CDP protein secretion inhibitors

文档序号：862844 发布日期：2021-03-16 浏览：14次中文

阅读说明：本技术 Cdp蛋白分泌抑制剂 (CDP protein secretion inhibitors ) 是由 D·麦克明 J·W·陶恩顿 P·P·夏普于 2019-03-29 设计创作，主要内容包括：本文提供例如经由抑制Sec61来抑制蛋白分泌的化合物。还提供所述抑制剂化合物的组合物和使用这些抑制剂的方法。本文公开的所述化合物可用于例如癌症、关节炎和/或炎症的所述治疗。(Provided herein are compounds that inhibit protein secretion, e.g., via inhibition of Sec 61. Compositions of the inhibitor compounds and methods of using these inhibitors are also provided. The compounds disclosed herein are useful, for example, in the treatment of cancer, arthritis, and/or inflammation.)

1. A compound, or a pharmaceutically acceptable salt thereof, having the structure of formula (I):

wherein R is¹Is H, C_0-3alkylene-CN or C_2-6An alkynyl group;

R²is isobutyl, C_2-8Alkenyl radical, C_1-8Haloalkyl, C_1-8Hydroxyalkyl radical, C_0-3alkylene-C_3-8Cycloalkyl radical, C_0-3alkylene-C_3-8Cycloalkenyl radical, C_0-3alkylene-C_3-6Heterocycloalkyl or C_0-3alkylene-C_3-6A heterocycloalkenyl group;

R³is C_1-6Alkyl radical, C_2-8Alkenyl radical, C_1-6Haloalkyl, C_1-8Hydroxyalkyl radical, C_0-3alkylene-C_3-8Cycloalkyl or C_0-3alkylene-C_3-8A cycloalkenyl group;

R⁴is C_1-8Alkyl radical, C_2-8Alkenyl radical, C_1-8Haloalkyl, C_1-8Hydroxyalkyl radical, C_0-3alkylene-C_3-8Cycloalkyl radical, C_0-3alkylene-C_3-8Cycloalkenyl group, or C_0-3alkylene-C_3-6A heterocycloalkyl group;

R⁵is C_3-8Cycloalkyl radical, C_3-8Cycloalkenyl, halo-substituted aryl, benzothienyl, tetrahydrobenzothienyl, triazolyl, quinolinyl, quinolinonyl, quinolonyl, tetrahydroquinolyl, tetrahydroquinolonyl or indolyl,

wherein the indolyl is N-substituted, and the quinolonyl or tetraquinolonyl is optionally N-substituted, and the N-substituent comprises C_3-8Alkynyl, C_0-2alkylene-C_3-8Cycloalkyl radical, C_0-2alkylene-C_3-8Cycloalkenyl radical, C_0-2Alkylene-substituted aryl or C_0-2Alkylene-heteroaryl, and the substituted aryl is substituted with one or more groups selected from halo, alkyl, haloalkyl, OH, and alkoxy; and is

Said benzothienyl is substituted with one or more groups selected from halo, haloalkyl, alkyl, OH, and alkoxy;

R⁶is C_1-8Alkyl radical, C_2-8Alkenyl radical, C_1-8Haloalkyl, C_1-8Hydroxyalkyl radical, C_0-3alkylene-C_3-8Cycloalkyl radical, C_0-3alkylene-C_3-8Cycloalkenyl radical, C_0-3alkylene-C_3-6Heterocycloalkyl or C_0-3alkylene-C_3-6A heterocycloalkenyl group;

R⁷is C_1-8An alkyl group; and is

R^N2、R^N3、R^N4、R^N5、R^N6And R^N7Each independently is H or C_1-3An alkyl group.

2. The compound or salt of claim 1 having the structure of formula (IA):

3. a compound or salt according to claim 1 or 2, wherein R¹Is CH₂CN。

4. A compound or salt according to claim 1 or 2, wherein R¹Is (CH)₂)_0-2C≡CH。

5. A compound or salt according to claim 1 or 2, wherein R¹Is H.

6. A compound or salt according to any one of claims 1-5, wherein R²Is C_1-8A haloalkyl group.

7. A compound or salt according to any one of claims 1-5, wherein R²Is C_0-3alkylene-C_3-8Cycloalkenyl or C_0-3alkylene-C_3-8A cycloalkyl group.

8. A compound or salt according to any one of claims 1-5, wherein R²Is CH₂CF₃、CH₂CH₂CF₃、CH₂-1-cyclohexenyl, CH₂-1-cyclopentenyl, CH₂-cyclopropyl, CH₂-cyclopentyl, CH₂-cyclohexyl or CH₂-4-tetrahydropyranyl.

9. A compound or salt according to any one of claims 1-5, wherein R²Is an isobutyl group.

10. The compound or salt of any one of claims 1-9, wherein R³Is CH (CH)₃)₂Or a phenyl group.

11. The compound or salt of any one of claims 1-9, wherein R³Is C_1-8A haloalkyl group.

12. The compound or salt of any one of claims 1-9, wherein R³Is C_0-3alkylene-C_3-8Cycloalkyl or C_0-3alkylene-C_3-8A cycloalkenyl group.

13. A compound or salt according to claim 12, wherein R³Is 1-cyclopentenyl or 1-cyclohexenyl.

14. The compound or salt of any one of claims 1-13, wherein R⁴Is CH (CH)₃)₂、C(CH₃)₃、CH₃、CH₂CH₃、CF₃、CH₂CF₃Tetrahydropyranyl or cyclopentyl.

15. The compound or salt of any one of claims 1-13, wherein R⁴Is C_1-8A haloalkyl group.

16. The compound or salt of any one of claims 1-13, wherein R⁴Is C_0-3alkylene-C_3-8Cycloalkyl or C_0-3alkylene-C_3-8A cycloalkenyl group.

17. A compound or salt according to claim 16, wherein R⁴Is 1-cyclopentenyl or 1-cyclohexenyl.

18. The compound or salt of any one of claims 1-17, wherein R⁵Comprising a cyclohexyl, cyclopentyl or cyclopentenyl group.

19. A compound or salt according to claim 18, wherein R⁵Comprises a cyclohexyl group.

20. The compound or salt of any one of claims 1-17, wherein R⁵Comprising quinolyl, quinolonyl, tetrahydroquinolyl, tetrahydroquinolonyl or tetrahydroquinolonyl, and R⁵Substituted with one or more of: c_3-8Alkynyl, C_0-2alkylene-C_3-8Cycloalkyl radical, C_0-2alkylene-C_3-8Cycloalkenyl radical, C_0-2Alkylene-aryl, or C_0-2Alkylene-heteroaryl.

21. The compound or salt of any one of claims 1-17, wherein R⁵Is composed ofAnd R' is C₄Alkynyl, CH₂-fluoropyridyl, CH₂-methylpyridyl, CH₂-methoxyphenyl, CH₂-methylphenyl, CH₂-fluorophenyl, CH₂-chlorophenyl, CH₂-bromophenyl, CH₂-difluorophenyl, CH₂-fluoromethoxyphenyl or CH₂-methylisoxazolyl.

22. The compound or salt of claim 21 wherein R' is p-fluorobenzyl, p-chlorobenzyl, p-methylbenzyl, or p-bromobenzyl.

23. The compound or salt of any one of claims 1-17, wherein R⁵Comprising a benzothienyl group, and which benzothienyl group is substituted with one or more of chlorine, fluorine, bromine, methyl and methoxy.

24. A compound or salt according to claim 23, wherein R⁵Contains a chlorobenzothiophenyl group.

25. The compound or salt of any one of claims 1-24, wherein R⁶Is CH₃、CH₂CH(CH₃)₂、CH₂CH(CH₃)(CH₂)₃CH₃、CH₂CF₃Or CH₂CH₂CF₃。

26. The compound or salt of any one of claims 1-24, wherein R⁶Is CH₂CH(CH₃)₂。

27. The compound or salt of any one of claims 1-24, wherein R⁶Is C_1-8A haloalkyl group.

28. The compound or salt of any one of claims 1-24, wherein R⁶Is C_0-3alkylene-C_3-8A cycloalkenyl group.

29. A compound or salt according to claim 28, wherein R⁶Is CH₂-1-cyclopentenyl or CH₂-1-cyclohexenyl.

30. The compound or salt of any one of claims 1-29, wherein R⁷Is CH₃。

31. The compound or salt of any one of claims 1-30, wherein R^N2、R^N3、R^N4、R^N5、R^N6And R^N7At least one of them is H.

32. The compound or salt of any one of claims 1-31, wherein R^N2、R^N3、R^N4、R^N5、R^N6And R^N7At least one of them is CH₃。

33. The compound or salt of any one of claims 1-30, wherein R^N2Is H, R^N3Is CH₃，R^N4Is H, R^N5Is CH₃，R^N6Is H, and R^N7Is CH₃。

34. A compound, or a pharmaceutically acceptable salt thereof, as set forth in table a.

35. A pharmaceutical composition comprising a compound or salt according to any one of claims 1-34, and a pharmaceutically acceptable excipient.

36. A method of inhibiting protein secretion in a cell comprising contacting the cell with a compound or salt according to any one of claims 1-34 in an amount effective to inhibit protein secretion.

37. The method of claim 36, wherein the protein is TNF α, PD1, Her3, VCAM, IL7, Prl, or FLT 3.

38. A method of treating cancer in a subject comprising administering to the subject a compound or salt of any one of claims 1-34 in an amount effective to treat the cancer.

39. The method of claim 38, wherein the cancer is breast or melanoma.

40. The method of claim 39, wherein the cancer is breast, and R⁵Containing a benzothienyl group.

41. A method of treating arthritis in a subject comprising administering to the subject a compound or salt according to any one of claims 1-34 in an amount effective to treat arthritis.

42. A method of treating inflammation in a subject, comprising administering to the subject a compound or salt according to any one of claims 1-34 in an amount effective to treat inflammation.

Background

Translocation of proteins into the endoplasmic reticulum ("ER") is the first step in protein secretion. Introduction of ER proteins is essential in all eukaryotic cells and is particularly important in rapidly growing tumor cells. Thus, the protein secretion process can serve as both a potential cancer drug and a target for bacterial virulence factors. See Kalies and《Traffic》,16(10):1027-1038(2015)。

when the N-terminal hydrophobic signal peptide is extended from the ribosome, protein transport to the ER begins in the cytosol. Binding of a signal recognition particle ("SRP") to the signal sequence targets the ribosome-nascent strand-SRP complex to the ER membrane, where contact of the SRP with its receptor triggers the handoff of the signal peptide to Sec 61. Sec61 is an ER membrane protein translocator (also known as a translocator) and is circular with 3 major subunits (heterotrimer). It includes a "plug" that blocks transport into or out of the ER. When the hydrophobic region of the nascent polypeptide interacts with the "seam" region of Sec61, the plug is displaced, allowing the polypeptide to translocate into the ER lumen. In mammals, only short proteins (<160 amino acids) can enter the ER post-translationally, and proteins smaller than 120 amino acids must use this pathway. Certain translocation ability is maintained by calmodulin binding to the signal sequence. Upon reaching the Sec61 channel, a signal peptide or signal anchor was inserted between the transmembrane domains ("TMD") 2 and 7 of Sec61 α, which formed the lateral portion of the gate, allowing the channel to be opened for soluble secreted proteins. Since the Sec61 channel consists of 10 TMDs (Sec61 α) surrounded by hydrophobic clamps formed by Sec61 γ, the channel opening depends on the conformational change, which in fact involves all TMDs.

Inhibition of protein transport across the ER membrane has the potential to treat or prevent diseases such as cancer cell growth and inflammation. Known secretion inhibitors, from broad spectrum to high substrate specificity, can interfere with virtually any stage of this multistep process, and even interfere with the transport of endocytic antigens into the cytosol for cross-presentation. These inhibitors interact with signal peptides, chaperones or the Sec61 channel to block substrate binding or prevent conformational changes required for protein introduction into the ER. Examples of protein secretin inhibitors include calmodulin inhibitors (e.g., E6 Berbamine and Ophiobolin a), lanthanum, sterols, cyclic dipeptides (e.g., HUN-7293, CAM741, NFI028, cotrinsin, aplha toxin A, Decatransin, valinomycin), CADA, bacteriolactones, Eeyarestatin I ("ESI"), and exotoxin a. However, the above secretion inhibitors suffer from one or more of the following: lack of selectivity for the Sec61 channel poses manufacturing challenges due to structural complexity, and application, bioavailability and partitioning of molecular weight limitations.

Thus, there is a need for new small molecule inhibitors of protein secretion.

Disclosure of Invention

Provided herein are compounds that inhibit Sec 61. Specifically, provided are compounds having the structure of formula (I):

wherein R is¹Is H, C_0-3alkylene-CN or C_2-6An alkynyl group; r²Is isobutyl, C_2-8Alkenyl radical, C_1-8Haloalkyl, C_1-8Hydroxyalkyl radical, C_0-3alkylene-C_3-8Cycloalkyl radical, C_0-3alkylene-C_3-8Cycloalkenyl radical, C_0-3alkylene-C_3-6Heterocycloalkyl or C_0-3alkylene-C_3-6A heterocycloalkenyl group; r³Is C_1-6Alkyl radical, C_2-8Alkenyl radical, C_1-6Haloalkyl, C_1-8Hydroxyalkyl radical, C_0-3alkylene-C_3-8Cycloalkyl or C_0-3alkylene-C_3-8A cycloalkenyl group; r⁴Is C_1-8Alkyl radical, C_2-8Alkenyl radical, C_1-8Haloalkyl, C_1-8Hydroxyalkyl radical, C_0-3alkylene-C_3-8Cycloalkyl radical, C_0-3alkylene-C_3-8Cycloalkenyl group, or C_0-3alkylene-C_3-6A heterocycloalkyl group; r⁵Is C_3-8Cycloalkyl radical, C_3-8Cycloalkenyl, halo-substituted aryl, benzothienyl, tetrahydrobenzothienyl, triazolyl, quinolinyl, quinolinonyl, quinolonyl, tetrahydroquinolyl, tetrahydroquinolonyl or indolyl, wherein the indolyl is N-substituted and the quinolonyl or tetraquinolonyl is optionally N-substituted, and the N-substituent comprises C_3-8Alkynyl, C_0-2alkylene-C_3-8Cycloalkyl radical, C_0-2alkylene-C_3-8Cycloalkenyl radical, C_0-2Alkylene-substituted aryl or C_0-2Alkylene-heteroaryl, and substituted aryl is substituted with one or more groups selected from halo, alkyl, haloalkyl, OH, and alkoxy; and benzothienyl is substituted with one or more groups selected from halo, haloalkyl, alkyl, OH, and alkoxy; r⁶Is C_1-8Alkyl radical, C_2-8Alkenyl radical, C_1-8Haloalkyl, C_1-8Hydroxyalkyl radical, C_0-3alkylene-C_3-8Cycloalkyl radical, C_0-3alkylene-C_3-8Cycloalkenyl radical, C_0-3alkylene-C_3-6Heterocycloalkyl or C_0-3alkylene-C_3-6A heterocycloalkenyl group; r⁷Is C_1-8An alkyl group; and R is^N2、R^N3、R^N4、R^N5、R^N6And R^N7Each independently is H or C_1-3An alkyl group. Also provided are compounds as set forth in table a below. Also provided are pharmaceutical compositions comprising a compound disclosed herein and a pharmaceutically acceptable excipient.

Further provided are methods of inhibiting protein secretion in a cell comprising contacting a cell with a compound as disclosed herein. Also provided are methods of treating cancer in a subject comprising administering to the subject a compound as disclosed herein in an amount effective to treat the cancer. Further provided are methods of treating arthritis in a subject comprising administering to the subject a compound as disclosed herein in an amount effective to treat arthritis. Also provided are methods of treating inflammation in a subject comprising administering to the subject a compound as disclosed herein in an amount effective to treat inflammation.

Detailed Description

Provided herein are compounds that inhibit protein secretion. The compounds described herein are useful for treating or preventing diseases associated with excessive protein secretion, such as inflammation and cancer, and improving the quality of life of affected individuals.

Provided herein are compounds, or pharmaceutically acceptable salts thereof, having the structure of formula (I):

wherein R is¹Is H, C_0-3alkylene-CN or C_2-6An alkynyl group;

R³is C_1-6Alkyl radical, C_2-8Alkenyl radical, C_1-6Haloalkyl, C_1-8Hydroxyalkyl radical, C_0-3alkylene-C_3-8Cycloalkyl or C_0-3alkylene-C_3-8A cycloalkenyl group;

wherein indolyl is N-substituted, and quinolonyl or tetraquinolonyl is optionally N-substituted, and the N-substituent comprises C_3-8Alkynyl, C_0-2alkylene-C_3-8Cycloalkyl radical, C_0-2alkylene-C_3-8Cycloalkenyl radical, C_0-2Alkylene-substituted aryl or C_0-2Alkylene-heteroaryl, and substituted aryl is substituted with one or more groups selected from halo, alkyl, haloalkyl, OH, and alkoxy; and is

Benzothienyl is substituted with one or more groups selected from halo, haloalkyl, alkyl, OH, and alkoxy;

R⁷is C_1-8An alkyl group; and is

R^N2、R^N3、R^N4、R^N5、R^N6And R^N7Each independently is H or C_1-3An alkyl group.

In some cases, the compound or salt of formula (I) has the structure of formula (IA):

in each case R¹Is H. In some cases, R¹Is CH₂And (C) CN. In some cases, R¹Is (CH)₂)_0-2C ≡ CH (e.g. CH)₂C ≡ CH or CH₂CH₂C≡CH)。

In each case R²Is C_1-8Haloalkyl or isobutyl. In each case R²Is CH₂CF₃、CH₂CH₂CF₃、CH₂-1-cyclohexenyl, CH₂-1-cyclopentenyl, CH₂-cyclopropyl, CH₂-cyclopentyl, CH₂-cyclohexyl or CH₂-4-tetrahydroA pyranyl group.

In each case R³Is C_1-8Haloalkyl or CH (CH)₃)₂. In each case R⁴Is C_1-8Haloalkyl or CH (CH)₃)₂. In each case R³Is C_0-3alkylene-C_3-8Cycloalkyl radicals, or C_0-3alkylene-C_3-8A cycloalkenyl group. In some cases, R³Is 1-cyclopentenyl or 1-cyclohexenyl. In each case R⁴Is CH (CH)₃)₂、C(CH₃)₃、CH₃、CH₂CH₃、CF₃、CH₂CF₃Tetrahydropyranyl or cyclopentyl. In each case R⁴Is C_0-3alkylene-C_3-8Cycloalkyl radicals, or C_0-3alkylene-C_3-8A cycloalkenyl group. In some cases, R⁴Is 1-cyclopentenyl or 1-cyclohexenyl.

In some cases, R³And R⁴The same is true. In some cases, R³And R⁴Each is CH (CH)₃)₂。

In each case R⁵Comprising a cyclohexyl, cyclopentyl or cyclopentenyl group.

In some cases, R⁵Comprising quinolyl, quinolonyl, tetrahydroquinolyl, tetrahydroquinolonyl. In some cases, R⁵Comprises And R' is C_3-8Alkynyl, C_0-2alkylene-C_3-8Cycloalkyl radical, C_0-2alkylene-C_3-8Cycloalkenyl radical, C_0-2Alkylene-aryl, or C_0-2Alkylene-heteroaryl, wherein aryl or heteroaryl may be unsubstituted or further substituted with one or more (e.g. 1,2 or) selected from halo, alkyl, haloalkyl, OH and alkoxy3) groups. The quinolinyl, quinolonyl, tetrahydroquinolinyl, or tetrahydroquinolonyl groups may be unsubstituted or further substituted with one or more (e.g., 1,2, or 3) groups selected from halo, alkyl, haloalkyl, OH, and alkoxy.

At R⁵In the case of containing an indolyl group, R⁵Can be of the structureWherein R' is C₄Alkynyl, CH₂-fluoropyridyl, CH₂-methylpyridyl, CH₂-methoxyphenyl, CH₂-methylphenyl (CH)₂Tolyl), CH₂-fluorophenyl, CH₂-chlorophenyl, CH₂-bromophenyl, CH₂-difluorophenyl, CH₂-fluoromethoxyphenyl or CH₂-methylisoxazolyl. In some cases, R' is p-fluorobenzyl or p-bromobenzyl.

In the case of (A), R⁵Comprising benzothienyl, which may be substituted with one or more of chloro, fluoro, bromo, methyl and methoxy. In some cases, R⁵Comprising a chlorobenzothienyl group (e.g., 5-chlorobenzothienyl).

In some cases, R⁶Is CH₃、CH₂CH(CH₃)₂、CH₂CH(CH₃)(CH₂)₃CH₃、CH₂CF₃Or CH₂CH₂CF₃. In each case R⁶Is C_1-8A haloalkyl group. In some cases, R⁶Is CH₂CH(CH₃)₂. In each case R⁶Is C_0-3alkylene-C_3-8A cycloalkenyl group. In some cases, R⁶Is CH₂-1-cyclopentenyl or CH₂-1-cyclohexenyl.

In each case R⁷Is methyl. In each case R^N2、R^N3、R^N4、R^N5、R^N6And R^N7At least one ofAnd each is H. In some cases, R^N2、R^N3、R^N4、R^N5、R^N6And R^N7Is at least one of CH₃. In some cases, R^N2Is H, R^N3Is CH₃，R^N4Is H, R^N5Is CH₃，R^N6Is H, and R^N7Is CH₃。

The term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds provided herein. These salts can be prepared in situ during the final isolation and purification of the compounds provided herein, or by separately reacting the free base form of the compound with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate, dodecylsulfonate, and amino acid salts and the like. (see, e.g., Berge et al, (1977) "Pharmaceutical Salts", journal of Pharmaceutical sciences (J.pharm.Sci.) 66:1-19.)

In some embodiments, the compounds provided herein may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these instances, the term "pharmaceutically acceptable salt" refers to the relatively non-toxic inorganic and organic base addition salts of the compounds provided herein. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compounds in their free acid form with a suitable base (such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation), with ammonia or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

Without being bound by any particular theory, the compounds described herein inhibit protein secretion by binding and disabling components of the transposon (including but not limited to Sec61), and in some cases, disrupt the interaction between the nascent signaling sequence of the translated protein and components of the transposon, including but not limited to Sec61, in a sequence-specific manner.

The compounds described herein may be IC₅₀Inhibition of secretion of the protein of interest (e.g. TNF α, PD1, Her3, VCAM, Prl, IL7 or FLT3) at most 5 μ M, or at most 3 μ M, or at most 1 μ M. In each case, the compounds disclosed herein can be IC₅₀At most 5. mu.M, or at most 3. mu.M, or at most 1. mu.M, inhibits the secretion of TNF α. In each case, the compounds disclosed herein can be IC₅₀At most 5. mu.M, or at most 3. mu.M, or at most 1. mu.M, inhibits the secretion of VCAM. In some cases, a compound disclosed herein may be IC₅₀Inhibits secretion of Her3 at up to 5. mu.M, or up to 3. mu.M, or up to 1. mu.M. In each case, the compounds disclosed herein can be IC₅₀At most 5. mu.M, or at most 3. mu.M, or at most 1. mu.M, inhibits secretion of Prl. In each case, the compounds disclosed herein can be IC₅₀Inhibits secretion of IL7 at a level of at most 5. mu.M, or at most 3. mu.M, or at most 1. mu.M. In each case, the compounds disclosed herein can be IC₅₀At most 5. mu.M, or at most 3. mu.M, or at most 1. mu.M inhibits secretion of FLT 3.

In some cases, a compound as disclosed herein is a compound as shown in table a below, or a pharmaceutically acceptable salt thereof. In some cases, the compound or salt is as shown in table a, or is C-01, having the structure:(C-01)。

TABLE A

Chemical definition

As used herein, the term "alkyl" refers to straight and branched chain saturated hydrocarbon groups containing from one to thirty carbon atoms (e.g., from one to twenty carbon atoms or from one to ten carbon atoms). Term C_nMeaning that the alkyl group has "n" carbon atoms. E.g. C₄Alkyl refers to an alkyl group having 4 carbon atoms. C_1-7Alkyl refers to alkyl groups having a full range of carbon numbers (i.e., 1 to 7 carbon atoms) as well as all subgroups (e.g., 1-6, 2-7, 1-5, 3-6, 1,2, 3,4, 5, 6, and 7 carbon atoms). Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), tert-butyl (1, 1-dimethylethyl), 3-dimethylpentyl, and 2-ethylhexyl. Unless otherwise specified, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group. "haloalkyl" indicates an alkyl group wherein one or more hydrogens of the alkyl group are replaced with a halogen atom (e.g., F, Br, Cl, or I). In some cases, all hydrogen atoms of the alkyl group are replaced with halogen atoms. "hydroxyalkyl" indicates an alkyl group wherein one or more hydrogen atoms of the alkyl group are replaced by an-OH moiety.

As used herein, surgeryThe term "alkylene" refers to a divalent saturated aliphatic radical. Term C_nMeaning that the alkylene group has "n" carbon atoms. For example, C_1-6Alkylene refers to alkylene groups having a number of carbon atoms that spans the entire range and all subgroups, as previously described for "alkyl" groups.

As used herein, the term "alkenyl" is defined identically to "alkyl" except for having at least one carbon-carbon double bond and having from two to thirty carbon atoms, such as two to twenty carbon atoms, or two to ten carbon atoms. Term C_nMeaning that the alkenyl group has "n" carbon atoms. For example, C₄Alkenyl means alkenyl having 4 carbon atoms. C_2-7Alkenyl refers to alkenyl groups having the full range of carbon numbers (i.e., 2 to 7 carbon atoms) and all subgroups (e.g., 2-6, 2-5, 3-6, 2,3, 4, 5, 6, and 7 carbon atoms). Specifically contemplated alkenyl groups include ethenyl, 1-propenyl, 2-propenyl, and butenyl. Unless otherwise indicated, alkenyl groups may be unsubstituted alkenyl groups or substituted alkenyl groups.

As used herein, the term "alkynyl" is defined identically to "alkyl" except for containing at least one carbon-carbon triple bond and having from two to thirty carbon atoms, for example two to twenty carbon atoms, or two to ten carbon atoms. Term C_nMeaning that the alkynyl group has "n" carbon atoms. For example, C₄Alkynyl means alkynyl having 4 carbon atoms. C_2-6Alkynyl refers to alkynyl groups having a full range of carbon numbers (i.e., 2 to 6 carbon atoms) as well as all subgroups (e.g., 3-5, 2-5, 3-6, 2,3, 4, 5, and 6 carbon atoms). Specifically contemplated alkynyl groups include ethynyl, 1-propynyl, 2-propynyl and butynyl. Unless otherwise indicated, alkynyl groups may be unsubstituted alkynyl groups or substituted alkynyl groups.

As used herein, the term "cycloalkyl" refers to an aliphatic cyclic hydrocarbon group containing from three to eight carbon atoms (e.g., 3,4, 5, 6,7, or 8 carbon atoms). Term C_nMeaning that the cycloalkyl group has "n" carbon ring atoms. For example, C₅Cycloalkyl means having 5 carbon atoms in the ringA cycloalkyl group of (a). C_5-8Cycloalkyl refers to cycloalkyl groups having the full range of carbon numbers (i.e., 5 to 8 carbon atoms) as well as all subgroups (e.g., 5-6, 6-8, 7-8, 5-7, 5, 6,7, and 8 carbon atoms). Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise specified, cycloalkyl groups may be unsubstituted cycloalkyl groups or substituted cycloalkyl groups.

As used herein, the term "cycloalkenyl" is defined similarly to "cycloalkyl" except that it contains at least one carbon-carbon double bond in the ring, but is not aromatic. Term C_nMeaning that the cycloalkenyl group has "n" carbon atoms. For example, C₅Cycloalkenyl refers to cycloalkenyl groups having 5 carbon atoms in the ring. In some cases cycloalkenyl is C_5-8A cycloalkenyl group. C_5-8Cycloalkenyl refers to cycloalkenyl groups having the full range of carbon numbers (i.e., 5 to 8 carbon atoms) and all subgroups (e.g., 5-6, 6-8, 7-8, 5-7, 5, 6,7, and 8 carbon atoms). Non-limiting examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Unless otherwise indicated, cycloalkenyl groups can be unsubstituted cycloalkenyl or substituted cycloalkenyl.

As used herein, the term "heterocycloalkyl" is defined similarly to cycloalkyl, and the term "heterocycloalkenyl" is defined similarly to cycloalkenyl, except that the ring contains one to three heteroatoms independently selected from oxygen, nitrogen, or sulfur. Term C_nMeaning that the heterocycloalkyl or heterocycloalkenyl group has "n" ring carbon atoms-with the remainder (1-3) being heteroatoms. The heterocycloalkyl and heterocycloalkenyl rings can be 3-to 10-membered rings having 1-3 ring heteroatoms. Non-limiting examples of heterocycloalkyl groups include piperidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, oxazepanyl, and the like. Cycloalkyl, cycloalkenyl, heterocycloalkyl and heterocycloalkenyl can optionally be chosen, for example, independently from alkyl, alkylene OH, C (O) NH₂、NH₂Oxygen (═ O), aryl, haloalkyl, halo, and OH. The heterocycloalkyl and heterocycloalkenyl radicals having a ring nitrogen may furthermore beSubstituted with N-, or the nitrogen ring atoms may be present as "NH". In some cases, the ring nitrogen atom is substituted with an alkyl group. In some cases, the ring nitrogen is substituted with C_3-8Alkynyl, C_0-2alkylene-C_3-8Cycloalkyl radical, C_0-2alkylene-C_3-8Cycloalkenyl radical, C_0-2Alkylene-aryl, or C_0-2Alkylene-heteroaryl substituted.

As used herein, the term "aryl" refers to a monocyclic or polycyclic (e.g., fused bicyclic and fused tricyclic) carbocyclic aromatic ring system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, phenanthryl, biphenylene, indanyl, indenyl, anthracenyl, fluorenyl, and tetralinyl. Unless otherwise indicated, aryl groups may be unsubstituted aryl groups or substituted aryl groups. The aryl group may be substituted, for example, with one or more (e.g., 1,2, or 3) substituents selected from halo, haloalkyl, alkyl, OH, and alkoxy. In some cases, substituted aryl groups are substituted with one or more groups selected from halo, alkyl, haloalkyl, OH, and alkoxy.

As used herein, the term "heteroaryl" refers to a monocyclic or polycyclic (e.g., fused bicyclic and fused tricyclic) aromatic ring system wherein one to four ring atoms are selected from oxygen, nitrogen, or sulfur and the remaining ring atoms are carbon, the ring system being connected to the remainder of the molecule through any available ring atoms. Term C_nMeaning that the heteroaryl group has "n" ring carbon atoms, with the remainder of the ring atoms (1-4) being heteroatoms. The heteroaryl group may be a 5-to 10-membered ring having 1-4 ring heteroatoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, furanyl, thienyl, quinolinyl, isoquinolinyl, benzoxazolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, triazinyl, triazolyl, purinyl, pyrazinyl, purinyl, indolinyl, phthalazinyl, indazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl, pyridopyridyl, indolyl, 3H-indolyl, pteridinyl, and quinoxalinyl, unless otherwise statedThe heteroaryl group can be unsubstituted heteroaryl or substituted heteroaryl. Heteroaryl groups may be substituted, for example, with one or more (e.g., 1,2, or 3) substituents selected from halo, haloalkyl, alkyl, OH, and alkoxy. In some cases, substituted heteroaryl is substituted with one or more groups selected from halo, alkyl, haloalkyl, OH, and alkoxy.

As used herein, the term "alkoxy" or "alkoxy" refers to an "-O-alkyl" group.

The term "halo", as used herein, is defined as fluoro, chloro, bromo and iodo.

A "substituted" functional group (e.g., a substituted alkyl, alkylene, cycloalkyl, aryl, or heteroaryl) is a functional group having at least one hydrogen group substituted with a non-hydrogen group (i.e., substituent). Examples of non-hydrogen radicals (or substituents) include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, ether, aryl, heteroaryl, heterocycloalkyl, hydroxyl, oxy (or oxy), alkoxy, ester, thioester, acyl, carboxyl, cyano, nitro, amino, sulfhydryl, and halo. When the substituted alkyl group includes more than one non-hydrogen group, the substituents may be bonded to the same carbon or two or more different carbon atoms.

Pharmaceutical formulations and administration

Further disclosed are the manufacture and use (alternatively referred to generically as formulations) of pharmaceutical compositions comprising one or more of the compounds provided herein. Also included are the pharmaceutical compositions themselves. The pharmaceutical composition typically includes a pharmaceutically acceptable carrier. Accordingly, provided herein are pharmaceutical formulations comprising a compound described herein (e.g., a compound of formula (I), a compound listed in table a or B, or a pharmaceutically acceptable salt of the foregoing) as previously described herein, and one or more pharmaceutically acceptable carriers.

The phrase "pharmaceutically acceptable" is employed herein to refer to those ligands, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. As used herein, the phrase "pharmaceutically acceptable carrier" includes buffers compatible with pharmaceutical administration, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted β -cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) phosphoric acid buffer solution; and (21) other non-toxic compatible materials employed in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions provided herein are non-pyrogenic, i.e., do not induce a significant temperature increase when administered to a patient.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, mold release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

As is well known in the art, compositions prepared as described herein may be administered in various forms depending on the condition to be treated and the age, condition and weight of the patient. For example, in the case of oral administration of the compositions, they may be formulated as tablets, capsules, granules, powders or syrups; or for parenteral administration, they may be formulated as injections (intravenous, intramuscular or subcutaneous), drip preparations or suppositories. For administration by the ocular mucosal route, they may be formulated as eye drops or eye ointments. These formulations may be prepared by conventional means in conjunction with the methods described herein, and the active ingredient may be mixed with any conventional additive or excipient, such as a binder, disintegrant, lubricant, corrigent, solubilizer, suspending agent, emulsifier, or coating agent, if desired.

The actual dosage level of the active ingredient in the pharmaceutical compositions provided herein can be varied so as to obtain a "therapeutically effective amount," which is an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

The concentration of a compound provided herein in a pharmaceutically acceptable mixture will vary depending on several factors, including the dose of the compound to be administered, the pharmacokinetic properties of the compound(s) employed, and the route of administration. In some embodiments, the compositions provided herein can be provided as an aqueous solution containing about 0.1% -10% w/v of a compound disclosed herein, as well as other materials, for parenteral administration. A typical dosage range may include about 0.01 to about 50mg/kg body weight per day, given in 1-4 divided doses. Each divided dose may contain the same or different compounds. The dosage will be a therapeutically effective amount depending on several factors, including the overall health of the patient and the formulation and route of administration of the compound(s) selected.

Dosage forms or compositions can be prepared containing in the range of 0.005% to 100% of a compound as described herein, with the remainder consisting of a non-toxic carrier. Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain from 0.001% to 100%, in one embodiment, from 0.1% to 95%, and in another embodiment, from 75% to 85% of the active ingredient. Although the dosage will vary depending on the symptoms, age and weight of the patient, the nature and severity of the condition to be treated or prevented, the route of administration and the form of the drug, in general, a daily dosage of 0.01 to 2000mg of the compound is recommended for adult patients, and this may be administered in a single dose or in divided doses. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

In the jurisdictions where patenting methods for practicing on the human body is prohibited, the meaning of "administering" a composition to a human subject should be limited to specifying a controlled substance that the human subject can self-administer by any technique (e.g., oral, inhalation, topical administration, injection, insertion, etc.). Is intended to be the broadest reasonable interpretation consistent with the law or law defining the subject matter of the patent. In the right to not forbid the patent rights granted to the method of practicing the human body, "administering" a composition includes both the method of practicing the human body and the aforementioned activities.

Application method

The compounds disclosed herein can inhibit protein secretion of a protein of interest. The compounds disclosed herein can interfere with the Sec61 protein secretion machinery of cells. In some cases, a compound as disclosed herein inhibits secretion of one or more of TNF α, PD1, Her3, VCAM, Prl, IL7, and FLT3, or each of TNF α, PD1, Her3, VCAM, Prl, IL7, and FLT 3. Protein secretion activity can be assessed as described in the examples section below.

As used herein, the term "inhibitor" is intended to describe a compound that blocks or reduces the activity of a pharmacological target (e.g., a compound that inhibits the function of Sec61 in the protein secretion pathway). The inhibitor may exert competitive, non-competitive or non-competitive inhibition. Inhibitors may bind reversibly or irreversibly and, thus, the term includes compounds that are suicide substrates of proteins or enzymes. Inhibitors may modify one or more sites on or near the active site of the protein, or may cause conformational changes elsewhere on the enzyme. The term inhibitor is used more broadly herein than the scientific literature to also encompass other kinds of pharmaceutically or therapeutically useful agents, such as promoters, antagonists, stimulators, cofactors, and the like.

Accordingly, provided herein are methods of inhibiting protein secretion in a cell. In these methods, the cell is contacted with a compound described herein or a pharmaceutical formulation thereof in an amount effective to inhibit secretion of the protein of interest. In some embodiments, the cells are contacted in vitro. In various embodiments, the cells are contacted in vivo. In various embodiments, contacting comprises administering the compound or pharmaceutical formulation to the subject.

The biological consequences of Sec61 inhibition are numerous. For example, Sec61 inhibition has been proposed for use in treating or preventing inflammation and/or cancer in a subject. Thus, pharmaceutical formulations for Sec 61-specific compounds provide a means for subjects to administer drugs and treat these conditions. As used herein, the term "treating" or the like refers to the elimination, alleviation or amelioration of a disease or condition and/or symptoms associated therewith. Although not excluded, treating a disease or condition does not require complete elimination of the disease, condition, or symptom associated therewith. As used herein, the term "treating" or the like may include "prophylactic treatment," which refers to reducing the likelihood of a relapse of a disease or condition, or reducing the likelihood of a relapse of a previously controlled disease or condition, in a subject who is not suffering from, but is at risk of, or susceptible to, a relapse of the disease or condition. The terms "treatment" and synonyms contemplate administration of a therapeutically effective amount of a compound of the present aspect to an individual in need of such treatment. Within the meaning of the present invention, "treatment" includes relapse prevention or stage prevention as well as treatment of acute or chronic signs, symptoms and/or dysfunctions. Symptomatic treatment may be used, for example, to suppress symptoms. It may be achieved within a short period of time, targeted within a medium period, or may be a long-term treatment, for example in the context of maintenance therapy. As used herein, the terms "patient" and "subject" are used interchangeably and refer to animals (such as dogs, cats, cows, horses, and sheep (i.e., non-human animals)) and humans. A particular patient is a mammal (e.g., a human). The term patient includes both males and females.

Sec 61-mediated inhibition of the secretion of inflammatory proteins (e.g., TNF α) can disrupt inflammatory signaling. Accordingly, provided herein are methods of treating inflammation in a subject by administering to the subject a therapeutically effective amount of a compound described herein. In some cases, a compound disclosed herein or a pharmaceutically acceptable salt thereof can be used to treat arthritis.

In addition, the viability of cancer cells is dependent on increased protein secretion into the ER for survival. Thus, non-selective or partially selective inhibition of protein secretion mediated by Sec61 may inhibit tumor growth. Alternatively, in the context of immune oncology, it is known that selective secretory inhibitors of secreted immune checkpoint proteins (e.g., PD-1, TIM-3, LAG3, etc.) can lead to activation of the immune system to combat various cancers.

Accordingly, also provided herein are methods of treating cancer in a subject by administering to the subject a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof. Particularly contemplated cancers that may be treated using the compounds and compositions described herein include, but are not limited to, melanoma, multiple myeloma, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), squamous cell cancer, leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, lymphoma, NPM/ALK-transformed anaplastic large cell lymphoma, renal cell carcinoma, rhabdomyosarcoma, ovarian cancer, endometrial cancer, small cell carcinoma, adenocarcinoma, gastric carcinoma, hepatocellular carcinoma, pancreatic cancer, thyroid cancer, anaplastic large cell lymphoma, hemangioma, head and neck cancer, bladder cancer, and colorectal cancer. In some cases, the cancer is multiple myeloma, prostate cancer, lung cancer, bladder cancer, or colorectal cancer. In some cases, the cancer is breast cancer or melanoma.

The compounds described herein are also contemplated for use in the prevention and/or treatment of a variety of diseases, including but not limited to proliferative diseases, neurotoxic/degenerative diseases, ischemic conditions, autoimmune and autoinflammatory disorders, inflammation, immune related diseases, HIV, cancer, organ transplant rejection, septic shock, viral and parasitic infections, conditions associated with acidosis, macular degeneration, pulmonary conditions, muscle wasting diseases, fibrotic diseases, bone and hair growth diseases.

Examples of proliferative diseases or conditions include diabetic retinopathy, macular degeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy, liver cirrhosis, biliary atresia, congestive heart failure, scleroderma, radiation-induced fibrosis and pulmonary fibrosis (idiopathic pulmonary fibrosis, collagen vascular disease, sarcoidosis, interstitial lung disease and extrinsic lung disorders).

Inflammatory diseases include acute (e.g., bronchitis, conjunctivitis, myocarditis, pancreatitis) and chronic diseases (e.g., chronic cholecystitis, bronchiectasis, aortic stenosis, restenosis, psoriasis, and arthritis), as well as diseases associated with inflammation, such as fibrosis, infection, and ischemia.

Immunodeficiency disorders occur when a portion of the immune system fails to function properly or is absent. They may affect B lymphocytes, T lymphocytes or phagocytes and may be inherited (e.g., IgA deficiency, Severe Complex Immunodeficiency (SCID), thymic dysplasia and chronic granuloma) or acquired (e.g., acquired immunodeficiency syndrome (AIDS), Human Immunodeficiency Virus (HIV) and drug-induced immunodeficiency). Immune related conditions include allergic disorders such as allergy, asthma and atopic dermatitis such as eczema. Other examples of such immune-related conditions include lupus, rheumatoid arthritis, scleroderma, ankylosing spondylitis, dermatomyositis, psoriasis, multiple sclerosis, and inflammatory bowel disease (e.g., ulcerative colitis and crohn's disease).

When the immune system mistakenly attacks cells introduced into the host, tissue/organ transplant rejection occurs. Allograft-induced graft-versus-host disease (GVHD) occurs when T cells from the donor tissue continue to attack and attack the host's tissue. In all three cases, autoimmune diseases, transplant rejection, and GVHD, it may be beneficial to modulate the immune system by treating a subject with a compound or composition of the disclosure.

Also provided herein are methods of treating an autoimmune disease in a patient comprising administering a therapeutically effective amount of a compound described herein. As used herein, an "autoimmune disease" is a disease or disorder that arises from and is directed against an individual's own tissue. Examples of autoimmune diseases include, but are not limited to, inflammatory reactions, such as inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel diseases (such as crohn's disease and ulcerative colitis); respiratory distress syndrome (including Adult Respiratory Distress Syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving T cell infiltration and chronic inflammatory reactions; atherosclerosis; insufficient leukocyte adhesion; rheumatoid arthritis; systemic Lupus Erythematosus (SLE); diabetes (e.g., type I diabetes or insulin dependent diabetes); multiple sclerosis; raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; sjogren's syndrome; juvenile onset diabetes mellitus; and immune responses associated with acute and delayed-type hypersensitivity reactions mediated by cytokines and T lymphocytes commonly found in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (addison's disease); diseases involving leukocyte extravasation; central Nervous System (CNS) inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or coom's positive anemia); myasthenia gravis; antigen-antibody complex mediated diseases; resistance to glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; graves' disease; lambert-eaton myasthenia syndrome; bullous pemphigus bullous; pemphigus; autoimmune polyendocrine adenopathy; raitch's disease; stiff person syndrome; beset's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune Thrombocytopenic Purpura (ITP) or autoimmune thrombocytopenia. The compounds provided herein may be useful in the treatment of conditions associated with inflammation, including but not limited to COPD, psoriasis, asthma, bronchitis, emphysema, and cystic fibrosis.

Also provided herein are methods of treating neurodegenerative diseases using compounds as disclosed herein. Neurodegenerative diseases and conditions include, but are not limited to, stroke, ischemic injury to the nervous system, neurotrauma (e.g., impinging brain injury, spinal cord injury and traumatic injury to the nervous system), multiple sclerosis and other immune-mediated neuropathies (e.g., Guillain-Barre syndrome and variants thereof, acute motor axonal neuropathy, acute inflammatory demyelinating polyneuropathy and Fisher syndrome), HIV/AIDS dementia, axonal disease, diabetic neuropathy, Parkinson's disease, Huntington's disease, multiple sclerosis, bacteria, parasites, fungi and viral meningitis, encephalitis, vascular dementia, multi-infarct dementia, Lewy body dementia, frontal dementia (e.g., pick's disease), subcortical dementia (e.g., Huntington's or progressive supranuclear palsy), focal cortical atrophy syndrome (e.g., primary aphasia syndrome), Metabolic toxic dementia (e.g., chronic hypothyroidism or B12 deficiency) and infection-induced dementia (e.g., syphilis or chronic meningitis).

Further guidance for inhibiting protein secretion using the compounds and compositions described herein, or pharmaceutically acceptable salts thereof, can be found in the examples section below.

Synthesis of the Compounds disclosed herein

The compounds as disclosed herein can be prepared via various synthetic means. Guidance is provided to the synthetic organic chemistry workers in view of the general discussion below and the specific procedures provided in the examples section.

For example, compounds having suitable R can be prepared according to the following scheme⁵Amino acids of substituents:

other building block amino acids having the desired side chain of any one of R1-R7 can be purchased or prepared according to the examples section below. The amino acids may be reacted together in parallel to provide a small peptide, or sequentially on, for example, a resin used for peptide synthesis. Suitable protecting groups may be selected according to the desired reaction and order of peptide coupling.

The peptide may then be macrocyclized to form the compounds of the present disclosure. Macrocyclization can occur via peptide bond formation conditions.

Examples of the invention

Synthesis of the Compounds:

example 1:

to a solution of diethyl 2-acetamidomalonate (5g, 0.023mol) in DMF (25mL) at 0 deg.C was added t-BuOK (2.84g, 0.025 mol). The mixture was stirred at room temperature for 1 h. 3- (bromomethyl) -5-chlorobenzo [ b ] thiophene (6.0g, 0.023mol) was added and the reaction mixture was stirred overnight. Water (60mL) was added, and the resulting precipitate was collected by filtration and dried to give diethyl 2-acetamido-2- ((5-chlorobenzo [ b ] thiophen-3-yl) methyl) malonate.

To a solution of 2-acetamido-2- ((5-chlorobenzo [ b ] thiophen-3-yl) methyl) malonic acid diethyl ester (20g, 50mmol) in EtOH (100mL) was added 4n naoh aqueous solution (44 mL). The reaction mixture was heated at reflux for 3 h. The mixture was adjusted to pH 3-5 with 6N aqueous HCl and then refluxed overnight. The solvent was removed under reduced pressure to half the volume. The resulting precipitate was collected by filtration and dried to give the compound 2-acetamido-3- (5-chlorobenzo [ b ] thiophen-3-yl) propionic acid.

2-acetamido-3- (5-chlorobenzo [ b ] thiophen-3-yl) propionic acid (15g, 51mmol) was suspended in distilled water (450mL), and the mixture was adjusted to pH 8-9 with 2N aqueous LiOH solution. L-acylase (1.5g) was added and the mixture was heated at 35 ℃ to 40 ℃ for 36 h. Activated carbon (1.5g) was added and the resulting mixture was heated at 60 ℃ for 1 h. The activated carbon was filtered off and the filtrate was adjusted to pH 1. The aqueous solution was washed with ethyl acetate (500mL × 2), and then concentrated to give the compound (S) -2-amino-3- (5-chlorobenzo [ b ] thiophen-3-yl) propionic acid.

To the compound (S) -2-amino-3- (5-chlorobenzo [ b ]]To a solution of thien-3-yl) propionic acid (13g, 51mmol) in water (80mL) was added Boc₂O(16.5g，76mmol) in acetone (30 mL). The pH was adjusted to 13 with NaOH mixture and then stirred overnight. The mixture was adjusted to pH 1-2 with HCl and extracted with ethyl acetate (200 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was washed with hexane to give the compound (S) -2- ((tert-butoxycarbonyl) amino) -3- (5-chlorobenzo [ b ]]Thiophen-3-yl) propionic acid.

To (S) -2- ((tert-butoxycarbonyl) amino) -3- (5-chlorobenzo [ b ] at 0 deg.C]Thien-3-yl) propionic acid (2.20g, 6.18mmol) in THF (6.5mL) was added portionwise sodium hydride (544mg, 13.6mmol, 60% dispersion in mineral oil) while maintaining the temperature<10 ℃. After 30min, iodomethane (1.93g, 13.6mmol) was added. The mixture was stirred for 6h then cooled to 0 ℃ and an additional portion of NaH (544mg, 13.6mmol) was added followed by MeI (1.93g, 13.6mmol) to warm the reaction mixture to room temperature and stir for an additional 16 h. The reaction was diluted with water (about 50mL) and the basic aqueous solution was extracted with diethyl ether (2X 25 mL). The aqueous solution was brought to pH 3 with citric acid (10%, aq) and extracted with EtOAc (3 × 50 mL). The combined organics were over MgSO₄Dried and concentrated in vacuo to afford (S) -2- ((tert-butoxycarbonyl) (methyl) amino) -3- (5-chlorobenzo [ b)]Thiophen-3-yl) propionic acid.

The flask was charged with (S) -2- ((tert-butoxycarbonyl) (methyl) amino) -3- (5-chlorobenzo [ b ]]Thiophen-3-yl) propionic acid (2.00g, 5.60mmol), Leu-OMe (1.18g, 6.50mmol) and HATU (2.58g, 6.78 mmol). The mixture was dissolved in DMF (15mL) under argon and cooled to 0 ℃, then DIPEA (2.95mL, 17.0mmol) was added and the mixture was stirred for 2h, diluted with EtOAc (20mL) and washed with water (4 × 10 mL). The combined aqueous phases were extracted with more EtOAc (10mL) and the combined organics were washed with brine (10mL) and dried (Na)₂SO₄) Filtered and concentrated to give a yellow solid (3.03g) which was subjected to the next reaction without further purification.

To a solution of the crude dipeptide ester (3.00g, 6.21mmol) in THF (10mL) and water (4mL) was added LiOH (7.5mL of a 1M solution in water, 7.45mmol) at 0 deg.C, and the reaction mixture was stirred for 1h, quenched dropwise with 1M HCl, and extracted with EtOAc (3X 10 mL). Combined organic matterWashed with brine (5mL) and dried (MgSO)₄) Filtered and concentrated to give ((S) -2- ((tert-butoxycarbonyl) (methyl) amino) -3- (5-chlorobenzo [ b ]]Thiophen-3-yl) propionyl) -L-leucine.

To ((S) -2- ((tert-butoxycarbonyl) (methyl) amino) -3- (5-chlorobenzo [ b)]Thien-3-yl) propionyl) -L-leucine (2.83g, 5.86mmol) in dioxane (5mL) was added HCl (5mL of a 4M solution in dioxane). After stirring for 1h, LCMS indicated only-50% deprotection occurred, and another aliquot of HCl (5mL, 4M solution in dioxane) was added. After a further 2h, deprotection was complete and the reaction mixture was concentrated and co-evaporated 3 times with toluene and then once with dioxane. The residue was dissolved in dioxane (37mL) and water (37mL) and Na was added₂CO₃(1.43g, 13.48mmol) followed by FmocOSu (2.57g, 7.62mmol) and the reaction mixture was stirred overnight. The dioxane was removed under reduced pressure and the reaction mixture was quenched to pH 3 with citric acid (10% aq) and extracted with EtOAc (3X 10 mL). The combined organic fractions were washed with water (5mL), then brine (5mL), then Na₂SO₄Dried, filtered and concentrated under reduced pressure. The crude material was flash chromatographed (silica, gradient elution, 0 to 5%, v/v, MeOH/DCM) to give ((S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) (methyl) amino) -3- (5-chlorobenzo [ b ])]Thiophen-3-yl) propionyl) -L-leucine. Lcms (esi): [ M-H ]]^-，603.2。

The following compounds were prepared from the corresponding aryl/heteroaryl bromides or commercially available/known amino acids using the procedure from example 1:

example 2

To a suspension of abrin base (2.00g, 9.16mmol) in water (17mL) and dioxane (17mL) was added K₂CO₃(3.60g, 26.12mmol), and then Boc was added dropwise₂A solution of O (2.40g, 11.00mmol) in dioxane (10 mL). The mixture was stirred at room temperature for 12h and then concentrated to remove dioxane. The reaction mixture was washed with hexane (2X 20mL), then acidified to pH-3 with citric acid (10% aq) and extracted with EtOAc (2X 20 mL). The combined organic phases were washed with water (10mL), brine (10mL) and then (Na)₂SO₄) Dried, filtered and concentrated under reduced pressure to give N^α- (tert-butoxycarbonyl) -N^α-methyl-L-tryptophan.

To a stirred solution of fresh powdered potassium hydroxide (4.32g, 75.38mmol) in dimethyl sulfoxide (anhydrous, 37mL) was added N under argon at room temperature^α- (tert-butoxycarbonyl) -N^α-methyl-L-tryptophan (6.00g, 18.85mmol) and the mixture was stirred for 1 h. 4-bromobenzyl bromide (5.18g, 20.73mmol) was then added and the mixture was stirred under argon for 16 h. The solution was diluted with water (10mL), washed with diethyl ether (2 × 5mL), and then acidified with citric acid (10% aq) until pH-3. The mixture was extracted with EtOAc (3X 20mL) and the combined fractions were washed with water (10mL) then brine (10mL) and then dried (Na)₂SO₄) And concentrated under reduced pressure to give 1- (4-bromophenylmethyl) -N^α- (tert-butoxycarbonyl) -N^α-methyl-L-tryptophan.

To 1- (4-bromophenyl-methyl) -N^α- (tert-butoxycarbonyl) -N^αTo a solution of (2.50g, 5.13mmol) of (E) -methyl-L-tryptophan in dioxane (1mL) was added HCl (5mL of a 4M solution in dioxane). The mixture was stirred at room temperature for 2h, then concentrated under reduced pressure, co-evaporated with dioxane several times and then redissolved in a mixture of dioxane (12mL) and water (12 mL). To this mixture was added Na₂CO₃(1.25g, 11.80mmol), followed by FmocOSu (2.25g, 6.67mmol), andthe mixture was stirred vigorously overnight. The mixture was concentrated to remove dioxane, acidified with citric acid (10mL of 10% aqueous solution) and extracted with EtOAc (3X 10mL), and the combined organic fractions washed with water then brine and dried (MgSO 2)₄) Filtered, and concentrated under reduced pressure. The residue was flash chromatographed (silica, gradient elution, 0-5% MeOH/DCM) to give N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-bromophenylmethyl) -N^α-methyl-L-tryptophan (2.83g, 91%).¹H-NMR(400MHz；CDCl₃Rotamers delta 7.80-7.73(m,3H),7.67-7.60(m,1H),7.54(t, J ═ 6.8Hz,1H),7.49-7.39(m,5H),7.36-7.31(m,2H),7.22-7.12(m,4H),6.97(s,1H),6.91-6.86(m,2H),5.18-5.15(m,2H),5.05-5.01(m,0.5H),4.87-4.82(m,0.5),4.51-4.35(m,2H),4.26-4.17(m,1.5H),4.05-4.01(m,0.5),3.56-3.50(m,1H),3.41-3.34(m,1H),2.86 (m, 3H). Lcms (esi): [2M-H ]]^-，1218.2。

The following compounds were prepared from the corresponding bromides using the procedure from example 2:

LCMS(ESI)：[2M-H]^-，1197.6。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (3-methoxybenzyl) -N^αmethyl-L-tryptophan

LCMS(ESI)：[2M-H]^-，1120.0。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- ((6-fluoropyridin-3-yl) methyl) -N^α-methyl-L-tryptophan

LCMS(ESI)：[2M-H]^-，1097.8。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- ((6-fluoropyridin-3-yl) methyl) -N^α-methyl-L-tryptophan

LCMS(ESI)：[2M-H]^-，1097.2。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -N^α-methyl-1- ((6 to methylpyridin-2-yl) methyl) -L-tryptophan

LCMS(ESI)：[2M-H]^-，1089.3。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (3, 4-difluorobenzyl) -N^α-methyl-L-tryptophan

LCMS(ESI)：[2M-H]^-，1131.3。

S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) (methyl) amino) -3- (benzo [ b ] thiophen-3-yl) propanoic acid

LCMS(ESI)：[2M-H]^-，913.1。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-methoxybenzyl) -N^α-methyl-L-tryptophan

LCMS(ESI)：[2M-H]^-，1119.7。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (2-methoxybenzyl) -N^α-methyl-L-tryptophan

LCMS(ESI)：[2M-H]^-，1120.0。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -N^α-methyl-1- ((3-methylisoxazol-5-yl) methyl) -L-tryptophan

LCMS(ESI)[2M-H]^-，1070.0。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-fluorophenylmethyl) -N^α-methyl-L-tryptophan

LCMS(ESI)：[2M-H]^-，1095.9。

Example 3

20mL scintillation vial filled with N^α- (tert-butoxycarbonyl) -N^α-methyl-L-tryptophan (500mg, 1.57mmol), iodobenzene (352mg, 1.72mmol), copper iodide (30mg, 0.16mmol), trans-N, N' -dimethylcyclohexane-1, 2-diamine (50. mu.L, 0.31mmol), potassium phosphate (733mg, 3.45mmol) and dioxane (5 mL). The mixture was degassed by bubbling nitrogen through the mixture for 10min, and then the reaction mixture was heated to 110 ℃ for 16 h. The reaction mixture was concentrated and treated with citric acid (2mL of 10% aqueous solution) and extracted with EtOAc (3 × 5mL) and the combined organics were washed with brine (5mL), filtered, concentrated under reduced pressure and chromatographed (silica, 0 to 10%, v/v MeOH/DCM) to give N^α- (tert-butoxycarbonyl) -N^α-methyl-1-phenyl-L-tryptophan.¹H-NMR(400MHz；CDCl₃Rotamers delta 7.70-7.68(m,1H),7.58(d, J ═ 7.8Hz,1H),7.55-7.48(m,4H),7.40-7.34(m,1H),7.26-7.16(m,3H),4.98-4.85(m,1H),3.58-3.39(m,1.5H),3.29-3.22(m,0.5H),2.89(s,1.5H),2.78(s,1.5H),1.45(s,4.5H),1.22(s, 4.5H). Lcms (esi): [2M-H ]]^-，789.6。

Method of using example 2Method from N^α- (tert-butoxycarbonyl) -N^α-methyl-1-phenyl-L-tryptophan to obtain N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -N^α-methyl-1-phenyl-L-tryptophan.

LCMS(ESI)：[2M-H]^-，1033.5。

Example 4

To a solution of L-leucine (2.50g, 19.06mmol) in allyl alcohol (25mL) was added p-TsOH monohydrate, and the mixture was stirred at 90 deg.C (oil bath) for 16 h. The next day, the solution was concentrated under reduced pressure to a solid residue, which was dissolved in DCM (25mL) and purified with NaHCO₃(10mL of a saturated aqueous solution). The aqueous fraction was extracted with DCM (2X 10mL) and dried (Na)₂SO₄) The organics were combined and filtered directly into HCl (4 equiv.,. about.19 mL of a 4M solution in dioxane). The solution was concentrated under reduced pressure and then Et₂And (4) precipitating O. The mixture was briefly sonicated, left to stand for a few minutes, and then the solution was decanted from the solid using a pipette. The product was dried in vacuo overnight to give the HCl salt of L-leucine allyl ester.¹H-NMR(400MHz；DMSO-d₆):δ8.61(t,J＝0.4Hz,3H),5.94(ddt,J＝17.2,10.6,5.4Hz,1H),5.39(dq,J＝17.3,1.6Hz,1H),5.29(dq,J＝10.5,1.3Hz,1H),4.70(d,J＝5.4Hz,2H),4.01-3.98(m,1H),1.81-1.73(m,1H),1.67(t,J＝7.3Hz,2H),0.91(dd,J＝6.5,1.2Hz,6H)。

(S) -2-amino-4, 4, 4-trifluorobutyric acid allyl ester hydrochloride

Using the procedure described above;¹H-NMR(400MHz；CDCl₃):δ6.00-5.89(m,1H),5.39-5.29(m,2H),4.66(dd,J＝5.9,1.1Hz,1H),4.20-3.97(m,1H),3.51(dd,J＝8.6,5.1Hz,1H),2.38-2.21(m,1H),2.05(ddt,J＝13.7,10.8,5.4Hz,1H),1.83-1.74(m,1H),1.57-1.53(m,1H)。

example 5:

100mL round bottom flask was charged with N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-bromophenylmethyl) -N^α-methyl-L-tryptophan (6.50g, 10.66mmol), Leu-OAllyl (2.54g, 12.26mmol) and HATU (4.87g, 12.87 mmol). The mixture was dissolved in DMF (50mL) under argon and cooled to 0 ℃, then DIPEA (5.57mL, 32.00mmol) was added and the mixture was stirred for 2h, diluted with EtOAc (50mL) and washed with water (4 × 20 mL). The combined aqueous phases were extracted with more EtOAc (10mL) and the combined organics were washed with brine (10mL) and dried (Na)₂SO₄) Filtered and concentrated to give N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-bromophenylmethyl) -N^α-methyl-L-tryptanthyl-L-leucine allyl ester.¹H-NMR(400MHz；CDCl₃Rotamers delta 7.80-7.79(m,2H),7.73-7.63(m,2H),7.55-7.34(m,5H),7.18(d, J ═ 0.1Hz,3H),7.00(s,1H),6.90-6.88(m,2H),6.45-6.43(m,1H),6.04-5.86(m,1H),5.38-5.07(m,4H),4.68-4.60(m,3H),4.44-4.35(m,1H),4.23-4.14(m,1H),3.93-3.80(m,1H),3.52-3.43(m,1H),3.28-3.22(m,1H),2.94-2.90(m,3H), 1.69-1.88 (m, 0.51H), 99-0.51 (m, 0H). Lcms (esi): [ M + H ]]⁺，764.6。

The following compounds were prepared using the procedure as described in example 5.

LCMS(ESI)：[M+H]⁺，788.8。

LCMS(ESI)：[M+H]⁺，803.25。

LCMS(ESI)：[M+H]⁺，652.6。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-bromophenylmethyl) -N^α-methyl-L-tryptophanyl-L-leucine tert-butyl ester

¹H-NMR(400MHz；CDCl₃Rotamers Δ 7.80-7.77(m,1H),7.73-7.65(m,2H),7.54-7.51(m,1H),7.45-7.38(m,3H),7.35-7.33(m,2H),7.24-7.14(m,3H),7.02-6.99(m,1H),6.95-6.84(m,2H),6.43-6.40(m,1H),5.21-5.08(m,2H),4.51-4.47(m,1H),4.37-4.33(m,0.5H),4.22-4.18(m,0.5H),3.87-3.78(m,1H),3.57-3.42(m,1H),3.28-3.22(m,1H),2.93 (m,1H), 1.93-6.64 (m,1H), 1.00 (m,1H), 1H, 6.64 (m, 1H). Lcms (esi): [ M + H ]]⁺，780.2。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -N^α-methyl-1- (4- (trifluoromethyl) benzyl) -L-tryptophanyl-L-leucine tert-butyl ester

LCMS(ESI)：[M+H]⁺，770.2。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (3-methoxybenzyl) -N^α-methyl-L-tryptophanyl-L-leucine tert-butyl ester

LCMS(ESI)：[M+H]⁺，732.4。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- ((6-fluoropyridin-3-yl) methyl) -N^α-methyl-L-tryptophanyl-L-leucine tert-butyl ester

LCMS(ESI)：[M+H]⁺，719.3。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- ((5-fluoropyridin-3-yl) methyl) -N^α-methyl-L-tryptophanyl-L-leucine tert-butyl ester

LCMS(ESI)：[M+H]⁺，719.2。

N^α- (((9H-fluoren-9-yl) methoxy) carbonyl) -N^α-methyl-1- ((6-methylpyridin-2-yl) methyl) -L-tryptophanyl-L-leucine tert-butyl ester

LCMS(ESI)：[M+H]⁺，716.2。

LCMS(ESI)：[M+H]⁺，627.1。

LCMS(ESI)：[M-H]^-，608.5。

LCMS(ESI)：[M+H]⁺，736.0.

LCMS(ESI)：[M+Na]⁺，593.9。

LCMS(ESI)：[M+Na]⁺，739.4。

Example 6:

to a solution of N α - (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-bromophenylmethyl) -N α -methyl-L-tryptophanyl-L-leucine allyl ester (8.29g, 10.87mmol) in DCM (75mL) was added PhSiH under argon at room temperature₃(2.62mL, 21.74mmol) followed by Pd (PPh)₃)₄(628mg, 0.54 mmol). The mixture was stirred for 2H then concentrated onto silica and chromatographed (silica, 0-5%, MeOH/DCM) to give N α - (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-bromophenylmethyl) -Na-methyl-L-tryptophanyl-L-leucine.¹H-NMR(400MHz；CDCl₃Rotamers) < delta > 7.79(dd, J ═ 7.2,0.6Hz,2H),7.66(d, J ═ 1.4Hz,3H),7.50-7.39(m,6H),7.18-7.15(m,3H),6.97(d, J ═ 0.3Hz,1H),6.87-6.85(m,2H),5.18-5.06(m,3H),4.60-4.54(m,1H),4.39-4.31(m,1H),4.21-4.14(m,1H),3.54-3.40(m,2H),3.25-3.18(m,1H),2.94-2.84(m,3H),1.72-1.50(m,3H),0.94-0.87(m, 6H). Lcms (esi): [ M + H ]]⁺，722.6。

The following compounds were prepared using the procedure of example 6:

LCMS(ESI)：[2M+H]⁺，1222.5

LCMS(ESI)：[2M-H]^-，1526.3。

LCMS(ESI)：[2M-H]^-，1496.6。

example 7:

to a solution of na- (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-bromophenylmethyl) -na-methyl-L-tryptophanyl-L-leucine tert-butyl ester (98mg, 0.13mmol) in DCM (1mL) was added TFA (1 mL). The mixture was stirred at room temperature for 5h, then concentrated under reduced pressure. The crude residue was chromatographed (silica, 0-5%, MeOH/DCM) to give N α - (((9H-fluoren-9-yl) methoxy) carbonyl) -1- (4-bromophenylmethyl) N α -methyl-L-tryptophanyl-L-leucine.

The following compounds were prepared using the procedure of example 7:

LCMS(ESI)：[2M-H]^-，1424.8。

the next step was performed without further analysis.

The next step was performed without further analysis.

LCMS(ESI)：[M-H]^-，657.0。

LCMS(ESI)：[2M-H]^-，1140.3。

LCMS(ESI)：[M+H]⁺，679.9。

Example 8:

to a 10 ℃ solution of Boc-D-Gln-OH (30.0g, 122mmol) in pyridine (150mL) was added dropwise a solution of DCC (25.9g, 125mmol) in pyridine (75 mL). The mixture was stirred at room temperature for 3.5 hours followed by addition of solid DCC (1 g). After 30min, the reaction mixture was filtered and the pyridine-rinsed solid was collected into the filtrate. The filtrate was concentrated in vacuo, resuspended in EtOAc (500mL), washed with 5% aqueous citric acid (3 × 300mL), brine (3 × 300mL), dried over sodium sulfate, filtered and concentrated in vacuo to give (R) -2- ((tert-butoxycarbonyl) amino) -4-cyanobutyric acid.¹H NMR(400MHz,DMSO-d₆)δ＝12.69(br s,1H),7.17(d,J＝8.2Hz,1H),3.98-3.86(m,1H),2.58-2.51(m,2H),2.07-1.96(m,1H),1.83(m,1H),1.39(s,9H)。

To (R) -2- ((tert-butoxycarbonyl) amino) -4-cyanobutyric acid intermediate (23.2g, 91.5mmol) was added water (300mL), and the mixture was heated to 95 ℃ for 15 min. The suspension was filtered hot and the filtrate was concentrated in vacuo to give (R) -2-amino-4-cyanobutyric acid.

To a 0 ℃ solution of (R) -2-amino-4-cyanobutyric acid 2(11g, 86mmol) in water: AcOH, 4:1(860mL) was added NaNO with stirring₂Aqueous solution (2M, 86 mL). The reaction was stirred overnight and the solvent volume was reduced to 150mL in vacuo. Concentrated HCl brought the solution to pH 1-2 and the aqueous layer was extracted with EtOAc. The organic layer was concentrated and triturated with ether to give (R) -4-cyano-2-hydroxybutyric acid. The supernatant was concentrated to a residue. The residue was dissolved in 500ml of 4:1 water AcOH and reacted with 108mmol of NaNO at 0 deg.C₂In 50ml of H₂O overnight and allowed to warm to ambient temperature overnight. The reaction was treated as before to produce more product.¹H NMR(400MHz,D₂O)δ＝4.36(dd,J＝4.3,8.3Hz,1H),2.66-2.59(m,2H),2.20(m,1H),2.09-1.96(m,1H)。

Example 9:

resin loading: Cl-2-Cl-trityl resin (2.5g, 1.6 meq/g, 4.07mmol) was swollen with DCM (20mL, anhydrous) for 1h and filtered. To the resin was added (2R) -4-cyano-2-hydroxybutyric acid (789mg, 6.11mmol, 1.5 equivalents) and a solution of DIPEA (2.11mL, 12.23mmol, 3 equivalents) in DCM (20mL, anhydrous) and the mixture was stirred for 16 h. The resin was then filtered and washed with DCM (2X 20 mL. times.1 min) and then with toluene (anhydrous, 2X 20 mL. times.1 min with gentle shaking). To the filtered resin was added a solution of Fmoc-N-Me-Ala-OH (3.58g, 11.0mmol, 2.0 equiv.), DIC (1.72mL, 11.0mmol, 2 equiv.), and N-methylimidazole (0.88mL, 11.0mmol, 2 equiv.) in toluene (20mL, anhydrous), and the mixture was stirred for 1 h. The resin was filtered and the coupling procedure was repeated again. The resin was then washed using resin 'washing method a' described in example 10. The resin was then dried under high vacuum overnight to give a supported dry resin.

Example 10:

general procedure for peptide extension on resin

Resin washing method a: DMF (10mL/g resin) was added to the resin and the resin was agitated by gentle shaking for 1 min. The resin was filtered and the procedure repeated with i-PrOH (1X 10mL/g resin), then DMF (1X 10mL/g resin), then i-PrOH (1X 10mL), then DMF (1X 10mL/g resin), then DCM (3X 10mL/g resin).

Resin washing method B: DMF (10mL/g resin) was added to the resin and the resin was agitated by gentle shaking for 1 min. The resin was filtered and the procedure repeated with i-PrOH (1X 10mL/g resin), then DMF (1X 10mL/g resin), then i-PrOH (1X 10mL), then DMF (3X 10mL/g resin).

Resin washing method C: DMF (10mL/g resin) was added to the resin and the resin was agitated by gentle shaking for 1 min. The resin was filtered and the procedure repeated with i-PrOH (1X 10mL/g resin), then DMF (1X 10mL/g resin), then i-PrOH (1X 10mL), then DMF (1X 10mL/g resin), then toluene (3X 10mL/g resin).

Fmoc removal: a solution of 4-methylpiperidine in DMF (20%, 10mL/g resin) was added to the DMF (or DCM) swollen resin and the mixture was stirred for 5 min. The resin was filtered under suction and the procedure repeated two more times. The resin was filtered and washed using 'resin wash method B'.

Resin coupling method a: DIPEA (4 equiv.) was added to a solution of Fmoc-AA-OH (2 equiv.) and HATU (2 equiv.) in DMF (0.1M). The solution was mixed to homogeneity and added to the resin. The mixture was stirred at room temperature for 1h, then filtered and washed using 'resin wash method B'.

Resin coupling method B: to a suspension of Fmoc-AA-OH (2 equiv.) and EEDQ (2 equiv.) in toluene (0.35M) was added 2 volumes of DMF. The solution was quickly mixed to homogeneity and added to toluene washed resin. The mixture was stirred at room temperature for 2h, then filtered and washed using 'resin wash method B'.

Synthesis of linear peptide: a resin of 2-Cl-trityl- (R) -2- ((N- (((9H-fluoren-9-yl) methoxy) carbonyl) -N-methyl-L-propylaminoyl) oxy) -4-cyanobutyrate (3.00g, 2.46mmol) was swollen with DCM (25mL) for 1H. The resin was then filtered and the Fmoc group removed using the 'Fmoc removal' procedure. The resin was then washed using 'resin wash method C', then coupled with Fmoc-N-Me-Trp (4-BrBn) -Leu-OH using 'resin coupling method B', and washed with 'resin wash method B', then Fmoc was removed using 'Fmoc removal' procedure, and the resin was washed with 'resin wash method B'. The peptides were elongated in a similar manner using:

1) Fmoc-Gly (CH) Using ` resin coupling method B `₂CF₃)-OH

2) Fmoc-N-Leu-OH Using' resin coupling method A

3) Fmoc-Gly (CH) Using ` resin coupling method B `₂CF₃)-OH

After removal of the last N-terminal Fmoc group, the resin was washed using 'washing method a'.

Resin compositionCracking: to a hypothetical 2.46mmol of loaded resin was added HFIP (15mL of a 20% solution in DCM) and the mixture was stirred for 15 min. The solution was filtered and collected into a 250mL round bottom flask containing HCl (0.25M in EtOAc, 40 mL-4 equiv.). The lysis procedure was repeated again and the solution was collected in the same flask of HCl. The remaining resin was washed with DCM (2 × 10mL) into HCl solution, and the combined solution was concentrated under reduced pressure. The residue was dissolved in a minimal volume of EtOAc and then treated with Et₂And (4) precipitating O. Decant, sonicate the mixture for-10 seconds, and allow the precipitate to settle for a few minutes, then carefully remove the liquid using a pipette. Et for residual solid₂O trituration and drying under high vacuum to give (3R,6S,9S,12S,15S,18S,21S) -12- ((1- (4-bromophenylmethyl) -1H-indol-3-yl) methyl) -3-carboxy-1-cyano-23, 23, 23-trifluoro-9, 18-diisobutyl-6, 7,13, 19-tetramethyl-5, 8,11,14,17, 20-hexaoxy-15- (2,2, 2-trifluoroethyl) -4-oxa-7, 10,13,16, 19-pentaazaeicosatriene-21-ammonium chloride. Hrms (esi): [ M-H ]]^-C₅₃H₈₇N₈O₉Calculated value, 1101.3878; found 1101.3868.

The following compounds were prepared using the procedures of examples 9 and 10:

LCMS(ESI)：[M-H]^-，841.0。

LCMS(ESI)：[M+H]⁺，1116.3。

LCMS(ESI)：[M-H]^-，963.8。

LCMS(ESI)：[M-H]^-，937.4。

LCMS(ESI)：[M+H]⁺,984.1。

LCMS(ESI)：[M-H]^-，1114.1。

LCMS(ESI)：[M-H]^-，1098.9。

LCMS(ESI)：[M+H]⁺，958.4。

LCMS(ESI)：[M-H]^-，986.5。

LCMS：(ESI)：[M-H]^-，988.0。

LCMS(ESI)：[M-H]^-，802.1。

LCMS(ESI)：[M-H]^-，949.2。

LCMS(ESI)：[M-H]^-，961.2。

LCMS(ESI)：[M-H]^-，1037.6。

LCMS：[M+H]⁺，1040.6。

LCMS：(ESI)：[M-H]^-，1000.2。

LCMS(ESI)：[M-H]^-，1000.2。

LCMS(ESI)：[M-H]^-，1000.1。

LCMS(ESI)：[M-H]^-，1049.6。

LCMS(ESI)：[M-H]^-，974.6。

LCMS：(ESI)：[M-H]^-，1005.5。

LCMS(ESI)：[M-H]^-，884.5。

macrocyclization of the linear peptide was not performedAnd (4) performing spectral analysis.

The linear peptide was macrocyclized without spectroscopic analysis.

LCMS(ESI)：[M-H]^-，1047.4。

LCMS(ESI)：[M+H]⁺，1064.6。

LCMS(ESI)：[M+H]⁺，1050.6。

LCMS(ESI)：[M+H]⁺，1076.7。

LCMS(ESI)：[M-H]^-，969.6。

LCMS(ESI)：[M-H]^-，879.3。

Example 11C-58:

reacting (3R,6S,9S,12S,15S,18S,21S) -12- ((1- (4-bromophenyl-methyl) -1H-indol-3-yl) methyl) -3-carboxy-1-cyano-23, 23, 23-trifluoro-9, 18-diisobutyl-6, 7,13, 19-tetramethyl-5, 8,11,14,17, 20-hexa-oxy-15- (2,2, 2-trifluoroethyl)) A solution of-4-oxa-7, 10,13,16, 19-pentaazaeicosatriane-21-ammonium chloride (930mg, 0.816mmol) and DIPEA (427. mu.L, 2.45mmol, 3 equiv.) in DCM (313mL) was added via dropping funnel (at a rate of-1 drop/sec) to a rapidly stirred solution of HATU (316mg, 0.858mmol), DMF (6mL) and DCM (1.31L). After complete addition, the funnel was rinsed into the reaction mixture with DCM (-10 mL) and the reaction was stirred for 18 h. An additional portion of HATU (158mg, 429mmol) was added and the reaction was stirred for an additional 2 h. The reaction mixture was washed with HCl (500mL of 0.2M aqueous solution) followed by NaHCO₃(500mL of a 33% saturated aqueous solution). The DCM layer was then dried (MgSO)₄) Filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, step gradient elution, 5% -50% acetone: hexanes). Concentration of the appropriate fractions afforded 3- ((2R,5S,8S,11S,14S,17S,20S) -14- ((1- (4-bromophenyl-methyl) -1H-indol-3-yl) methyl) -8, 17-diisobutyl-7, 13,19, 20-tetramethyl-3, 6,9,12,15,18, 21-heptaoxy-5, 11-bis (2,2, 2-trifluoroethyl) -1-oxa-4, 7,10,13,16, 19-hexaazacycloheneicosane-2-yl) propionitrile.¹H-NMR (400 MHz; acetone-d)₆):δ8.45(d,J＝10.1Hz,1H),8.25(d,J＝9.6Hz,1H),8.01(d,J＝7.0Hz,1H),7.71(dd,J＝7.1,0.9Hz,1H),7.50-7.47(m,2H),7.41(t,J＝5.9Hz,1H),7.36(s,1H),7.18-7.09(m,4H),5.45-5.34(m,2H),5.26-5.06(m,3H),4.76-4.71(m,1H),4.37(dd,J＝10.9,3.6Hz,1H),3.99-3.94(m,1H),3.38-3.32(m,1H),3.29(s,3H),3.17-3.09(m,1H),2.94(s,3H),2.58(s,3H),2.52-2.48(m,2H),2.37-2.18(m,4H),2.07(dt,J＝4.4,2.2Hz,2H),1.96-1.82(m,2H),1.57-1.45(m,8H),1.04-0.94(m,10H),0.90-0.85(m,2H),0.49-0.42(m,1H)。LCMS(ESI)：C₄₉H₆₂BrF₆N₈O₈[M+H]⁺Calculated value, 1083.4; found value, 1083.2

The following compounds were prepared using the procedure of example 11:

C-37 ¹H-NMR (400 MHz; acetone-d)₆):δ8.73(d,J＝10.0Hz,1H),7.99(d,J＝9.6Hz,1H),7.73(d,J＝6.7Hz,1H),7.36-7.33(m,2H),7.31-7.25(m,3H),5.10-4.97(m,3H),4.88(dt,J＝9.5,7.1Hz,1H),4.52(ddd,J＝11.7,7.0,3.6Hz,1H),4.43(dd,J＝10.9,4.2Hz,1H),3.92(q,J＝6.8Hz,1H),3.20(s,3H),3.12-3.09(m,2H),2.87(s,3H),2.60(t,J＝3.7Hz,2H),2.54(s,3H),2.31-2.25(m,1H),2.08-1.97(m,1H),1.81(dtd,J＝13.9,12.5,5.4Hz,2H),1.70-1.38(m,13H),1.08(d,J＝6.4Hz,3H),1.01-0.90(m,16H),0.71-0.66(m,6H),-0.18(ddd,J＝14.1,10.7,3.4Hz,1H)。LCMS(ESI)：C₄₄H₇₀N₇O₈[M+H]⁺Calculated value, 824.5; found value, 824.6

C-48 ¹H-NMR (400 MHz; acetone-d)₆):δ8.88(d,J＝9.9Hz,1H),8.18(d,J＝9.4Hz,1H),7.75(t,J＝8.4Hz,2H),7.49-7.43(m,3H),7.29(s,1H),7.23-7.18(m,2H),7.16-7.09(m,2H),5.39(s,2H),5.23-5.12(m,2H),5.06(dt,J＝10.2,3.7Hz,2H),4.45-4.39(m,1H),4.30(dd,J＝11.1,4.1Hz,1H),4.01(q,J＝6.8Hz,1H),3.30(s,4H),3.25-3.10(m,2H),2.94(s,3H),2.57(s,3H),2.56-2.52(m,2H),2.35-2.18(m,3H),2.16-2.15(m,1H),2.01-1.93(m,2H),1.87-1.80(m,1H),1.75-1.61(m,2H),1.58-1.46(m,4H),1.38-1.26(m,2H),1.02(d,J＝6.4Hz,3H),0.99-0.95(m,3H),0.87-0.84(m,1H),0.37-0.27(m,3H),0.01(t,J＝6.0Hz,3H),-0.55(s,1H)。LCMS(ESI)：C₅₀H₆₄BrF₆N₈O₈[M+H]⁺Calculated, 1097.4; found value, 1097.2

C-49 ¹H-NMR (400 MHz; acetone-d)₆):δ8.92(d,J＝10.0Hz,1H),8.20(d,J＝9.5Hz,1H),8.02(dd,J＝16.7,7.9Hz,2H),7.79-7.78(m,1H),7.51-7.40(m,3H),5.18(ddd,J＝16.4,9.6,4.6Hz,2H),5.09(td,J＝11.3,3.7Hz,2H),4.39-4.31(m,1H),4.28(t,J＝5.5Hz,1H),4.04(qd,J＝9.1,5.3Hz,1H),3.47(dd,J＝15.0,10.8Hz,2H),3.33(s,3H),3.19-3.11(m,1H),2.96(s,3H),2.57(s,4H),2.34-2.20(m,3H),2.19-2.01(m,4H),1.98-1.88(m,1H),1.87-1.80(m,1H),1.74-1.64(m,2H),1.49(d,J＝6.8Hz,4H),1.37(d,J＝10.1Hz,2H),1.22(s,1H),1.01(d,J＝6.4Hz,3H),0.95(d,J＝6.6Hz,3H),0.49-0.48(m,3H),-0.08(d,J＝6.6Hz,2H),-0.41--0.48(m,1H)。LCMS(ESI)：C₄₃H₅₈F₆N₇O₈S[M+H]⁺Calculated value, 947.4; found value, 947.3

C-50 ¹H-NMR (400 MHz; acetone-d)₆) δ 8.96(d, J ═ 10.0Hz,1H),8.05-7.95(m,3H),7.70(d, J ═ 6.7Hz,1H),7.52-7.40(m,3H),5.17-5.04(m,2H),4.88-4.82(m,1H),4.36(ddd, J ═ 17.9,9.5,4.1Hz,2H),4.01 (quintuple, J ═ 7.0Hz,1H),3.47-3.36(m,2H),3.31(s,3H),2.96(s,3H),2.52(s,4H),2.27-2.21(m,3H),1.94-1.73(m,4H),1.66-1.45(m,8H),1.39 (m, 1.88H), 3.7 (m,6H), 3H), 3.83-6H, 3H), 3.6 (d, 3H), 3.0, 3H), 3.7, 3H, 3.6, 3H, 3H, -0.07(d, J ═ 6.6Hz,3H), -0.42(ddd, J ═ 14.3,11.1,3.4Hz,1H). lcms (esi): c₄₅H₆₅F₃N₇O₈S[M+H]⁺Calculated value, 920.5; found value, 920.9

C-51 ¹H-NMR (400 MHz; acetone-d)₆):δ8.70(d,J＝10.1Hz,1H),8.28(d,J＝9.5Hz,1H),7.84(dd,J＝7.0,1.3Hz,1H),7.71(d,J＝6.9Hz,1H),7.64-7.53(m,5H),7.42-7.38(m,1H),7.36(d,J＝11.9Hz,1H),7.28-7.20(m,2H),5.22(dt,J＝9.4,6.2Hz,1H),5.13-5.06(m,3H),4.41(ddd,J＝11.5,7.4,3.7Hz,1H),4.31(dd,J＝10.9,4.3Hz,1H),3.97(q,J＝6.8Hz,1H),3.37-3.31(m,5H),3.16(ddd,J＝14.9,11.2,6.3Hz,1H),2.95(s,3H),2.59(s,3H),2.52(t,J＝6.2Hz,2H),2.32(ddd,J＝15.1,11.0,5.8Hz,1H),2.23-2.16(m,1H),1.97(dtd,J＝9.8,7.5,5.4Hz,1H),1.83(ddd,J＝14.1,10.8,3.4Hz,1H),1.68-1.34(m,12H),1.03-0.94(m,13H),0.41(d,J＝6.5Hz,3H),-0.06(d,J＝6.6Hz,3H),-0.38(td,J＝9.6,4.1Hz,1H)。LCMS(ESI)：C₅₀H₆₈F₃N₈O₈[M+H]⁺Calculated value, 965.5; found value, 966.0

C-57 ¹H-NMR (400 MHz; acetone-d)₆):δ8.31(dd,J＝9.8,5.1Hz,2H),7.91(d,J＝7.0Hz,1H),7.80-7.77(m,1H),7.49-7.44(m,3H),7.34(d,J＝2.9Hz,1H),7.19-7.16(m,4H),5.35(d,J＝4.0Hz,1H),5.23(dd,J＝10.5,4.8Hz,1H),5.13(td,J＝9.9,3.9Hz,2H),4.36(dd,J＝11.0,4.3Hz,1H),4.13-4.09(m,1H),4.00(q,J＝6.8Hz,1H),3.34(s,3H),3.23-3.15(m,2H),2.89(s,3H),2.66(ddd,J＝11.7,7.9,4.9Hz,2H),2.59(s,3H),2.36-2.25(m,2H),2.16-2.13(m,1H),1.82-1.77(m,2H),1.68(td,J＝11.5,4.3Hz,1H),1.53-1.47(m,9H),1.03(d,J＝6.5Hz,3H),0.99-0.93(m,9H),0.86(dd,J＝9.6,6.6Hz,2H),0.06-0.05(m,1H)。LCMS(ESI)：C₅₀H₆₄BrF₆N₈O₈[M+H]⁺Calculated, 1097.4; found 1097.0.

A mixture of C-781: 4 epimers;¹H-NMR (400 MHz; acetone-d)₆Major epimers δ 8.97(d, J ═ 10.1Hz,1H),8.15(d, J ═ 9.6Hz,1H),7.80-7.77(m,2H),7.49-7.43(m,3H),7.30(d, J ═ 9.7Hz,1H),7.23-7.09(m,5H),5.51(td, J ═ 9.8,4.3Hz,1H),5.39-5.35(m,2H),5.25(dd, J ═ 6.2,3.3Hz,1H),5.16-5.05(m,2H),4.44-4.35(m,2H),4.05-3.99(m,1H),3.31(s,3H),3.23-3.07(m, 3.90H), 4.44-4.35(m,2H),4.05-3.99(m,1H),3.31(s,3H),3.23-3.07(m, 90H), 2H), 1.58 (m,1H), 1.51 (1H), 1H), 2H), 1.37-1.28(m,4H),1.02(d, J ═ 6.4Hz,3H),0.98-0.91(m,4H),0.90-0.84(m,3H),0.33(d, J ═ 6.4Hz,3H), -0.03(d, J ═ 6.6Hz,3H), -0.55-0.62 (m, 1H). Lcms (esi): c₄₉H₆₂BrF₆N₈O₈[M+H]⁺Calculated value, 1083.4; found 1083.2.

C-67 ¹H-NMR (400 MHz; d 6-propanone): delta 8.76(d, J ═ J)

10.1Hz,1H),8.30(d,J＝9.4Hz,1H),8.16(d,J＝1.9Hz,1H),8.01(d,J＝8.6Hz,1H),7.77(d,J＝6.7Hz,1H),7.47(s,1H),7.43(dd,J＝8.6,2.0Hz,1H),5.18(dt,J＝9.4,6.2Hz,1H),5.12-5.02(m,3H),4.38-4.25(m,2H),3.98(q,J＝6.8Hz,1H),3.45(dd,J＝15.1,11.1Hz,1H),3.34-3.30(m,3H),3.27(d,J＝3.7Hz,1H),3.20-3.08(m,2H),2.91(s,3H),2.59-2.56(m,5H),1.99-1.93(m,1H),1.82(ddd,J＝14.1,10.9,3.4Hz,1H),1.67-1.61(m,2H),1.59-1.29(m,12H),1.01-0.91(m,15H),0.46(d,J＝6.5Hz,3H),-0.04(d,J＝6.6Hz,3H),-0.55(ddd,J＝13.9,10.7,3.2Hz,1H)。HRMS(ESI)：C₄₄H₆₀ClF₃N₇O₈S[M-H]^-Calculated value, 938.3870; found 938.3849.

C-29 ¹H-NMR (400 MHz; acetone-d)₆):δ8.78(d,J＝10.1Hz,1H),8.06-7.99(m,3H),7.69-7.67(m,1H),7.52-7.41(m,2H),7.38(s,1H),5.04(t,J＝5.0Hz,3H),4.89-4.83(m,1H),4.40-4.37(m,1H),4.33-4.28(m,1H),3.96(q,J＝6.8Hz,1H),3.48-3.39(m,3H),3.33(s,3H),2.93(s,3H),2.54(d,J＝2.3Hz,5H),2.23-2.18(m,1H),1.98-1.89(m,1H),1.83-1.76(m,2H),1.64-1.57(m,2H),1.53-1.43(m,9H),1.41-1.31(m,3H),1.05(d,J＝6.4Hz,3H),0.98-0.89(m,17H),0.84(dd,J＝12.5,5.6Hz,1H),0.51(d,J＝6.4Hz,3H),-0.07(d,J＝6.5Hz,3H),-0.37--0.45(m,1H)。LCMS(ESI)：C₄₆H₇₀N₇O₈S[M+H]⁺Calculated value, 880.5; found value, 880.4

The following compounds were prepared using the procedures in examples 9-11 in combination:

C-68 LC-MS(ESI)：m/z 887.5[M+H]⁺

C-66 LC-MS(ESI)：m/z 874.5[M+H]⁺

C-64 LC-MS(ESI)：m/z 872.6[M+H]⁺

C-63 LC-MS(ESI)：m/z 886.6[M+H]⁺

C-62 LC-MS(ESI)：m/z 872.6[M+H]⁺

C-61 LC-MS(ESI)：m/z 872.6[M+H]⁺

C-60 LC-MS(ESI)：m/z 886.6[M+H]⁺

C-56 LC-MS(ESI)：m/z 910.6[M+H]⁺

C-55 LC-MS(ESI)：m/z 896.6[M+H]⁺

C-54 LC-MS(ESI)：m/z 898.6[M+H]⁺

C-53 LC-MS(ESI)：m/z 872.6[M+H]⁺

C-52 LC-MS(ESI)：m/z 886.6[M+H]⁺

C-47 LC-MS(ESI)：m/z 898.6[M+H]⁺

C-45 LC-MS(ESI)：m/z 912.6[M+H]⁺

C-44 LC-MS(ESI)：m/z 900.6[M+H]⁺

C-43 LCMS(ESI)：m/z 886.6[M+H]⁺

C-26 LCMS(ESI)：m/z 892.5[M+H]⁺

C-25 LC-MS(ESI)：m/z 838.5[M+H]⁺

C-24 LC-MS(ESI)：m/z 927.6[M+H]⁺

C-23 LC-MS(ESI)：m/z 898.6[M+H]⁺

C-22 LC-MS(ESI)：m/z 900.6[M+H]⁺

C-21 LC-MS(ESI)：m/z 872.6[M+H]⁺

C-20 LC-MS(ESI)：m/z 858.6[M+H]⁺

C-19 LC-MS(ESI)：m/z 858.6[M+H]⁺

C-18 LC-MS(ESI)：m/z 914.7[M+H]⁺

C-17 LC-MS(ESI)：m/z 927.6[M+H]⁺

C-10 LC-MS(ESI)：m/z 892.5[M+H]⁺

C-09 LC-MS(ESI)：m/z 892.5[M+H]⁺

C-08 LC-MS(ESI)：m/z 827.6[M+H]⁺

C-07 LC-MS(ESI)：m/z 813.5[M+H]⁺

C-06 LC-MS(ESI)：m/z 799.5[M+H]⁺

C-05 LC-MS(ESI)：m/z 841.6[M+H]⁺

C-04 LC-MS(ESI)：m/z 855.6[M+H]⁺

C-03 LC-MS(ESI)：m/z 853.5[M+H]⁺

example 12C 12:

meanwhile, a solution of HATU (20mg, 0.052mmol) and HOAt (5.4mg, 0.040mmol) in both DMF (2.5mL) and linear depsipeptide (44mg, 0.040mmol) was added to a solution of DIPEA (21. mu.L, 0.12mmol) in DCM (35mL) via a syringe pump at 1mL/h under argon at room temperature. After the addition was complete, the reaction was stirred for 16h and then concentrated under reduced pressure. The crude material was purified by reverse phase HPLC.¹H-NMR (400 MHz; acetone-d)₆):δ8.73(d,J＝9.9Hz,1H),8.03(d,J＝9.6Hz,1H),7.73(d,J＝7.6Hz,1H),7.61(d,J＝6.7Hz,1H),7.46(d,J＝8.1Hz,1H),7.32(td,J＝6.0,2.7Hz,2H),7.27(s,1H),7.18-7.05(m,4H),5.43-5.37(m,3H),5.09-5.01(m,3H),4.89-4.83(m,1H),4.41-4.32(m,2H),3.94(d,J＝6.8Hz,2H),3.31(s,3H),3.28-3.24(m,1H),2.91(s,3H),2.54(s,3H),2.50(t,J＝6.3Hz,2H),2.22-2.16(m,1H),1.97-1.93(m,1H),1.84-1.75(m,2H),1.64-1.56(m,3H),1.56-1.44(m，9H)，1.38-1.26(m，3H)，1.05(d，J＝6.3Hz，3H)，0.98-0.89(m，18H)，0.36(d,J＝6.5Hz,3H),-0.01(d,J＝6.6Hz,3H),-0.52--0.58(m,1H)。LCMS(ESI)：C₅₃H₇₆FN₈O₈[M+H]⁺Calculated value, 971.6; found value, 971.9

The following compounds were prepared using the procedure of example 12:

C-34 ¹H-NMR(400MHz；DMSO-d₆):δ8.77(d,J＝9.9Hz,1H),8.36-8.34(m,1H),7.69-7.67(m,1H),7.30(t,J＝7.1Hz,2H),7.25-7.20(m,3H),4.84-4.77(m,3H),4.68-4.66(m,1H),4.33-4.28(m,1H),4.19-4.14(m,1H),3.92-3.87(m,2H),3.19-3.14(m,1H),2.94(s,3H),2.74(s,3H),2.41(s,3H),1.71-1.63(m,3H),1.51-1.43(m,5H),1.34-1.23(m,10H),1.20(d,J＝7.0Hz,4H),0.98(dd,J＝8.8,5.2Hz,6H),0.89-0.82(m,15H),0.69-0.67(m,3H),0.58-0.57(m,3H)。LCMS(ESI)：C₄₂H₆₉N₆O₈[M+H]⁺calculated value, 785.5; found 784.5.

C-111H-NMR (400 MHz; acetone-d 6): delta 8.85-8.83(m,1H),7.80-7.77(m,1H),7.59-7.54(m,2H),7.46(d, J ═ 8.3Hz,1H),7.23-7.12(m,4H),7.07-7.03(m,1H),6.89-6.86(m,2H),5.32-5.31(m,2H),5.01-4.84(m,3H),4.46-4.42(m,1H),4.38-4.33(m,1H),3.77(s,4H),3.46-3.39(m,1H),3.21-3.15(m,1H),3.07(s,3H),2.90(s,3H),2.53 (m,1H), 9.42H), 3.42(m,1H), 3.54H, 3.42H, 1H), 4H) 1.23(d, J ═ 7.1Hz,3H),1.06(d, J ═ 6.4Hz,3H),0.99 to 0.89(m,17H),0.50(d, J ═ 6.5Hz,3H),0.11(d, J ═ 6.5Hz,3H),0.01 to 0.07(m, 1H). Lcms (esi): c₅₂H₇₈N₇O₉[M+H]⁺Calculated value, 944.6; found 945.2.

C-30 ¹H-NMR (400 MHz; acetone-d)₆):δ8.90-8.86(m,1H),7.83-7.79(m,1H),7.60-7.55(m,2H),7.47-7.45(m,1H),7.33-7.29(m,2H),7.29-7.24(m,1H),7.17-7.13(m,1H),7.11-7.04(m,3H),5.43-5.39(m,2H),5.04-4.83(m,4H),4.45-4.41(m,1H),4.35-4.32(m,1H),3.84-3.79(m,2H),3.14(m,5H),2.90(s,3H),2.53(s,2H),1.81-1.72(m,2H),1.63-1.56(m,3H),1.54-1.42(m,7H),1.31-1.20(m,6H),1.06(d,J＝6.3Hz,3H),0.99-0.88(m,18H),0.45-0.43(m,3H),0.07-0.06(m,3H),-0.15--0.21(m,1H)。LCMS(ESI)：C₅₁H₇₅FN₇O₈[M+H]⁺Calculated value, 932.6; found 933.1.

C-42 ¹H-NMR (400 MHz; acetone-d)₆):δ8.75-8.72(m,1H),8.04-8.02(m,1H),7.77-7.75(m,1H),7.71-7.60(m,3H),7.47-7.43(m,2H),7.40-7.32(m,2H),7.19-7.10(m,2H),5.54(s,2H),5.10-5.02(m,3H),4.90-4.83(m,1H),4.42-4.35(m,1H),3.97-3.92(m,1H),3.31(s,2H),3.27-3.01(m,4H),2.92(s,2H),2.54-2.53(m,2H),2.50(d,J＝6.2Hz,1H),2.23-2.16(m,1H),2.00-1.92(m,1H),1.80-1.75(m,2H),1.64-1.57(m,3H),1.56-1.45(m,9H),1.39-1.28(m,4H),1.06-0.85(m,12H),0.55-0.51(m,1H),0.37-0.36(m,2H),0.02-0.00(m,2H),-0.45--0.53(m,1H)。LCMS(ESI)：C₅₄H₇₆F₃N₈O₈[M+H]⁺Calculated value, 1021.6; found value, 1021.9

C-46 ¹H-NMR (400 MHz; acetone-d)₆):δ8.75-8.72(m,1H),8.05-8.01(m,1H),7.77-7.75(m,1H),7.67-7.60(m,2H),7.47-7.43(m,2H),7.32(s,1H),7.18-7.10(m,2H),5.54(s,2H),5.11-5.02(m,2H),4.81-4.74(m,1H),4.41-4.33(m,2H),3.97-3.92(m,1H),3.31(s,2H),3.27-3.22(m,2H),2.92(s,2H),2.55-2.53(m,2H),2.51-2.50(m,1H),2.23-2.16(m,1H),2.01-1.90(m,1H),1.82-1.74(m,1H),1.66-1.57(m,2H),1.57-1.47(m,5H),1.40-1.22(m,5H),1.04-0.87(m,10H),0.37-0.35(m,3H),0.01(dd,J＝6.2,2.6Hz,3H),-0.45--0.53(m,1H)。LCMS(ESI)：C₅₄H₇₆F₃N₈O₈[M+H]⁺The value of the calculated value is calculated,1021.6, respectively; found 1022.4.

C-31 ¹H-NMR (400 MHz; acetone-d)₆):δ8.65-8.63(m,1H),8.05-8.03(m,1H),7.91-7.89(m,1H),7.62-7.59(m,2H),7.32-7.30(m,1H),7.16-7.12(m,2H),7.07-7.05(m,2H),6.87-6.84(m,2H),5.64(s,1H),5.15-5.04(m,4H),4.90-4.86(m,2H),4.44-4.40(m,1H),4.34-4.28(m,2H),3.77(s,5H),3.32(s,3H),3.30-3.26(m,2H),2.94(s,3H),2.58-2.56(m,3H),2.54(s,2H),2.23-2.17(m,2H),2.00-1.94(m,2H),1.81-1.73(m,4H),1.64-1.54(m,6H),1.54-1.42(m,10H),1.36-1.29(m,8H),1.06(d,J＝6.4Hz,2H),1.01-0.85(m,18H),0.52-0.51(m,3H),-0.03--0.05(m,3H),-0.29--0.36(m,1H)。LCMS(ESI)：C₅₄H₇₉N₈O₉[M+H]⁺Calculated value, 983.6; found value, 984.2

C-32 ¹H-NMR (400 MHz; acetone-d)₆):δ8.74-8.71(m,1H),8.04-8.01(m,1H),7.73-7.71(m,1H),7.59-7.57(m,1H),7.49(d,J＝8.2Hz,1H),7.29-7.21(m,2H),7.17-7.06(m,2H),7.04-7.01(m,2H),6.84(td,J＝7.5,0.9Hz,1H),5.37-5.26(m,2H),5.10-5.01(m,3H),4.89-4.83(m,2H),4.40-4.33(m,2H),3.93(s,5H),3.30(d,J＝6.7Hz,3H),3.26-3.23(m,2H),2.92(s,2H),2.54(s,3H),2.49(t,J＝6.2Hz,2H),2.22-2.15(m,2H),1.97-1.89(m,2H),1.83-1.75(m,3H),1.63-1.57(m,4H),1.52-1.39(m,9H),1.39-1.19(m,7H),1.05(d,J＝6.3Hz,4H),1.00-0.88(m,18H),0.28(d,J＝6.5Hz,3H),-0.08(d,J＝6.6Hz,3H),-0.54--0.61(m,1H)。LCMS(ESI)：C₅₄H₇₉N₈O₉[M+H]⁺Calculated value, 983.6; found value, 984.1

C-13 ¹H-NMR (400 MHz; acetone-d)₆):δ8.71-8.68(m,1H),8.03-8.00(m,1H),7.74-7.72(m,1H),7.63-7.61(m,1H),7.48-7.46(m,1H),7.27-7.20(m,2H),7.18-7.08(m,2H),6.88-6.82(m,2H),5.36-5.35(m,2H),5.07-5.00(m,3H),4.90-4.83(m,1H),4.42-4.32(m,2H),3.92(t,J＝6.8Hz,2H),3.77(s,4H),3.28(m,5H),2.91(s,3H),2.54(s,3H),2.48-2.45(m,2H),2.23-2.14(m,2H),1.98-1.90(m,2H),1.84-1.73(m,3H),1.67-1.59(m,4H),1.52-1.47(m,8H),1.38-1.30(m,5H),1.05(d,J＝6.3Hz,3H),1.00-0.89(m,19H),0.41(d,J＝6.4Hz,2H),0.01(d,J＝6.6Hz,2H),-0.38--0.46(m,1H)。LCMS(ESI)：C₅₄H₇₉N₈O₉[M+H]⁺Calculated value, 983.6; found value, 983.9

C-14 ¹H-NMR (400 MHz; acetone-d)₆):δ8.73(d,J＝10.0Hz,1H),8.02(d,J＝9.7Hz,1H),7.74(d,J＝7.6Hz,1H),7.61(d,J＝6.9Hz,1H),7.50-7.47(m,2H),7.44(d,J＝8.1Hz,1H),7.27(s,1H),7.20(t,J＝9.7Hz,2H),7.18-7.09(m,2H),5.39(s,2H),5.11-5.01(m,3H),4.89-4.83(m,1H),4.41-4.33(m,2H),3.30-3.21(m,5H),2.91(s,3H),2.54(s,3H),2.50(t,J＝6.3Hz,2H),2.22-2.15(m,1H),1.98-1.90(m,1H),1.83-1.71(m,3H),1.64-1.44(m,12H),1.39-1.29(m,3H),1.05(d,J＝6.3Hz,3H),0.94(dt,J＝21.9,7.8Hz,19H),0.36(d,J＝6.4Hz,3H),0.00(d,J＝6.6Hz,3H),-0.48--0.55(m,1H)。LCMS(ESI)：C₅₃H₇₆BrN₈O₈[M+H]⁺Calculated value, 1031.5; found value, 1031.2

C-15 ¹H-NMR (400 MHz; acetone-d)₆):δ8.71-8.69(m,1H),8.03-8.00(m,1H),7.74-7.72(m,1H),7.62(ddt,J＝6.3,1.3,0.4Hz,1H),7.51(d,J＝8.2Hz,1H),7.24(s,1H),7.22-7.18(m,1H),7.14-7.11(m,1H),5.96(d,J＝0.3Hz,1H),5.39(d,J＝1.8Hz,2H),5.07-5.01(m,2H),4.89-4.85(m,1H),4.42-4.32(m,2H),3.97-3.91(m,1H),3.29(s,4H),3.27-3.25(m,2H),2.91(s,3H),2.54(s,2H),2.47(dd,J＝6.4,6.2Hz,2H),2.34(d,J＝2.9Hz,3H),2.23-2.14(m,2H),1.96-1.90(m,2H),1.81-1.74(m,2H),1.74-1.66(m,2H),1.64-1.56(m,3H),1.52-1.47(m,7H),1.39-1.30(m,6H),1.05(d,J＝6.3Hz,3H),0.98-0.89(m,18H),0.43(d,J＝6.4Hz,2H),0.23-0.18(m,1H),0.01(d,J＝6.6Hz,2H),-0.41--0.49(m,1H)。LCMS(ESI)：C₅₁H₇₆N₉O₉[M+H]⁺Calculated value, 958.6; found value, 958.9

C-28 ¹H-NMR (400 MHz; acetone-d)₆):δ8.75-8.73(m,1H),8.03(dd,J＝9.6,0.2Hz,1H),7.76-7.74(m,1H),7.65-7.63(m,1H),7.47(d,J＝8.0Hz,1H),7.31-7.22(m,3H),7.19-7.10(m,2H),5.45-5.39(m,2H),5.10-5.01(m,3H),4.89-4.83(m,1H),4.41-4.32(m,2H),3.97-3.92(m,1H),3.32(s,3H),3.25-3.24(m,2H),2.91(s,3H),2.54(s,3H),2.52(t,J＝6.3Hz,2H),2.23-2.16(m,2H),2.00-1.90(m,1H),1.83-1.76(m,3H),1.64-1.40(m,12H),1.40-1.26(m,4H),1.06-1.00(m,3H),1.03-0.87(m,17H),0.83-0.77(m,1H),0.34(d,J＝6.5Hz,3H),-0.03(d,J＝6.6Hz,3H),-0.54--0.61(m,1H)。LCMS(ESI)：C₅₃H₇₅F₂N₈O₈[M+H]⁺Calculated value, 989.6; found value, 989.5

C-33 ¹H-NMR (400 MHz; acetone-d)₆):δ8.74-8.71(m,1H),8.04-8.01(m,1H),7.74(dt,J＝7.7,0.4Hz,1H),7.62-7.57(m,2H),7.47-7.45(m,1H),7.31(s,1H),7.18-7.11(m,3H),6.81-6.79(m,1H),5.47-5.42(m,2H),5.10-5.01(m,3H),4.90-4.83(m,1H),4.42-4.32(m,2H),3.97-3.92(m,1H),3.31(s,3H),3.26(t,J＝0.3Hz,2H),2.92(s,3H),2.55(s,3H),2.50(t,J＝6.3Hz,2H),2.23-2.14(m,2H),1.97-1.90(m,2H),1.83-1.75(m,2H),1.65-1.45(m,11H),1.39-1.27(m,4H),1.05(d,J＝6.3Hz,2H),0.99-0.84(m,16H),0.84-0.76(m,1H),0.41-0.39(m,2H),0.01--0.01(m,2H),-0.45--0.52(m,1H)。LCMS(ESI)：C₅₃H₇₈N₉O₈[M+H]⁺Calculated value, 968.6; found value, 968.5

C-36 ¹H-NMR (400 MHz; acetone-d)₆):δ8.75(d,J＝10.0Hz,1H),8.25-8.24(m,1H),8.03(d,J＝9.7Hz,1H),7.87(td,J＝8.1,2.5Hz,1H),7.75(d,J＝7.6Hz,1H),7.64-7.62(m,1H),7.53(d,J＝8.1Hz,1H),7.34(d,J＝2.9Hz,1H),7.20-7.10(m,2H),7.02(dd,J＝8.4,2.9Hz,1H),5.48(s,2H),5.07-5.01(m,2H),4.89-4.83(m,2H),4.40-4.31(m,2H),3.95(q,J＝6.8Hz,1H),3.32(s,3H),3.25-3.20(m,2H),2.91(s,3H),2.54-2.51(m,3H),2.50(s,1H),2.23-2.15(m,1H),1.99-1.89(m,1H),1.83-1.75(m,2H),1.64-1.55(m,3H),1.55-1.44(m,8H),1.37-1.21(m,3H),1.05(d,J＝6.3Hz,3H),0.98-0.89(m,18H),0.29(d,J＝6.5Hz,3H),-0.04(d,J＝6.6Hz,3H),-0.62--0.69(m,1H)。LCMS(ESI)：C₅₂H₇₅FN₉O₈[M+H]⁺Calculated value, 972.6; found value, 973.6

C-35 ¹H-NMR (400 MHz; acetone-d)₆):δ8.74(d,J＝10.0Hz,1H),8.48-8.44(m,2H),8.04(d,J＝9.7Hz,1H),7.77-7.75(m,1H),7.66-7.64(m,1H),7.52(d,J＝8.3Hz,2H),7.36(s,2H),7.21-7.11(m,2H),5.55(s,2H),5.11-5.01(m,3H),4.89-4.83(m,1H),4.41-4.33(m,2H),3.95(q,J＝6.8Hz,1H),3.38-3.33(s,3H),3.32-3.25(m,2H),2.92(s,3H),2.54(s,3H),2.51(d,J＝6.2Hz,2H),2.23-2.15(m,1H),1.99-1.90(m,1H),1.84-1.74(m,2H),1.71-1.56(m,3H),1.56-1.44(m,8H),1.38-1.26(m,3H),1.05(d,J＝6.3Hz,3H),1.01-0.87(m,18H),0.31(d,J＝6.4Hz,3H),-0.03(d,J＝6.5Hz,2H),-0.55--0.61(m,1H)。LCMS(ESI)：C₅₂H₇₅FN₉O₈[M+H]⁺Calculated value, 972.6; found value, 973.6

C-38 ¹H-NMR (400 MHz; acetone-d)₆):δ8.74-8.71(m,1H),8.06-8.02(m,1H),7.76-7.73(m,1H),7.64-7.61(m,1H),7.50-7.43(m,3H),7.28(d,J＝2.6Hz,1H),7.24-7.21(m,1H),7.21-7.09(m,3H),5.43-5.38(m,2H),5.10-5.02(m,2H),4.95-4.89(m,2H),4.40-4.36(m,2H),3.96-3.91(m,2H),3.30(s,3H),3.24-3.20(m,3H),2.91(s,3H),2.55(s,3H),2.51-2.48(m,2H),2.22-2.15(m,2H),2.02-1.97(m,1H),1.62-1.54(m,4H),1.47(d,J＝6.8Hz,3H),1.39-1.28(m,3H),1.06(d,J＝6.2Hz,2H),0.99-0.94(m,8H),0.74-0.70(m,1H),0.37-0.34(m,3H),0.19-0.14(m,1H),0.14-0.05(m,2H),0.01--0.00(m,2H),-0.49--0.56(m,1H)。LCMS(ESI)：C₅₃H₇₄BrN₈O₈[M+H]⁺Calculated value, 1029.5; found value, 1029.4

C-39 ¹H-NMR (400 MHz; acetone-d)₆):δ8.69-8.66(m,1H),8.08-8.05(m,1H),7.77-7.73(m,1H),7.61-7.59(m,1H),7.50-7.43(m,3H),7.27(dd,J＝1.3,0.4Hz,1H),7.24-7.20(m,1H),7.17-7.09(m,2H),5.40-5.38(m,2H),5.08-4.99(m,2H),4.89-4.84(m,1H),4.47-4.39(m,2H),4.37-4.34(m,1H),3.97-3.92(m,1H),3.46-3.43(m,2H),3.29(s,3H),2.91(s,3H),2.53(s,3H),2.49(td,J＝6.2,0.5Hz,1H),2.42-2.37(m,3H),2.23-2.16(m,2H),1.98-1.88(m,2H),1.82-1.76(m,2H),1.67-1.63(m,3H),1.56-1.51(m,2H),1.51-1.45(m,3H),1.41-1.28(m,4H),1.09-1.07(m,2H),1.02-0.85(m,12H),0.37-0.35(m,3H),0.01--0.01(m,3H),-0.48--0.52(m,1H)。LCMS(ESI)：C₅₄H₇₈BrN₈O₈[M+H]⁺Calculated value, 1045.5; found value, 1046.3

C-40 ¹H-NMR (400 MHz; acetone-d)₆):δ8.73(dd,J＝9.9,0.2Hz,1H),8.03(d,J＝9.8Hz,1H),7.74(dd,J＝7.6,0.3Hz,1H),7.62-7.61(m,1H),7.50-7.47(m,2H),7.45-7.43(m,1H),7.27(s,1H),7.22(d,J＝8.4Hz,2H),7.16-7.11(m,2H),5.39(s,2H),5.09-5.02(m,3H),4.80-4.74(m,1H),4.38-4.32(m,3H),3.96-3.91(m,2H),3.30-3.21(m,5H),3.01(d,J＝10.4Hz,1H),2.91(s,3H),2.54(s,3H),2.50(t,J＝6.2Hz,2H),2.21-2.15(m,1H),1.96-1.87(m,2H),1.82-1.75(m,1H),1.61(t,J＝10.9Hz,2H),1.53-1.47(m,7H),1.36-1.26(m,7H),1.03(d,J＝6.4Hz,3H),0.98-0.87(m,12H),0.81-0.79(m,1H),0.36(d,J＝6.4Hz,3H),0.00(d,J＝6.6Hz,3H)。LCMS(ESI)：C₅₃H₇₆BrN₈O₈[M+H]⁺Calculated value, 1031.5; found value, 1031.6

C-41 ¹H-NMR (400 MHz; acetone-d)₆):δ8.70(d,J＝10.0Hz,1H),8.27(d,J＝9.5Hz,1H),7.75(dt,J＝7.9,0.9Hz,1H),7.71-7.69(m,1H),7.49-7.43(m,3H),7.27(s,1H),7.23-7.18(m,2H),7.16-7.09(m,2H),5.39(d,J＝3.1Hz,2H),5.24-5.19(m,1H),5.11-5.03(m,3H),4.41-4.35(m,1H),4.30(dd,J＝10.9,4.3Hz,1H),3.99-3.94(m,1H),3.32-3.28(m,3H),3.27-3.22(m,1H),3.21-3.11(m,2H),2.91(s,3H),2.59(s,3H),2.52(dd,J＝7.0,5.6Hz,2H),2.35-2.28(m,1H),2.27-2.16(m,1H),2.00-1.92(m,1H),1.86-1.79(m,1H),1.67-1.44(m,8H),1.38-1.28(m,3H),1.03-0.93(m,13H),0.89-0.85(m,3H),0.54(dd,J＝9.5,6.5Hz,1H),0.34(d,J＝6.5Hz,3H),-0.01(d,J＝6.6Hz,3H),-0.52--0.58(m,1H)。LCMS(ESI)：C₅₁H₆₉BrF₃N₈O₈[M+H]⁺Calculated value, 1057.4; found value, 1057.0

C-16 ¹H-NMR (400 MHz; acetone-d)₆):δ8.72(dd,J＝10.4,0.6Hz,1H),8.03-8.01(m,1H),7.73(dd,J＝7.7,0.2Hz,1H),7.63-7.61(m,1H),7.45(d,J＝8.0Hz,1H),7.31-7.25(m,6H),7.13(tdd,J＝14.8,6.9,1.1Hz,2H),5.48-5.41(m,2H),5.40(d,J＝1.7Hz,2H),5.12-5.01(m,3H),4.89-4.83(m,1H),4.41-4.33(m,2H),3.93(q,J＝6.8Hz,1H),3.29(s,3H),3.02(t,J＝7.4Hz,1H),2.91(s,3H),2.54(s,3H),2.23-2.14(m,2H),1.96-1.89(m,1H),1.81-1.75(m,2H),1.65-1.64(m,3H),1.52-1.47(m,7H),1.41-1.30(m,3H),1.05(d,J＝6.4Hz,2H),0.99-0.89(m,15H),0.40(d,J＝6.4Hz,3H),0.02(d,J＝6.6Hz,3H),-0.41--0.48(m,1H)。LCMS(ESI)：C₅₃H₇₇N₈O₈[M+H]⁺Calculated value, 953.6; found 953.5.

C-71 ¹H-NMR(400MHz；acetone-d₆):δ10.19-10.19(m,1H),8.69-8.67(m,1H),8.03-8.01(m,1H),7.71(d,J＝7.7Hz,1H),7.59-7.58(m,1H),7.43(d,J＝8.1Hz,1H),7.15-7.06(m,3H),5.09-5.02(m,3H),4.90-4.84(m,1H),4.42-4.38(m,1H),4.35-4.30(m,1H),3.94(q,J＝6.8Hz,1H),3.30(s,3H),3.30-3.22(m,3H),2.91(s,3H),2.53-2.51(m,4H),2.22-2.16(m,1H),2.01-1.91(m,1H),1.80-1.74(m,3H),1.65-1.56(m,3H),1.55-1.44(m,8H),1.39-1.30(m,3H),1.05(d,J＝6.3Hz,3H),0.99-0.89(m,17H),0.50(d,J＝6.4Hz,3H),0.02(d,J＝6.6Hz,3H),-0.39--0.47(m,1H)。LCMS(ESI)：C₄₆H₇₀N₈O₈[M-H]^-Calculated value, 862.5; found 861.4.

Under argon, a 4mL Invitrogen vial was charged with indole (6.6mg, 0.076mmol), DMF (0.5mL, anhydrous) was added, and sodium hydride (600. mu.L of 100mg/mL fine suspension in DMF) was added. After stirring for 30min, 4- (bromomethyl) -2-fluoro-1-methoxybenzene (120 μ L of a 7mM solution in DMF, 8.4 μ M) was added and the solution was stirred overnight. The reaction mixture was purified with acetonitrile water: a solution of TFA (0.5mL, 94.9: 5:0.1) was quenched and the solution passed through a PTFE filter and directly subjected to HPLC purification to give benzyl indole C-27.¹H-NMR (400 MHz; acetone-d)₆):δ8.75-8.71(m,1H),8.04-8.01(m,1H),7.75-7.73(m,1H),7.64-7.62(m,1H),7.52(dd,J＝8.1,0.4Hz,1H),7.30(s,1H),7.21-7.14(m,2H),7.12-7.01(m,2H),6.88-6.84(m,1H),5.34(d,J＝3.0Hz,2H),5.10-5.01(m,3H),4.89-4.83(m,1H),4.40-4.36(m,1H),4.32-4.27(m,1H),3.97-3.89(m,1H),3.88-3.83(s,3H),3.32(s,3H),2.92(s,3H),2.54(s,4H),2.23-2.15(m,2H),1.99-1.90(m,2H),1.82-1.69(m,4H),1.52(s,5H),1.52-1.47(m,6H),1.26-1.16(m,2H),1.09-1.02(m,3H),0.98-0.85(m,18H),0.27-0.25(m,3H),-0.10(d,J＝6.5Hz,3H),-0.64--0.71(m,1H)。LCMS(ESI)：C₅₄H₇₈FN₈O₉[M+H]⁺Calculated value, 10016; found value, 1001.5

Example 13-C-80-C-99

Compounds C-80 to C-99 were prepared in a similar manner to that described above in examples 1-12. LCMS (ESI) M/z [ M + H ] was measured and is shown in the following table.

Compound (I)	LC-MS(ESI)：M/Z[M+H]
		C-80	978.5
C-81	964
		C-82	1101.1
C-83	1019.5
		C-84	1039.4
C-85	976.5
		C-86	976.4
C-87	990.3
		C-88	978.3
C-89	964.3
		C-90	966.4
C-91	967.3
		C-92	940.4
C-93	914.3
		C-94	940.3
C-95	1013.4
		C-96	964.3
C-97	978.3
		C-98	938.4
C-99	952

Measurement of

Constitutive Activity VCAM-ss-Gluc assay

Flp-In 293 T-REx^TMpcDNA for cells^TM5/FRT plasmid, which is inserted with a cDNA (N-MPGKMVVILGASNILWIMFAASQAFKIETTPESR-C) encoding Gauss luciferase plus 10 amino acids fused to the 3' end of the cDNA encoding the VCAM signal sequence (SEQ ID NO: 1). Transfected cells were selected against the selectable markers hygromycin and blasticidin to generate stable cell lines constitutively expressing the VCAMss +10 aa/Gaussian luciferase fusion protein. The day before assay, cells were trypsinized and plated in 384-well tissue culture plates. The following day, the medium was removed from the wells and replaced with fresh medium. Dilutions of compounds in DMSO/medium were added to wells and 5% CO at 37 ℃₂And (4) incubating. After 24 hours, coelenterazine substrate was added to each well and luciferase signal was quantified using Tecan infiinite M1000 Pro for titer determination.

The results of selecting the compounds provided herein are shown below.

Dox-induced VCAM-ss-Gluc assay

Flp-In 293 T-REx^TMpcDNA for cells^TM5/FRT/TO plasmid, which is inserted with a cDNA (N-MPGKMVVILGASNILWIMFAASQAFKIETTPESR-C) encoding Gauss luciferase plus 10 amino acids fused TO the 3' end of the cDNA encoding the VCAM signal sequence (SEQ ID NO: 2). Selection of transfected cells against the selection markers hygromycin and blasticidin to generate stable cell lines containing the VCAMss +10 aa/Gauss luciferase cDNA insert expressed at T-REx^TMAnd (5) adjusting under the system. The day before assay, cells were trypsinized and plated in 384-well tissue culture plates. The following day, dilutions of compounds in DMSO/doxycycline-containing medium were added to wells and at 37 ℃, 5% CO₂And (4) incubating. After 24 hours, coelenterazine substrate was added to each well and luciferase signal was quantified using Tecan infiinite M1000 Pro for titer determination.

The results of selecting the compounds provided herein are shown below.

Dox-induced TNF alpha-FL-Gluc assay

Flp-In 293 T-REx^TMpcDNA for cells^TM5/FRT/TO plasmid transfected with the cDNA encoding Gauss luciferase fused TO the 3' end of the cDNA encoding full length TNF α (amino acids 1-233). Selection of transfected cells against the selection markers hygromycin and blasticidin to generate stable cell lines containing the TNF α -FL/Gauss luciferase cDNA insert expressed at T-REx^TMAnd (5) adjusting under the system. The day before assay, cells were trypsinized and plated in 384-well tissue culture plates. The following day, dilutions of compounds in DMSO/doxycycline-containing medium were added to wells and at 37 ℃, 5% CO₂And (4) incubating. After 24 hours, coelenterazine substrate was added to each well and luciferase signal was quantified using Tecan infiinite M1000 Pro for titer determination.

The results of selecting the compounds provided herein are shown below.

Dox-induced PD1-ss-Gluc assay

Flp-In 293 T-REx^TMpcDNA for cells^TM5/FRT/TO plasmid, which is inserted with a cDNA encoding Gauss luciferase plus 10 amino acids fused TO the 3' end of the cDNA encoding the PD1 signal sequence (N-MQIPQAPWPVVWAVLQLGWRPGWFLDSPDR-C) (SEQ ID NO: 3). Selecting transfected cells against the selectable markers hygromycin and blasticidin to generate stable cell lines containing the PD1-ss +10 aa/Gaussian luciferase cDNA insert expressed at T-REx^TMAnd (5) adjustment under the system. The day before assay, cells were trypsinized and plated in 384-well tissue culture plates. The following day, dilutions of compounds in DMSO/doxycycline-containing medium were added to wells and at 37 ℃, 5% CO₂And (4) incubating. After 24 hours, coelenterazine substrate was added to each well and luciferase signal was quantified using Tecan infiinite M1000 Pro for titer determination.

The results of selecting the compounds provided herein are shown below.

Constitutive activityPrl-ss-Gluc assay for sex

Flp-In 293 T-REx^TMpcDNA for cells^TM5/FRT plasmid, the plasmid insert encoding and Prl signal sequence cDNA 3' end fusion of Gaussian luciferase plus 10 amino acids cDNA (N-MNIKGSPWKGSLLLLLVSNLLLCQSVAPLPICPGGAAR-C) (SEQ ID NO: 4). Transfected cells were selected against the selectable markers hygromycin and blasticidin to generate stable cell lines of constitutively expressed Prlss +10 aa/Gaussian luciferase fusion protein. The day before assay, cells were trypsinized and plated in 384-well tissue culture plates. The following day, the medium was removed from the wells and replaced with fresh medium. Dilutions of compounds in DMSO/medium were added to wells and 5% CO at 37 ℃₂And (4) incubating. After 24 hours, coelenterazine substrate was added to each well and luciferase signal was quantified using Tecan infiinite M1000 Pro for titer determination.

The results of selecting the compounds provided herein are shown below.

24hr Dox inducible Prl-ss-Gluc assay

Flp-In 293 T-REx^TMpcDNA for cells^TM5/FRT/TO plasmid, which is inserted with a cDNA encoding Gauss luciferase plus 10 amino acids fused TO the 3' end of the cDNA encoding the Prl signal sequence (N-MNIKGSPWKGSLLLLLVSNLLLCQSVAPLPICPGGAAR-C) (SEQ ID NO: 5). Selection of transfected cells against the selection markers hygromycin and blasticidin to generate stable cell lines containing the-ss +10 aa/Gaussian luciferase cDNA insert expressed at T-REx^TMAnd (5) adjustment under the system. The day before assay, cells were trypsinized and plated in 384-well tissue culture plates. The following day, dilutions of compounds in DMSO/doxycycline-containing medium were added to wells and at 37 ℃, 5% CO₂And (4) incubating. After 24 hours, coelenterazine substrate was added to each well and luciferase signal was quantified using Tecan infiinite M1000 Pro for titer determination.

The following table shows the results of selecting the compounds provided herein.

IL7Rss assay

The mature domain of luciferase secreted from Gauss princeps (gluc) was cloned into pcDNA 5/FRT/TO. The signal sequence from the mature domain of IL7R (MTILGTTFGMVFSLLQVVSGESGY) (SEQ ID NO:6) plus four amino acids was cloned upstream of the GLuc mature domain and used in transient transfection experiments. At 5% CO₂HEK-293T T-rex cells were cultured in Dulbecco's modified eagle's medium with 10% FBS. Cells were plated at 1 × 10e6 cells per well in 6-well plates and incubated for 6 h. Cells were transiently transfected with IL7Rss-Gluc using Lipofectamine 2000 (Invitrogen). After overnight incubation, transfected cells were replated in clear-bottomed black 96-well plates at a density of 20,000 cells per well. Six hours after plating, cells were treated with doxycycline (1 μ g/ml) and with increasing concentrations of the indicator compound for 24 h. Secreted luciferase in conditioned medium was quantified by luminescence using the BioLux gaussian luciferase assay kit (New England Biolabs, Ipswich, MA, USA) and the SpectraMax M5 microplate reader (Molecular Devices).

C-01 showed an IC50 of 23nM, and C-16 showed an IC50 of 10 nM.

FLT3ss measurement

The mature domain of luciferase secreted from Gauss princeps (gluc) was cloned into pcDNA 5/FRT/TO. The signal sequence from the mature domain of FLT3(MPALARDGGQLPLLVVFSAMIFGTITNQDL) (SEQ ID NO:7) plus four amino acids was cloned upstream of the GLuc mature domain and used in transient transfection experiments. At 5% CO₂HEK-293T T-rex cells were cultured in Dulbecco's modified eagle's medium with 10% FBS. Cells were plated at 1 × 10e6 cells per well in 6-well plates and incubated for 6 h. Cells were transiently transfected with FLT3ss-Gluc using Lipofectamine 2000 (Invitrogen). After overnight incubation, transfected cells were replated in clear-bottomed black 96-well plates at a density of 20,000 cells per well. Six hours after plating, cells were treated with doxycycline (1. mu.g/ml) and concentrated with increasing concentrationsDegree indicator compound treatment for 24 h. The secreted luciferase in conditioned medium was quantified by luminescence using the BioLux gauss luciferase assay kit (new england biologies laboratories, ipwick, ma) and the SpectraMax M5 microplate reader (molecular instruments corporation).

C-01 showed an IC50 of 52nM, and C-16 showed an IC50 of 32 nM.

HER3ss assay

The mature domain of luciferase secreted from Gauss princeps (gluc) was cloned into pcDNA 5/FRT/TO. The signal sequence from the mature domain of HER3(MRANDALQVLGLLFSLARGSEVG) (SEQ ID NO:8) plus four amino acids was cloned upstream of the GLuc mature domain and used in transient transfection experiments. At 5% CO₂HEK-293T T-rex cells were cultured in Dulbecco's modified eagle's medium with 10% FBS. Cells were plated at 1 × 10e6 cells per well in 6-well plates and incubated for 6 h. Cells were transiently transfected with HER3ss-Gluc using Lipofectamine 2000 (Invitrogen). After overnight incubation, transfected cells were replated in clear-bottomed black 96-well plates at a density of 20,000 cells per well. Six hours after plating, cells were treated with doxycycline (1 μ g/ml) and with increasing concentrations of the indicator compound for 24 h. The secreted luciferase in conditioned medium was quantified by luminescence using the BioLux gauss luciferase assay kit (new england biologies laboratories, ipwick, ma) and the SpectraMax M5 microplate reader (molecular instruments corporation).

The following table shows the results of selecting the compounds provided herein.

BT474 or JJN-3 assay

BT474 is a human breast tumor cell line. JJN3 is a human plasma cell leukemia cell line. Cell lines (BT474 or JJN3 cells) were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin and L-glutamine at 5% CO₂The cells were cultured at 37 ℃. Cells were seeded at 100,000 cells/mL. An aliquot of 5000 cells (50 μ L) was added to each well of a black clear bottom 96-well plate. Compounds or controls (50uL of 1% DMSO in cell culture medium) were added to the cells to the final concentrations indicated, and the cells were incubated for 72h (unless otherwise indicated). After incubation was complete, Alamar blue (10uL of a 1mg/ml solution) was added to each well and the plates were incubated for 5 h. Fluorescence was then measured (excitation 545nm, emission 590nm) and reported as Relative Luminescence Units (RLU) normalized to DMSO control.

The following table shows the results of selecting the compounds provided herein.

U266 determination

Human multiple myeloma cell line U266B1 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM glutamine and 1 xpicillin/streptomycin. Cells were plated in 384-well tissue culture plates and treated with compound dilutions in DMSO/medium. The plates were incubated at 37 ℃ with 5% CO₂Incubation was performed for 48 hours. After 48 hours, the mixture is(Promega) was added to each well and luciferase signal was quantified using Tecan Infinite M1000 Pro for titer determination.

The following table shows the results of selecting the compounds provided herein.

H929 assay

Human multiple myeloma cell line NCI-H929 in Advanced RPMI 1640 medium supplemented with 6% fetal bovine serum, 2mM glutamine and 1 XPicillin/streptomycinCulturing in medium. On the day of assay, cells were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM glutamine and 1 x penicillin/streptomycin and plated in 384-well tissue culture plates and treated with compound dilutions in DMSO/medium. The plates were incubated at 37 ℃ with 5% CO₂Incubation was performed for 48 hours. After 48 hours, the mixture is(Promega) was added to each well and luciferase signal was quantified using Tecan Infinite M1000 Pro for titer determination.

The following table shows the results of selecting the compounds provided herein.

Watch (A)

I.a. indicates IC50 > 25 μ M.

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