8. + -. 8.6% ID/g at 24h p.i.) is evident, but¹⁷⁷Lu-RPS-066 (6.2. + -. 0.8% ID/g at 24h p.i.) and¹⁷⁷Lu-RPS-067 (4.6. + -. 0.6% ID/g at 24h p.i.) is notable (p<0.001) and more quickly cleared. Uptake of these two ligands was significantly lower than other RPS ligands (p)<0.001) but not significantly different from each other (p)<0.14)。

Combined with continued tumor accumulation, faster renal clearance resulted at 24h p.i. time¹⁷⁷Lu-RPS-066 and¹⁷⁷the tumor to kidney ratio of Lu-RPS-067 was 1.92. + -. 0.30 and 1.25. + -. 0.20. These ratios are significantly higher than other RPS ligands (p)<0.001) but reflects low and fast renal clearance, rather than high and sustained tumor uptake. For the same reason, at all time points of the study,¹⁷⁷the tumor to kidney ratio of Lu-PSMA-617 was significantly higher than that of the other ligands (p)<0.001). By 96 hours, each member of the RPS series showed a tumor to kidney ratio substantially in excess of 1 (range 1.56-3.32).

Uptake was negligible in other tissues than spleen, which showed moderate, possibly PSMA-mediated uptake at 4h p.i. and then cleared to background levels (fig. 8). As expected, all RPS series had significantly higher blood activity than the other RPS series¹⁷⁷Lu-PSMA-617(p<0.05)。¹⁷⁷Lu-RPS-063、¹⁷⁷Lu-RPS-061 and¹⁷⁷Lu-RPS-068 showed the highest blood activity at 4h p.i. (FIG. 9), while¹⁷⁷Lu-RPS-069、¹⁷⁷Lu-RPS-066 and¹⁷⁷Lu-RPS-067 showed lower blood retention at the same time point. By 24h p.i., all ligands had blood activity below 0.3% ID/g, and by 96h, blood activity dropped below 0.1% ID/g. Interestingly, although all RPS ligands contained the same albumin binding group N⁶- (2- (4-iodophenyl) acetyl) -L-lysine, but in shorter PEG compounds¹⁷⁷Lu-RPS-068 and¹⁷⁷Lu-RPS-063 and longer PEG compounds¹⁷⁷Lu-RPS-066 and¹⁷⁷a significant difference (p) was observed between Lu-RPS-067<0.001). This indicates that the linker affects binding to and/or clearance of plasma proteins.

The negative correlation between PEG length and affinity for PSMA is consistent with findings reported to date for PSMA constructs and other targeting ligands. Small PEG linkers (e.g., PEG3 or PEG4) have been incorporated into PSMA-targeting small molecule drug conjugates [22,23]However, to date, constructs of this nature have shown low affinity and/or poor tumor uptake. One SAR study did confirm that the PEG2 and PEG4 linkers best retained PSMA affinity in the PSMA-targeted contrast family, whereas PEG12 and PEG24 resulted in a large decrease in affinity [24 ]]. These results are consistent with the following observations: in small molecule GCPII ligands, the PEG12 linker has reduced affinity relative to PEG 8[ 25]. About PEG linker pair⁶⁸SAR studies of the effects of Ga-labeled bombesin antagonists found that with each incremental extension of the PEG linker, the affinity decreased slightly [26 ]]. This study also identified minor differences in the biodistribution of the ligands.

The area under the curve (AUC) of the time-activity curve (TAC) of tumor uptake,¹⁷⁷lu-labelled RPS-061, -063, 066, -068 and-069 ligands with¹⁷⁷Lu-PSMA-617 delivered significantly larger doses to tumors than did the tumor (fig. 10). This can be confirmed by comparing the dose integrals in the tumors.¹⁷⁷Lu-RPS-068 and¹⁷⁷Lu-RPS-063 ratio¹⁷⁷Lu-PSMA-617 is approximately 4 times higher, and¹⁷⁷Lu-RPS-061、⁷⁷Lu-RPS-069 and¹⁷⁷Lu-RPS-066 is also at least twice as high (FIG. 11).

It is possible that¹⁷⁷Tumor uptake of Lu-labeled RPS ligands is also greater than other reports to date¹⁷⁷Lu-labeled PSMA targeting ligands are higher, and the other ligands include¹⁷⁷Lu-PSMA I&T (reported uptake in LNCaP tumors at 1h p.i. 7.96. + -. 1.76[27 ]]) And recently reported¹⁷⁷Lu-CTT1403 (reported to reach 46% ID/g at 72h p.i. in PC3-PIP tumors [23 ]]). PSMA expression in PC3-PIP tumors was higher than that common in human prostate cancer by ten times than in LNCaP cells [28]This means that¹⁷⁷The uptake of Lu-CTT1403 was likely to be much lower in LNCaP tumors.¹⁷⁷Lu-RPS-063 and¹⁷⁷uptake of Lu-RPS-068 in LNCaP tumors and reports¹³¹Uptake of I-MIP-1095 [29]Rather, to our knowledge, the uptake of this small molecule in LNCaP xenograft tumors is greatest in reports to date.¹⁷⁷Lu-RPS-063、¹⁷⁷Lu-RPS-068 and¹³¹comparison of TAC for I-MIP-1095 showed that AUC was similar over the 96 hours of the study (FIG. 10).

It has previously been reported that prolonged blood retention leads to increased tumor accumulation over time [23,30]Presumably as a result of an increased number of ligand passages through the tumor bed.¹⁷⁷Lu-RPS-068 appears to increase from 4 hours to 24 hours, but this difference is not statistically significant (p ═ 0.26). This phenomenon was also not evident in other trifunctional RPS ligand series after 4h p.i., although a delay in blood clearance for the first 4 hours may increase tumor uptake within this time interval. However, the clearance rate of the ligand from the tumor is very slow and thus is comparable to¹⁷⁷Lu-PSMA-617 can deliver more activity to a target tissue than it does. Further modification of albumin binding by substitution of albumin binding groups can be used to subtly alter blood clearance and enhance high and durable tumor accumulation.

Although the clinical outcome for mCRPC using radioligand therapy targeting PSMA is encouraging, (1) overcoming resistance to beta-particle radiation, (2) being suitable for the treatment of diffuse metastatic lesions (especially in bone), and (3) the next generation of ligands that provide a longer progression-free survival are crucial for continued improvement of treatment. Although at present and¹⁷⁷Lu-PSMA-617 or¹³¹Single dose-related toxicity of I-MIP-1095 is minimal, but the incidence of hematologic toxicity and persistent dry mouth may increase during subsequent treatment cycles [3]And biochemical reactions may be reduced [3,5 ]]. Alpha particle mediated therapy has been proposed as a means to overcome resistance to beta particles and reduce hematologic toxicity [31]. Early use²¹³Bi-PSMA I&Preclinical studies performed by T have established the formation of DNA double strand breaks in tumors in vivo [32]To human patients²²⁵Ac-PSMA-617 or²¹³Primary treatment of Bi-PSMA-617 has elicited a tremendous response in refractory cancers [31,33]. However, multiple treatment cycles are required for therapeutic efficacy, leading to irreversible dry mouth and keratoconjunctivitis sicca [33]。

These early findings have demonstrated the therapeutic potential of alpha-particle radiotherapy, but underscore the need for radioligands with higher therapeutic indices that can deliver high doses to tumors. In LNCaP xenograft tumors,¹⁷⁷each of Lu-RPS-061, -063, -066, -068, and-069 exhibits a ratio¹⁷⁷Lu-PSMA-617 significantly higher tumor uptake and a corresponding increase in AUC correlated with an increase in the radioactive dose delivered to the tumor. Three ligands with highest tumor absorption rate¹⁷⁷Lu-RPS-063、¹⁷⁷Lu-RPS-068 and¹⁷⁷the tumor to kidney ratio of Lu-RPS-061 was 2.75 + -0.17, 1.56 + -0.23 and 3.64 + -0.29 at 96h p.i. respectively. On the contrary, the present invention is not limited to the above-described embodiments,¹⁷⁷Lu-CTT1403 never reached 1.0[23 ]]. Although at the same point in time¹⁷⁷The tumor to kidney ratio of Lu-PSMA-617 was 14.39 ± 2.2 and the ratio of tumor dose integral to kidney dose integral was 1.95 over 96 hours, but this was due to very low kidney uptake and high non-tumor uptake. It has been widely demonstrated that PSMA expression in the kidneys of nude mice is higher than that in the human kidneys [34,35,36]This means that preclinical studies consistently overestimate the dose delivered to this organ. Several PSMA-targeted therapeutics (including¹³¹I-MIP-1095(29)、¹⁷⁷Lu-DKFZ-617(21) and¹⁷⁷Lu-PSMA I&t (37)) all showed early kidney concentrations equal to or higher than 100% ID/g in nude mice, but were safely switched to clinical and acceptable; despite the different renal doses.

In addition, other kidney protection regimens, including pharmacological replacement with 2-PMPA, have been shown to further reduce activity in the kidney [37,38 ]. Taken together, these observations indicate that trifunctional RPS ligands exhibit both high and sustained tumor uptake and a broad therapeutic index, which is desirable for alpha particle radiation therapy.

Reference is made to section 1.3

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Section 1.4

Materials and instruments. The synthesis of RPS-074 is described below. All solvents and reagents were purchased from commercial suppliers and used without further purification. The intermediates (((S) -1- (tert-butoxy) -6- (3- (3-ethynylphenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamic acid di-tert-butyl ester (406) and macropa-NCS were synthesized as described above. Is used inHigh Purity Silica GelCompounds were purified by silica gel chromatography on silica gel, preparative tlc (analtech) on silica-coated glass plates, or flash chromatography using CombiFlash Rf + (Teledyne Isco) system. Using Xbridge^TMPrep C18 5μm OBD^TMPreparative HPLC was performed on a 19X 100mm column (Waters) on a two-pump Agilent ProStar HPLC equipped with an Agilent ProStar 325Dual wavelet UV-Vis detector. At 220nm and 280nmUV absorption was monitored. Using a binary solvent system, wherein solvent A comprises H₂O + 0.01% TFA, solvent B contained 90% v/v MeCN/H₂O + 0.01% TFA. Purification was performed using the following gradient HPLC method: 0-1 minute of 0% B, 1-28 minutes of 0-100% B, 28-30 minutes of 100-0% B.

The final product was identified and characterized using thin layer chromatography, analytical HPLC and mass spectrometry. NMR spectroscopy was used to confirm the structures of Compound 406 and macropa-NCS. NMR analysis was performed using a Bruker Avance III 500MHz spectrometer. Spectrum in CDCl₃Or in DMSO-d 6. Using XSelect^TMCSH^TMAnalytical HPLC was performed on C185 μm 4.6X 50mm columns (Waters). Use of Waters ACQUITY connected to Waters SQ Detector 2Mass determination was performed by LCMS analysis. All compounds evaluated in the biological assay were greater than 95% pure as judged by analytical HPLC.

Synthesis of RPS-074.

N²- (1- (9H-fluoren-9-yl) -3-oxo-2, 7,10,13,16,19,22,25, 28-nonaoxa-4-azatriundecane-31-acyl) -N⁶Preparation of t-butyl (- (benzyloxy) carbonyl) -L-lysine (402). To Fmoc-N-amino-PEG-8-acid (663mg, 1.0mmol), L-N in DMF (10mL)^εTo a stirred mixture of-Z-Lys-OtBu hydrochloride (446mg, 1.2mmol) and HATU (456mg, 1.2mmol) was added DIPEA (260mg, 2.0mmol) and the reaction was allowed to react at room temperature under N₂Stirring was continued overnight. The solvent was removed under reduced pressure and the crude residue was purified by flash chromatography (0-10% MeOH in CH)₂Cl₂Solution) to give compound 2 as a colorless oil (845mg, 86%). Mass (ESI +: 983.0[ M + H ]]⁺.Calc.Mass:981.5。

N²- (1- (9H-fluoren-9-yl) -3-oxo-2, 7,10,13,16,19,22,25, 28-nonaoxa-4-azatriundecane-31-acyl) -N⁶- (4- (4-iodophenyl) butanoyl) -L-lysinePreparation of tert-butyl ester of amino acid (403). Compound 402(1.45g, 1.48mmol) was dissolved in MeOH (25 mL). 10% Palladium on charcoal (15mg) was added and the suspension was stirred in a three-necked flask at room temperature for 10 minutes. The flask was evacuated and then placed in H₂Under an atmosphere. The suspension was then stirred at room temperature for 5 hours and then filtered through celite. The filter cake was washed with MeOH, and the combined filtrates were concentrated under reduced pressure to give the amine as a yellow oil (1.17g, 93%), which was used without further purification. Mass (ESI +: 849.4[ M + H ]]⁺Mass 848.0. To in CH₂Cl₂To a solution of amine (865mg, 1.01mmol) and 4- (4-iodophenyl) butanoic acid 2, 5-dioxopyrrolidin-1-yl ester (387mg, 1.00mmol) (20mL) was added TEA (167. mu.L, 1.20 mmol). The resulting solution was stirred at room temperature under Ar for 4 hours. The solution was then treated sequentially with 1% v/v AcOH/H₂O and brine wash. The organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure to give a yellow oil. By flash chromatography (0-30% MeOH in CH)₂Cl₂Solution) and compound 3 was isolated as a yellow oil (360mg, 32%). Mass (ESI +: 1120.9[ M + H ]]⁺.Calc.Mass:1119.5。

N²- ((S) -10- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2, 2-dimethyl-4, 11-dioxo-3, 15,18,21,24,27,30,33, 36-nonaoxa-5, 12-diaza-trinexadecane-39-yl) -N-acetyl⁶Preparation of tert-butyl- (4- (4-iodophenyl) butanoyl) -L-lysine (404). Will be in CH at room temperature₂Cl₂A solution of 403(360mg, 0.32mmol) and diethylamine (0.67mL, 6.48mmol) in (2mL) was stirred for 7 hours. The solution was concentrated under reduced pressure and the crude residue was purified by flash chromatography (0-30% MeOH in CH)₂Cl₂A solution). The product containing fractions were combined and concentrated to give the amine as a yellow oil (96mg, 33%). Mass (ESI +: 899.2[ M + H ]]⁺Mass 897.4. To in CH₂Cl₂TEA (28. mu.L, 200. mu. mol) was added to a solution of amine (96mg, 107. mu. mol) and Fmoc-L-Lys (Boc) -OSu (62mg, 110. mu. mol) (5 mL). The mixture was stirred at room temperature under Ar overnight. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (0-30% MeOH in CH)₂Cl₂A solution). The desired product was co-eluted with a small amount of impurities, so the mixture was passed through preparative TLC (10% v/v MeOH/CH)₂Cl₂) A second purification is performed. Compound 404 was isolated as a colorless oil (78mg, 51%). Mass (ESI +: 1349.0[ M + H ]]⁺.Calc.Mass:1347.6。

N²- ((S) -10-amino-2, 2-dimethyl-4, 11-dioxo-3, 15,18,21,24,27,30,33, 36-nonaoxa-5, 12-diazatrinonadecane-39-acyl) -N⁶Preparation of tert-butyl- (4- (4-iodophenyl) butanoyl) -L-lysine (405). Will be in CH₂Cl₂A solution of 404(73mg, 54. mu. mol) and diethylamine (0.5mL, 4.83mmol) in (2mL) was stirred at room temperature overnight. The solvent was removed under reduced pressure and the crude product was dissolved in MeOH and purified by preparative TLC (10% v/v MeOH in CH)₂Cl₂A solution). Amine 405 was isolated as a light oil (25mg, 41%). Mass (ESI +: 1127.7[ M + H ]]⁺.Calc.Mass:1126.2。

Preparation of di-tert-butyl ((S) -1- (tert-butoxy) -6- (3- (3- (1- (2- (2- (2- (3- ((2, 5-dioxopyrrolidin-1-yl) oxy) -3-oxopropoxy) ethoxy) ethyl) -1H-1,2, 3-triazol-4-yl) phenyl) ureido) -1-oxohex-2-yl) carbamoyl) -L-glutamate (407). Mix 0.5M CuSO₄(100. mu.L) and 1.5M sodium ascorbate (100. mu.L) until the brown color turned orange. The mixture was then added to a solution of 406(315mg, 0.5mmol) and azido-PEG 3-NHS (177mg, 0.5mmol) in DMF (2 mL). The mixture was stirred at room temperature for 2 hours. Then using it with CH₂Cl₂Diluting and mixing with H₂And O washing. The organic layer was MgSO₄Dried, filtered and concentrated under reduced pressure to give a light-colored oil. The crude product was purified by flash chromatography (0-30% MeOH in CH)₂Cl₂Solution) to yield compound 407 as a clear oil (460mg, 95%). Mass (ESI +: 975.9[ M + H ]]⁺.Calc.Mass:974.5。

((S) -1- (tert-butoxy) -6- (3- (3- (1- ((14S,45S) -45- (tert-butoxycarbonyl) -14- (4- ((tert-butoxycarbonyl) amino) butyl) -54- (4-iodophenyl) -12,15,43, 51-tetraoxy-3, 6,9,19,22,25,28,31,34,37, 40-undecaoxa-13, 16,44, 50-tetraazapentatetradecyl) -1H-1,2, 3-triazole-4-carboxylic acidPreparation of (tert-butyl) -yl) phenyl) ureido) -1-oxyhex-2-yl) carbamoyl) -L-glutamic acid (408). To in CH₂Cl₂To a solution of amine 405(25mg, 22. mu. mol) in CH (4mL)₂Cl₂(1mL) of a solution of ester 407(24mg, 25. mu. mol) and TEA (7. mu.L, 50. mu. mol). The reaction was stirred at room temperature under Ar for 5 hours. The reaction was then concentrated under reduced pressure and the crude residue was dissolved in EtOAc (1mL) and purified by preparative TLC (90% EtOAc in hexanes) to give compound 408(33mg, 76%) as a pale yellow oil. Mass (ESI +) 994.3[ (M +2H)/2 +]⁺.Calc.Mass:1986.2。

((S) -1-carboxy-5- (3- (3- (1- ((14S,45S) -45-carboxy-14- (4- (3- (2-carboxy-6- ((16- ((6-carboxypyridin-2-yl) methyl) -1,4,10, 13-tetraoxa-7, 16-diazacyclooctadecan-7-yl) methyl) pyridin-4-yl) thioureido) butyl) -54- (4-iodophenyl) -12,15,43, 51-tetraoxa-3, 6,9,19,22,25,28,31,34,37, 40-undecaoxa-13, 16,44, 50-tetraazapentatetradecyl) -1H-1, preparation of 2, 3-triazol-4-yl) phenyl) ureido) pentyl) carbamoyl) -L-glutamic acid (RPS-074).

Compound 408(33mg, 16. mu. mol) was dissolved in CH₂Cl₂(2 mL). TFA (0.5mL) was then added and the reaction was stirred at room temperature overnight. In N₂The solvent was removed under air flow and the crude product was lyophilized to give a white residue (22mg, 83%). Mass (ESI +) 832.0[ (M +2H)/2]⁺Mass 1661.8. To a solution of free amine (13mg, 7.8. mu. mol) and TEA (0.22mL, 1.56mmol) in DMF (1mL) was added a solution of macropa-NHS (6mg, 10. mu. mol) in DMF (1 mL). The resulting mixture was stirred at room temperature for 90 minutes. The reaction was concentrated under reduced pressure and the crude product was purified by preparative HPLC. The peak corresponding to the desired product was collected and lyophilized to give RPS-074 as a white powder (4.5mg, 26%). Mass (ESI +) 1126.6[ (M +2H)/2 ]]⁺.Calc.Mass:2251.3。

Synthesis of DOTA-Lys-IPBA

Preparation of 2, 5-dioxopyrrolidin-1-yl 4- (4-iodophenyl) butanoate (409). Will be in CH₂Cl₂A solution of 4- (4-iodophenyl) butyric acid (1.16g, 4.0mmol), N-hydroxysuccinimide (483mg, 4.2mmol), EDC.HCl (768mg, 4.0mmol) and 4-DMAP (5.8mg, 47. mu. mol) in (30mL) was stirred for 20 h. The reaction mixture was then washed successively with 1M HCl, saturated NaHCO₃And a brine wash. The organic layer was MgSO₄Drying, filtration and concentration under reduced pressure gave NHS ester 409 as a white powder (1.29g, 83%).¹H NMR(CDCl₃,500MHz):δ7.61(d,2H,J＝7.2Hz).6.95(d,2H,J＝7.6Hz),2.83(s,4H),2.67(t,2H,J＝7.6Hz),2.59(t,2H,J＝7.3Hz),2.03(quint,2H,J＝7.3Hz)。

N²- (tert-butyloxycarbonyl) -N⁶Preparation of- (4- (4-iodophenyl) butyryl) -L-lysine (410). Boc-L-Lys-OH (871mg, 3.53mmol) was suspended in DMF (10mL) and stirred at room temperature. To the stirred suspension, NHS ester 409(1.29g, 3.33mmol) in DMF (5mL) and NEt were slowly added₃(557. mu.L, 4.00 mmol). The resulting suspension was stirred at room temperature overnight. The reaction was quenched with 1M HCl (2mL) and the solvent was removed under reduced pressure. The crude residue was dissolved in CH₂Cl₂In sequence with 1M HCl and saturated NaHCO₃The solution and brine washes. The organic fraction is over MgSO₄Drying, filtration and concentration under reduced pressure gave Boc-Lys-IPBA (410) as a clear foam (1.25g, 72%).¹H NMR(CDCl₃,500MHz):δ7.57(d,2H,J＝7.7Hz),6.91(d,2H,J＝7.8Hz),5.94(br s,1H),5.32(br s,1H),4.21(m,1H),3.21(m,2H),2.56(t,2H,J＝7.6Hz),2.15(t,2H,J＝7.1Hz),1.90(quint,2H,J＝7.5Hz),1.88(m,1H),1.69(m,1H),1.51(m,2H),1.42(s,9H),1.41(m,2H).Mass(ESI+):519.3(M+H)⁺.Calc.Mass:518。

N⁶Preparation of- (4- (4-iodophenyl) butyryl) -L-lysine (411). Boc-Lys-IPBA (518mg, 1.0mmol) was dissolved in 10mL of 20% v/v TFA/CH₂Cl₂To the solution and stirred at room temperature overnight. In N₂Removing the solvent under a gas stream and separatingLys-IPBA (411) (402 mg; 96%) was obtained as a colourless oil.¹H NMR(DMSO,500MHz):δ7.75(br s,1H),7.61(d,2H,J＝7.8Hz),6.99(d,2H,J＝7.8Hz),3.79(m,1H),2.99(m,2H),2.02(t,2H,J＝7.3Hz),1.74(quint,2H,J＝7.4Hz),1.37(m,4H),1.24(m,2H).Mass(ESI+):419.2(M+H)⁺.Calc.Mass:418.3。

Preparation of 2,2',2 ", 2"' - (2- (4- (3- ((S) -1-carboxy-5- (4- (4-iodophenyl) butyrylamino) pentyl) thioureido) benzyl) -1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetrayl) tetraacetic acid (DOTA-Lys-IPBA). To a solution of Lys-IPBA (11mg, 26. mu. mol) and DIPEA (17. mu.L, 100. mu. mol) in DMF (1mL) was added H₂p-SCN-Bn-DOTA.2.5 Cl.2.5H in O (1mL)₂O (8mg, 11.6. mu. mol). The reaction was stirred at room temperature for 4 hours. The solvent was removed under reduced pressure and the crude residue was purified by preparative HPLC. The peak corresponding to the product was collected and lyophilized to give DOTA-Lys-IPBA as a white powder (5mg, 43%).¹H NMR(DMSO,500MHz):δ9.72(br s,1H),7.89(d,2H,J＝7.6Hz),7.77(m,1H),7.61(d,2H,J＝7.1Hz),7.51(d,2H,J＝7.8Hz),7.24(m,2H),6.99(d,2H,J＝7.8Hz),4.84(m,1H),3.70-3.04(m,14H),3.01(m,4H),2.02(t,2H,J＝7.1Hz),1.76(m,4H),1.39(m,2H),1.31(m,2H).Mass(ESI+):971.0(M+H)⁺.Calc.Mass:969.9。

Radiochemistry. All reagents were purchased from Sigma Aldrich and were reagent grade unless otherwise indicated. Hydrochloric acid (HCl) is(>99.999%) with trace analytical mass. Aluminum-supported silica Thin Layer Chromatography (TLC) plates were purchased from Sigma Aldrich. By being atDilution in Water to prepare 0.05M HCl and 1M NH₄Stock solutions of OAc.

²²⁵Ac-RPS-074: to a solution in 0.05M HCl²²⁵Ac(NO₃)₃(Oak Ridge National Laboratory, USA) (950. mu.L of 16.7-21.0MBq) 20. mu.L of RPS-074 in DMSO was added in 1 mg/mL. By adding 90. mu.L of 1M NH₄OAc raises the pH to 5-5.5. The reaction was incubated at 25 ℃ under EppendorfC (VWR) for 20 minutes. Then, the reaction mixture is reacted with H₂O (9mL) was diluted and passed through a pre-activated Sep-Pak C18 Light column (Waters). Reaction vial and small column are washed with H₂O (5mL) wash, and then 500. mu.L EtOH followed by 500. mu.L physiological saline (in deionized H)₂0.9% NaCl in O; VWR). Diluting the eluate to 4mL in physiological saline to obtain stock solution with radioactivity concentration of 1.1-1.5 MBq/mL. An aliquot was taken from the final solution and spotted on an aluminum-supported silica TLC plate to identify radiochemical impurities. Will be in 0.05M HCl²²⁵Ac(NO₃)₃An aliquot of the solution was spotted in parallel lanes as a control. Immediately, the plate was run in 10% v/v MeOH/10mM EDTA mobile phase and then left for 8 hours to reach radiochemical equilibrium. After 3 minutes exposure on a fluorescent screen, the plate was observed on a Cyclone Plus storage phosphor System (Perkin Elmer). The radiochemical purity is expressed as²²⁵The ratio of Ac-RPS-074 to total activity was determined to be 98.1%. The plates were visualized again after 16 hours to confirm purity.

²²⁵Ac-DOTA-Lys-IPBA: to 2 in 0.05M HCl²²⁵Ac(NO₃)₃(Oak Ridge National Laboratory, USA) (5.0 MBq in 900. mu.L) to a solution of 30. mu.L DOTA-Lys-IPBA in DMSO at 1mg/mL was added. By adding 80. mu.L of 1M NH₄OAc raises the pH to 5-5.5, and then the reaction solution is placed in EppendorfC (VWR) at 95 ℃ for 25 minutes. The reaction mixture is then washed with H₂O (9mL) was diluted and passed through a pre-activated Sep-Pak C18 Light column (Waters). Reaction flask and small column are connected with H₂O (5mL) wash with 200. mu.L of 50% v/v EtOH/saline followed by 800. mu.L of physiological saline (in deionized H)₂0.9% NaCl in O; VWR) eluted product. Radiochemistry as described above, by radioTLCPurity (96%).

IC₅₀The in vitro assay of (1): according to the previously described method [2 ]]After a small change, by aiming at^99mTc- ((7S,12S,16S) -1- (1- (carboxymethyl) -1H-imidazol-2-yl) -2- ((1- (carboxymethyl) -1H-imidazol-2-yl) methyl) -9, 14-dioxo-2, 8,13, 15-tetraazaoctadecane-7, 12,16, 18-tetracarboxylic technetium tricarbonyl complex) ((technetium tricarbonyl complex)^99mTc-MIP-1427)(K_d＝0.64±0.46nM[1]) Screening in a multiple concentration competitive binding assay for binding to PSMA on LNCaP cells to determine the IC of unlabeled metal-free ligand₅₀The value is obtained. Briefly, LNCaP cells were plated 72 hours prior to the experiment to achieve approximately 5x 10 in RPMI-1640 medium supplemented with 0.25% bovine serum albumin⁵Density of individual cells/well (in triplicate). In RPMI-1640 medium [ 3] containing 0.00125% w/v bovine serum albumin]In the presence of 0.001-10,000nM test compound, the cells are incubated with 1nM^99mTc-MIP-1427 was incubated for 2 hours. The radioactive incubation medium was then removed by pipette and the cells were washed twice using 1mL of ice-cold PBS 1X solution. After treatment with 1mL of 1M NaOH, cells were harvested from the plate and transferred to tubes using 2470Wizard²Radioactivity was counted in an automated gamma counter (Perkin Elmer). A standard solution (10% activity added to each well) was prepared for decay correction. To is directed at^99mNon-specific binding of Tc-MIP-1427 corrects cell-specific activity. IC was determined by fitting data points to a sigmoidal Hills1 curve in Origin software₅₀The value is obtained.

Inoculation of mice with xenografts: all animal studies have been approved by the animal care and use committee of the wel kannel institute of medicine, and conducted according to the guidelines prescribed by the usps for the policy of humane care and use of experimental animals. Animals were housed in approved facilities under standard conditions for 12 hour light/dark cycles. Food and water were provided ad libitum throughout the study. Male BALB/c athymic nu/nu mice were purchased from Jackson laboratories. For inoculation in mice, LNCaP cells were plated at 4x 10⁷The ratio of individual cells/mL was suspended in a 1:1 mixture of PBS: Matrigel (BD Biosciences). The left flank of each mouse was injected with 0.25mL of cell suspension. When the tumor reaches about 200-800mm³The biodistribution is performed when the tumor is 50-900mm³Within range, a therapeutic study is initiated.

Biodistribution studies in LNCaP xenograft mice. LNCaP xenograft tumor bearing mice (4 per compound per time point) were injected intravenously with 105kBq and 320ng (142pmol)²²⁵Bolus of Ac-RPS-074. Mice were sacrificed at 4h, 24h, 7d, 14d and 21d post injection. Blood samples were taken and a complete biodistribution study was performed on the following organs (including contents): heart, lung, liver, small intestine, large intestine, stomach, spleen, pancreas, kidney, muscle, bone, and tumor. The tissue is weighed and weighed at 2470Wizard²Count on an automatic gamma counter (Perkin Elmer). Counts were corrected to reduce decay and injection activity and tissue uptake was expressed as percent injected dose per gram (% ID/g). The standard error for each data point was calculated.

Therapeutic studies in LNCaP xenograft mice. Mice bearing LNCaP xenograft tumors were randomly divided into 5 groups (7 per group). Set of intravenous injections 148kBq and 93ng (41pmol)²²⁵Bolus of Ac-RPS-074. The second treatment group was injected with 74kBq and 47ng (21pmol)²²⁵Ac-RPS-074. The third treatment group was injected with 37kBq and 23ng (10pmol)²²⁵Ac-RPS-074. The fourth group injected the same volume of vehicle. Fifth group injection 133kBq²²⁵Ac-DOTA-Lys-IPBA. Tumor size was measured and recorded three times per week with digital calipers and using a modified ellipseSphere equation V0.5 length width 4]Tumor volume was calculated. The tumor reaches 2000mm³After or if they show any visible signs of discomfort (including weight loss, loss of appetite, excessive sleepiness or sore and rash formation), the mice are sacrificed. Body weight was measured twice weekly using a digital balance and mice were monitored for signs of discomfort. Mice were photographed weekly to visually confirm changes in tumor volume.

The treated mice were imaged by μ PET/CT. As previously reported [5 ]]Preparation of⁶⁸Ga-PSMA-11 (also known as⁶⁸Ga-HBED-CC). At injection 138kBq or 74kBq²²⁵75 days after Ac-RPS-074, 8 mice were injected intravenously with 5.5MBq⁶⁸Ga-PSMA-11. After isoflurane inhalation anesthesia, mu PET/CT (Inveon) was used 1 hour after injection^TM(ii) a Siemens Medical Solutions, Inc.) mice were imaged. The total collection time was 30 minutes. A CT scan is performed immediately prior to acquisition for anatomical co-registration and attenuation correction. Using a supplier supplied Inveon^TMThe software reconstructs the image.

In vitro and in vivo evaluation of RPS-074

Use is directed to^99mMulti-concentration competitive binding assay for Tc-MIP-1427 determination of IC of RPS-074 in vitro₅₀Values showing affinity for PSMA on LNCaP cells. The results show that IC of RPS-074₅₀12.0 + -3.4 nM, which is consistent with the reported PSMA affinity of structurally similar trifunctional ligands [6 ]]. The biodistribution of RPS-074 was examined in mice carrying LNCaP xenografts. Mice were injected intravenously with 105kBq and 320ng (142pmol)²²⁵Bolus of Ac-RPS-074. Mice were sacrificed 4 hours, 24 hours, 7 days, 14 days, and 21 days post injection. FIG. 12 demonstrates that 4 hours after injection (p.i.) in blood (12.3. + -. 0.5% ID/g), lung (5.0. + -. 0.2% ID/g), kidney (6.7. + -. 0.4% ID/g) and tumor (5.8. + -. 0.3% ID/g)²²⁵The uptake of Ac-RPS-074 was evident. By 24 hours post-injection, non-target tissues including kidney (3.0 ± 0.3% ID/g) were cleared of activity concurrently with blood clearance, and activity in tumors increased to 12.7 ± 1.5% ID/g (fig. 12). By 7 days after injection, the activity in the tumor was still high (9.5. + -. 1.5% ID/g), while blood and itActivity in all his tissues was below 1% ID/g (FIG. 12). There was clearly persistent tumor uptake (11.9 ± 1.5% ID/g) at day 14 post-injection, with little difference from background for all other tissues. By day 21 after injection, the anti-tumor effect was clearly evident, and only 1 mouse had tumor. Despite the absence of tumor, activity in non-target tissues remained indistinguishable from background.

Even in the absence of a tumor,²²⁵Ac-RPS-074 still showed excellent complex stability within 3 weeks. Biodistribution studies have shown that there is no significant signal accumulation in the liver or bone, which are two normally ingested freedoms²²⁵Ac³⁺Organ of (7)]。²²⁵Ac-RPS-074 also shows good pharmacokinetic properties; tumor to kidney ratio and tumor to blood ratio rapidly favour tumors. By 24h post-injection, the tumor to kidney ratio reached 4.3 + -0.7, and at 7d and 14d was 15.0 + -2.9 and 62.2 + -9.5, respectively. Tumor to blood ratios at the same time points were 3.3 ± 0.5, 137.5 ± 30.4 and 995.8 ± 139.7. Significant differences in pharmacokinetic profiles indicate that the absorbed dose will be different for each tissue.

Evaluation of treatment in LNCaP xenograft mice

LNCaP xenografts were randomized into 5 groups and treated with 148kBq and 93ng (41pmol)²²⁵Ac-RPS-074, 74kBq and 47ng (21pmol)²²⁵Ac-RPS-074, 37kBq and 23ng (10pmol)²²⁵Ac-RPS-074、133kBq ²²⁵Treatment with a bolus of Ac-DOTA-Lys-IPBA, or vehicle control. Using 138kBq and 74kBq²²⁵Significant antitumor effects were observed in Ac-RPS-074 treated mice. In the 138 kBq-treated group, 75d 6/7 (86%) of tumors were not detectable after injection: (<0.5mm³) Whereas 1/7 (14%) tumors were not detectable in the 74kBq group. The distribution of the initial tumor volumes of the two groups was 100-624mm respectively³And 64-455mm³(FIG. 13). The tumor volume in the 74kB group decreased up to 42d post-injection, and then 6/7 (86%) began to increase again in tumor volume. Before collecting pathological sample, by using⁶⁸Ga-PSMA-11 was subjected to μ PET/CT imaging to confirm the absence of tumors (FIG. 14). Through the steps ofTumors that reappear in the 74kBq treated group showed PSMA expression. Physiological uptake also occurs apparently in the kidneys and salivary glands.

FIG. 13 shows that 37kBq treatment groups and acceptance of 133kBq²²⁵The positive control group of Ac-DOTA-Lys-IPBA showed initial effect relative to the vehicle group, but the tumor volumes were from 99-331mm³And 233-³Has an initial volume increase of more than 2000mm and a final volume³. In this study, a clear dose response was evident. Up to 42 days post-injection, 74kBq and 138kBq treated groups performed similarly, but the tumor volume of 5/7 (71%) mice in the current group was measured to be less than 1mm³In time, the tumor gradually recurs. In contrast, in the 37kBq treated group, tumors in mice appeared to grow at a similar rate as untreated tumors.

Each mouse survived 75 days in the 138kBq treated group (figure 15). In contrast, in each of the other groups, at least one mouse was sacrificed before study termination due to excessive tumor growth. 37kBq groups and 133kBq²²⁵The survival curves for the Ac-DOTA-Lys-IPBA positive control group were similar, with 100% of the mice surviving the first 21 days. In contrast, only 1/7 (14%) of untreated mice survived to this time point. No toxic effects were observed in all groups. During the 75 day study, the body weight change was 92-106% of the original measurement. The remaining mice were sacrificed 75 days after injection and the tumors (if present), kidneys, liver, parotid gland and sublingual glands were excised and examined for signs of injury.

Reference is made to section 1.4

[1]Hillier SM,Maresca KP,Lu G,Merkin RD,Marquis JC,Zimmerman CN,Eckelman WC,Joyal JL,Babich JW.^99mTc-Labeled Small-Molecule Inhibitors of Prostate-Specific Membrane Antigen for Molecular Imaging of Prostate Cancer.JNucl Med.2013；54:1369-76.

[2]Kelly JM,Amor-Coarasa A,Nikolopoulou A,Wüstemann T,Barelli P,Kim D,Williams C.Jr,Zheng X,Bi C,Hu B,Warren JD,Hage DS,DiMagno SG,BabichJW.Dual-Target Binding Ligands with Modulated Pharmacokinetics forEndoradiotherapy of Prostate Cancer.J Nucl Med.2017；58:1442-1449.

[3]M,Umbricht CA,Schibli R,Müller C.Albumin-Binding PSMA Ligands:Optimization of the Tissue Distribution Profile.Mol Pharm.2018；15:934-946.

[4]Jensen MM,JT,Binderup T,A.Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate andreproducible than determined by¹⁸F-FDG-microPET or external caliper.BMC Med Imaging 2008；8:16.

[5]Amor-Coarasa A,Kelly JM,Gruca M,Nikolopoulou A,Vallabhajosula S,Babich JW.Continuation of comprehensive quality control of the itG⁶⁸Ge/⁶⁸Ga generator and production of⁶⁸Ga-DOTATOC and ⁶⁸Ga-PSMA-HBED-CC for clinical research studies.Nucl Med Biol.2017；53:37-39.

[6]Kelly J,Amor-Coarasa A,Ponnala S,Nikolopoulou A,Williams C.,Jr,Schlyer D,Zhao Y,Kim D,Babich JW.Trifunctional PSMA-Targeting Constructs forProstate Cancer with Unprecedented Localization to LNCaP Tumors.Eur J NuclMed Mol Imaging 2018；In press.

[7]Miederer M,Scheinberg DA,McDevitt MR.Realizing the potential of the Actinium-225 radionuclide generator in targeted alpha-particle therapyapplications.Adv Drug Deliv Rev.2008；60:1371-1382。

While certain embodiments have been illustrated and described, modifications, substitutions of equivalents, and other types of changes to the compounds of the present technology or to salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers or racemic mixtures thereof described herein may be made by one of ordinary skill in the art upon reading the foregoing description. Each aspect and embodiment described above may also have included or incorporated such variations or aspects disclosed with respect to any or all of the other aspects and embodiments.

The present technology is also not limited to the specific aspects described herein, which are intended as single illustrations of individual aspects of the technology. As will be apparent to those skilled in the art, many modifications and variations can be made to the present technology without departing from the spirit and scope of the invention. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that the present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. It is therefore intended that the specification be considered as exemplary only, with a true scope, spirit and scope of the technology being indicated only by the following claims, their definitions and any equivalents.

The embodiments illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be read broadly and without limitation. Additionally, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase "consisting essentially of … …" will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase "consisting of … …" does not include any elements not specified.

In addition, where features or aspects of the disclosure are described in terms of markush groups, those skilled in the art will recognize that the disclosure is thereby also described in terms of any single member or subgroup of members of the markush group. Each of the narrower species and subclass groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

All publications, patent applications, issued patents, and other documents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. To the extent that they contradict definitions in this disclosure, definitions contained in the text incorporated by reference are excluded.

The present technology may include, but is not limited to, the features and combinations of features recited in the following letter paragraphs, it being understood that the following paragraphs should not be construed as limiting the scope of the appended claims or as requiring that all such features be included in such claims:

A. a compound of formula I

Or a pharmaceutically acceptable salt and/or solvate thereof, wherein

ABD is an antigen binding domain;

W¹is-C (O) -, - (CH)₂)_n-, or- (CH)₂)_o–NH₂-C(O)–；

R¹、R²And R³Is one of

And R is¹、R²And R³The other two are both H;

X¹is absent, O, S or NH;

tox is a cytotoxic-and/or imaging agent-containing domain;

alb is an albumin binding moiety;

m is 0 or 1;

n is 1 or 2;

o is 1 or 2;

p is 0,1, 2 or 3, with the proviso that X is when p is 0¹Is absent; and is

q is 1 or 2.

B. The compound of paragraph a, wherein the compound of formula I has formula II

Or a pharmaceutically acceptable salt and/or solvate thereof, wherein

P¹、P²And P³Each independently is H, methyl, benzyl, 4-methoxybenzyl, or tert-butyl;

R¹、R²and R³Is one of

And R is¹、R²And R³The other two are both H;

rad is a moiety capable of comprising a metal ion, optionally further comprising a metal ion.

C. The compound of paragraph B, wherein P¹、P²And P³Each independently is H or t-butyl.

D. The compound of paragraph B or paragraph C, wherein P¹、P²And P³Each independently is H.

E. The compound of any of paragraphs a-D, wherein Tox of formula I or Rad of formula II comprise a metal ion.

F. The compound of any of paragraphs B-E wherein the Rad of formula II comprises a chelating agent and a chelated metal ion.

G. The compound according to any of paragraphs a-F, wherein the metal ion is a radionuclide that:¹⁷⁷Lu³⁺、¹⁷⁵Lu³⁺、⁴⁵Sc³⁺、⁶⁶Ga³⁺、⁶⁷Ga³⁺、⁶⁸Ga³⁺、⁶⁹Ga³⁺、⁷¹Ga³⁺、⁸⁹Y³⁺、⁸⁶Y³⁺、⁸⁹Zr⁴⁺、⁹⁰Y³⁺、^99mTc⁺¹、¹¹¹In³⁺、¹¹³In³⁺、¹¹⁵In³⁺、¹³⁹La³⁺、¹³⁶Ce³⁺、¹³⁸Ce³⁺、¹⁴⁰Ce³⁺、¹⁴²Ce³⁺、¹⁵¹Eu³⁺、¹⁵³Eu³⁺、¹⁵²Dy³⁺、¹⁴⁹Tb³⁺、¹⁵⁹Tb³⁺、¹⁵⁴Gd³⁺、¹⁵⁵Gd³⁺、¹⁵⁶Gd³⁺、¹⁵⁷Gd³⁺、¹⁵⁸Gd³⁺、¹⁶⁰Gd³⁺、¹⁸⁸Re⁺¹、¹⁸⁶Re⁺¹、²¹³Bi³⁺、²¹¹At⁺、²¹⁷At⁺、²²⁷Th⁴⁺、²²⁶Th⁴⁺、²²⁵Ac³⁺、²³³Ra²⁺、¹⁵²Dy³⁺、²¹³Bi³⁺、²¹²Bi³⁺、²¹¹Bi³⁺、²¹²Pb²⁺、²¹²Pb⁴⁺、²⁵⁵Fm³⁺or uranium 230.

H. The compound of any of paragraphs a-G, wherein the metal ion is an alpha-emitting radionuclide selected from the group consisting of:²¹³Bi³⁺、²¹¹At⁺、²²⁵Ac³⁺、¹⁵²Dy³⁺、²¹²Bi³⁺、²¹¹Bi³⁺、²¹⁷At⁺、²²⁷Th⁴⁺、²²⁶Th⁴⁺、²³³Ra²⁺、²¹²Pb²⁺and²¹²Pb⁴⁺。

I. the compound of any of paragraphs a-H, wherein the albumin binding moiety is

Wherein Y is¹、Y²、Y³、Y⁴And Y⁵Independently at each occurrence is H, halogen or alkyl;

X³、X⁴、X⁵and X⁶Each independently is O or S;

a is independently at each occurrence 0,1 or 2;

b is independently at each occurrence 0 or 1;

c is independently at each occurrence 0 or 1, and

d is independently at each occurrence 0,1, 2,3 or 4, optionally wherein b and c cannot be the same value.

J. The compound of any of paragraphs a-I, where R¹、R²And R³Is one of

And R is¹、R²And R³The other two are both H;

L³is absent, -C (O) -, -C₁-C₁₂Alkylene-, -C₁-C₁₂alkylene-C (O) -, -C₁-C₁₂alkylene-NR¹⁰-, or-arylene-;

R¹⁰is H, alkyl or aryl; and is

CHEL is a covalently conjugated chelator, optionally including chelated metal ions.

K. The compound of any of paragraphs a-J, wherein the compound of formula I is a compound of formula III

Or a pharmaceutically acceptable salt and/or solvate thereof, wherein

R¹、R²And R³Is one of

And R is¹、R²And R³The other two are both H;

L³is absent, -C (O) -, -C₁-C₁₂Alkylene-, -C₁-C₁₂alkylene-C (O) -, -C₁-C₁₂alkylene-NR¹⁰-, or-arylene-;

R¹⁰is H, alkyl or aryl; and is

CHEL is a covalently conjugated chelator, optionally including chelated metal ions.

L. a compound according to any one of paragraphs A-K, wherein L¹Is that

–O(CH₂CH₂O)_r–CH₂CH₂C (o) -, an amino acid, a peptide of 2,3, 4,5, 6,7,8, 9, or 10 amino acids, or a combination of any two or more thereof.

M. the compound according to any one of paragraphs A-L, wherein L¹Is that

–O(CH₂CH₂O)_r–CH₂CH₂C (o) -, glycine, polyglycine consisting of 2,3, 4,5, 6,7,8, 9, or 10 glycine residues, or a combination of any two or more thereof.

A compound according to any one of paragraphs a-M, wherein L²Is that

–(CH₂CH₂O)_s–CH₂CH₂C (o) -, 2,3, 4,5, 6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 amino acid peptide, or a combination of any two or more thereof.

O. a compound according to any one of paragraphs a-N, wherein L²Is that

–(CH₂CH₂O)_s–CH₂CH₂C (O) -, 2,3, 4,5, 6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20An amino acid, or a combination of any two or more thereof.

P. a composition comprising a pharmaceutically acceptable carrier and the composition of any one of paragraphs a-O.

A pharmaceutical composition comprising

An effective amount of a compound of any one of paragraphs a-O for detecting cancer and/or mammalian tissue overexpressing prostate specific membrane antigen ("PSMA"); and

a pharmaceutically acceptable carrier.

R. the pharmaceutical composition of paragraph Q, wherein the cancer comprises one or more of glioma, breast cancer, adrenocortical cancer, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

S. the pharmaceutical composition of paragraph Q or paragraph R, wherein the mammalian tissue comprises one or more of glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

T. the pharmaceutical composition of any one of paragraphs Q-S, wherein the pharmaceutical composition is formulated for intravenous administration, optionally comprising sterile water, Ringer' S solution, or isotonic saline solution.

U. the pharmaceutical composition of any one of paragraphs Q-T, wherein the effective amount of the compound is from about 0.01 μ g to about 10mg of the compound per gram of the pharmaceutical composition.

The pharmaceutical composition of any of paragraphs Q-U, wherein the pharmaceutical composition is provided in an injectable dosage form.

W. A pharmaceutical composition comprising

An effective amount of a compound of any one of paragraphs a-O for use in treating cancer and/or mammalian tissue overexpressing prostate specific membrane antigen ("PSMA"); and

a pharmaceutically acceptable carrier.

X. the pharmaceutical composition of paragraph W, wherein the cancer comprises one or more of glioma, breast cancer, adrenocortical cancer, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

Y. the pharmaceutical composition of paragraph W or paragraph X, wherein the mammalian tissue comprises one or more of glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

The pharmaceutical composition of any one of paragraphs W-Y, wherein the pharmaceutical composition is formulated for intravenous administration, optionally comprising sterile water, ringer's solution, or isotonic saline solution.

The pharmaceutical composition of any of paragraphs W-Z, wherein the effective amount of the compound is from about 0.01 μ g to about 10mg of the compound per gram of the pharmaceutical composition.

A pharmaceutical composition according to any of paragraphs W-AA, wherein the pharmaceutical composition is provided in an injectable dosage form.

The pharmaceutical composition of any of paragraphs W-AB, wherein the effective amount of the compound for treating cancer and/or mammalian tissue overexpressing PSMA is also an effective amount for imaging cancer and/or mammalian tissue overexpressing PSMA.

AD. A method comprising

Administering to a subject an effective amount of a compound of any one of paragraphs a-O for imaging cancer and/or mammalian tissue overexpressing prostate specific membrane antigen ("PSMA"); and

after the administration, radiation from the compound is detected.

AE. the method of paragraph AD wherein after administration, the method comprises: one or more of positron emission, gamma rays from the positron emission and annihilation, and cerenkov radiation due to the positron emission are detected.

The method of paragraph AD or paragraph AE, wherein the cancer comprises one or more of glioma, breast cancer, adrenocortical cancer, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

The method of any one of paragraphs AD-AF, wherein the subject is suspected of having mammalian tissue overexpressing PSMA, optionally wherein the mammalian tissue comprises one or more of glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

AH., the method of any one of paragraphs AD-AG, wherein administering the compound comprises parenteral administration.

The method of any of paragraphs AD-AH, wherein administering the compound comprises intravenous administration.

The method of any one of paragraphs AD-AI, wherein the effective amount of the compound is from about 0.1 μ g to about 50 μ g per kilogram body weight of the subject.

AK. A method comprising

Administering to a subject an effective amount of a compound of any one of paragraphs a-O of a mammalian tissue that overexpresses prostate specific membrane antigen ("PSMA") for treating cancer.

AL. the method of paragraph AK, wherein the cancer comprises one or more of glioma, breast cancer, adrenocortical cancer, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma and prostate cancer.

AM., the method of paragraph AK or paragraph AL, wherein the subject is suspected of having mammalian tissue overexpressing PSMA, optionally wherein the mammalian tissue comprises one or more of glioma, breast cancer, adrenocortical carcinoma, cervical cancer, vulvar cancer, endometrial cancer, primary ovarian cancer, metastatic ovarian cancer, non-small cell lung cancer, bladder cancer, colon cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and prostate cancer.

AN. the method of any one of paragraphs AK-AM, wherein administering the compound comprises parenteral administration.

AO., the method according to any one of paragraphs AK-AN, wherein administering the compound comprises intravenous administration.

AP. the method according to any one of paragraphs AK-AO, wherein the effective amount of the compound for treating cancer and/or mammalian tissue overexpressing PSMA is from about 0.1 μ g to about 50 μ g per kilogram body weight of the subject.

AQ. the method according to any of the paragraphs AK-AP, wherein the effective amount of the compound is also an effective amount for imaging cancer and/or mammalian tissue overexpressing PSMA.

The method of any of paragraphs AK-AQ, wherein said method further comprises: after the administration, radiation from the compound is detected.

AS., the method according to any of paragraphs AK-AR, wherein the method further comprises: after administration, one or more of positron emission, gamma rays from the positron emission and annihilation, and cerenkov radiation due to the positron emission are detected.

AT. A method of achieving in vivo tissue distribution of a radiotherapeutic agent in a mammalian subject, wherein a ratio of tumor activity to renal activity of 1 or greater is observed within about 4 hours to about 24 hours of administration of the radiotherapeutic agent to the mammalian subject, wherein

The method comprises administering the radiotherapeutic agent to the mammalian subject; and is

AU., the method of paragraph AT, wherein the method further comprises: an image of the mammalian subject is obtained from about 4 hours to about 24 hours after administration of the radiotherapeutic agent.

AV. the method of paragraph AT or paragraph AU wherein the ratio of tumor activity to renal activity of 1 or greater persists for up to about 24 hours after administration of the radiotherapeutic agent.

AW., the method of any of paragraphs AT-AV, wherein substantially no radionuclide activity is observed in the salivary glands of the mammalian subject from about 24 hours to about 48 hours after administration of the radiotherapeutic agent.

AX. the method of any of paragraphs AT-AW, wherein the consecutive number of atoms associated with the first covalent linker is from about 10 atoms to about 30 atoms.

AY. the method of any of paragraphs AT-AX, wherein the consecutive number of atoms associated with the second covalent linker is from about 15 atoms to about 40 atoms.

AZ. the method of any one of paragraphs AT-AY, wherein the administering comprises intravenous administration.

The method of any of paragraphs AT-AZ, wherein the radiotherapeutic agent is the compound of any of paragraphs a-O.

Other embodiments are set forth in the following claims, with the full scope of equivalents to which such claims are entitled.

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Tri-functional constructs with tunable pharmacokinetics useful for imaging and anti-tumor therapy

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