Peptide conjugate of cytotoxin as therapeutic agent

Abstract

To provide a pharmaceutical composition that realizes more selective delivery of a topoisomerase I inhibitor to an affected tissue.SOLUTION: Provided is a peptide conjugate of cytotoxin such as a topoisomerase I inhibitor which is useful in treatment of a disease such as cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to peptide conjugates of cytotoxins, such as topoisomerase I inhibitors, that are useful in the treatment of diseases such as cancer. [Background technology]

[0002] Cancer is a group of diseases characterized by abnormal control of cell growth. The annual incidence of cancer is estimated to exceed 1.6 million cases in the United States alone. Although surgery, radiation, chemotherapy, and hormones are used to treat cancer, cancer remains the second leading cause of death in the United States. It is estimated that approximately 600,000 Americans die from cancer each year.

[0003] Systemic drug therapy for human cancers often works by slowing or terminating the uncontrolled replication characteristic of cancer cells. One class of such drugs is topoisomerase I inhibitors. The topoisomerase I enzyme relaxes supercoiled DNA, relaxes the helical structure of DNA, and is involved in transcriptional regulation. See Li, M., Genomics Proteomics Bioinformatics 14 (2016), 166-171. Topoisomerase I is essential for the development of the Mammalia system due to its dynamic function in DNA replication and transcription. However, due to its direct role in transcriptional regulation, topoisomerase I dysfunction can lead to abnormal cellular function. See Li, M., Genomics Proteomics Bioinformatics 14 (2016), 166-171. Therefore, several human diseases, including cancer, neurodegenerative disorders, and autoimmune diseases, are linked to the regulation and activity of topoisomerase I.

[0004] Inhibitors of topoisomerase I have been developed and continue to be developed as anticancer drugs. In particular, topoisomerase I inhibitors are widely used to treat colorectal cancer, gastric cancer, and other cancers. See Ogitani, Bioorg. Med. Chem. Lett. 26 (2016), 5069-5072. Although topoisomerase I inhibitors are useful in the treatment of cancer, these compounds also exhibit side effects such as neutropenia and severe diarrhea. Preferential delivery of topoisomerase inhibitors to these diseased tissues may avoid these serious side effects. Therefore, more selective delivery of topoisomerase I inhibitors to diseased tissues is needed. Summary of the Invention

[0005] The present disclosure relates, inter alia, to compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein the constituent variables are defined herein.

[0006] The present disclosure further provides pharmaceutical compositions comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0007] The present disclosure also provides methods of treating a disease or condition (e.g., cancer) by administering a therapeutically effective amount of a compound of the present disclosure to a human or other mammal in need of such treatment. In some embodiments, the disease or condition is characterized by acidic or hypoxic diseased tissue.

[0008] The present disclosure also provides the use of a compound described herein in the manufacture of a medicament for use in therapy.The present disclosure also provides a compound described herein for use in therapy.

[0009] The present disclosure also provides methods for synthesizing the disclosed compounds and intermediates useful in those methods. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows plots of plasma concentrations of Compound 11 and released exatecan after a single intravenous dose of 5 mg / kg of Compound 11 in rats (data are expressed as mean±SEM). [Figure 2] FIG. 1 shows plots of peptide concentrations in tumor and bone marrow after a single intraperitoneal administration of 10 mg / kg of compound 11 in mice (data are expressed as mean±SEM). [Figure 3] Figure 1 shows a graph of total bone marrow counts from femurs of tumor-bearing nude mice after 4 days of once-daily administration of 2.6 and 5.2 μmol / kg of either compound 11 (equivalent to 10, 20 mg / kg conjugate) or free exatecan (equivalent to 1.15 and 2.3 mg / kg exatecan) (data presented as mean ± SEM). [Figure 4A] FIG. 1 shows excised stomachs of tumor-bearing nude mice after administration of 5.2 μmol / kg of vehicle or either compound 11 (equivalent to 20 mg / kg conjugate) or free exatecan (equivalent to 2.3 mg / kg exatecan) once daily for 4 days. [Figure 4B] FIG. 1 shows the stomach of a tumor-bearing nude mouse in situ after administration of 5.2 μmol / kg of either compound 11 (equivalent to 20 mg / kg conjugate) or free exatecan (equivalent to 2.3 mg / kg exatecan) once daily for 4 days. [Figure 5A] Figure 1 shows a plot of the mean tumor volume resulting from the administration of equimolar amounts of either free exatecan or Compound 11 in nude mice bearing HCT116 colorectal lateral tumors. Animals were dosed parenterally once daily, four times per week, for three weeks. [Figure 5B] FIG. 1 shows Kaplan-Meier survival curves for administering equimolar amounts of either free exatecan or compound 11 in nude mice bearing HCT116 colorectal lateral tumors. [Figure 6A]Figure 1 shows the single-agent efficacy of compound 11 in nude mice bearing MKN45 HER2-negative gastric cancer flank tumors. Animals were dosed parenterally once daily, four times a week, for two weeks. [Figure 6B] FIG. 1 shows Kaplan-Meier survival curves for administering equimolar amounts of either free exatecan or compound 11 in nude mice bearing MKN45 HER2-negative gastric cancer flank tumors. [Figure 7A] Figure 1 shows a plot of the mean tumor volume resulting from administration of Compound 11 in SCID mice bearing JIMT-1 HER2 intermediate breast cancer flank tumors. Animals were dosed parenterally once daily, four times per week, for three weeks. [Figure 7B] FIG. 1 shows a plot of percent change in body weight in SCID mice bearing JIMT-1 HER2 intermediate breast cancer flank tumors administered compound 11. [Figure 8A] Figure 1 shows a plot of mean tumor volume in nude mice bearing MDA-MB-231 triple-negative breast cancer flank tumors treated with compound 11. Animals were dosed parenterally, once daily, four times a week for three weeks. [Figure 8B] FIG. 1 shows a plot of percent change in body weight relative to day 0 in nude mice bearing MDA-MB-231 triple-negative breast cancer flank tumors administered Compound 11. [Figure 9A] Figure 1 shows a plot of mean tumor volume in nude mice bearing MDA-MB-231 triple-negative breast cancer flank tumors treated with Compound 11 and talazoparib. Animals were treated parenterally with Compound 11 once daily, four times weekly, for three weeks, and orally with talazoparib once daily for 18 days. [Figure 9B] FIG. 1 shows a plot of the percent change in body weight relative to day 0 of nude mice bearing MDA-MB-231 triple-negative breast cancer flank tumors administered Compound 11 and talazoparib. [Figure 10] 10 is a graph of the degradation of Compound 11 and Compound 29 resulting from treatment with 10 mM glutathione over 16 hours. As shown in FIG. 10, Compound 29 was released much faster than Compound 11 under similar glutathione exposure. DETAILED DESCRIPTION OF THE INVENTION

[0011] Compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 7 is a peptide, R 8 is a small molecule topoisomerase I targeting moiety that binds topoisomerase I, and Q is the part R 7 and R 8 is a linker that is covalently attached to

[0012] Compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 7 R across cell membranes with acidic or hypoxic mantles with a pH below approximately 6.0 8 a peptide capable of selectively delivering Q-, R 8 is a small molecule topoisomerase I targeting moiety that binds topoisomerase I, and Q is the part R 7 and R 8 is a linker that is covalently attached to

[0013] Compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 7 is a peptide, R 8 teeth, [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of Q is the part R 7 and R 8 is a linker that is covalently attached to

[0014] Compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 7 is a peptide, R 8 teeth, [ka] [ka] [ka] [ka] [ka] [ka] and Q is the part R7 and R 8 is a linker that is covalently attached to

[0015] Compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 7 is a peptide, R 8 teeth, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of Q is [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 9 , R 10 , R 11 , and R 12 are independently H, C 1-4 Alkyl, C 1-4 Alkenyl, C 6-10 Aryl, C 3-10 Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR C1 C(O)NR c1 R d1 wherein C is selected from 1-4 Alkyl, C 1-4 Alkenyl, C 6-10 Aryl, C 3-10 Cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from halo, CN, NO, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1, N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 1 and R 2 along with the carbon atoms to which they are attached, C 3-14 A cycloalkyl group or a 4- to 14-membered heterocycloalkyl group, each of which is C 1-4 Alkyl, Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 1 and R 3 along with the carbon atoms to which they are attached, C 3-14 A cycloalkyl group or a 4- to 14-membered heterocycloalkyl group, each of which is represented by C 1-4 Alkyl, Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 2 and R 3 along with the carbon atoms to which they are attached, C 3-14 A cycloalkyl group or a 4- to 14-membered heterocycloalkyl group, each of which is C 1-4 Alkyl, Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 3 and R 4 along with the carbon atoms to which they are attached, C 3-14 A cycloalkyl group or a 4- to 14-membered heterocycloalkyl group, each of which is C 1-4 Alkyl, Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1, and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 5 and R 6 along with the carbon atoms to which they are attached, C 3-14 A cycloalkyl group or a 4- to 14-membered heterocycloalkyl group, each of which is represented by C 1-4 Alkyl, Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from R 13 is H or C 1-6 is alkyl, A is H or C 1-4 is alkyl, R a1 , R b1 , R c1 , and R d1 are independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 haloalkyl, OH, CN, NO, and COCH, wherein 1-6 Alkyl and C 2-6 Each alkenyl may be optionally substituted with OH, CN, NO, or COCH; [ka] is C 6-10 aryl or 5- to 10-membered heteroaryl, wherein the 5- to 10-membered heteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4 ring-forming heteroatoms independently selected from N, O, and S; Ring G is C 3-14 a cycloalkyl group or a 4- to 14-membered heterocycloalkyl group, each of which is C 1-4 Alkyl, Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from [N,O,S] is NH, O, or S; [N,O] is NH or O, [C,N,O] is CR X R Y , NH, or O, and Each R X and R Y are independently H and C 1-4 alkyl.

[0016] Compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 7 is a peptide, R 8 teeth, [ka] [ka] [ka] [ka] [ka] [ka] Q is [ka] [ka] [ka] [ka] is selected from the group consisting of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 9 , R 10 , R 11 , and R 12 are independently H, C 1-4 Alkyl, C 1-4 Alkenyl, C 6-10 Aryl, 5-10 membered heteroaryl, halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R.c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 wherein C is selected from 1-4 Alkyl, C 1-4 Alkenyl, C 6-10 Aryl and 5- to 10-membered heteroaryl are each halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 1 and R 2 along with the carbon atom to which they are attached, halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1C optionally substituted with 1, 2, or 3 substituents independently selected from 3-7 forming a cycloalkyl group, Or, R 1 and R 3 along with the carbon atoms to which they are attached, halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 C optionally substituted with 1, 2, or 3 substituents independently selected from 3-7 forming a cycloalkyl group, Or, R 2 and R 3 along with the carbon atoms to which they are attached, halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 C optionally substituted with 1, 2, or 3 substituents independently selected from 3-7 forming a cycloalkyl group, Or, R 3 and R 4along with the carbon atoms to which they are attached, halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 C optionally substituted with 1, 2, or 3 substituents independently selected from 3-7 forming a cycloalkyl group, Or, R 5 and R 6 along with the carbon atoms to which they are attached, halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 C optionally substituted with 1, 2, or 3 substituents independently selected from 3-7 forming a cycloalkyl group, R 13 is H or C 1-6 is alkyl, A is H or C 1-4 is alkyl, R a1 , R b1 , R c1, and R d1 are independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 haloalkyl, OH, CN, NO, and COCH, wherein 1-6 Alkyl and C 2-6 Each alkenyl may be optionally substituted with OH, CN, NO, or COCH; [ka] is C 6-10 aryl or 5- to 10-membered heteroaryl, wherein the 5- to 10-membered heteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4 ring-forming heteroatoms independently selected from N, O, and S; [N,O,S] is NH, O, or S; [N,O] is NH or O, [C,N,O] is CR X R Y , NH, or O, and Each R X and R Y are independently H and C 1-4 alkyl.

[0017] In some embodiments, the left side of Q is R 8 and the right side of Q is R 7 Combine with.

[0018] In some embodiments, the sulfur atom of the disulfide moiety of Q is R 7 It is part of the cysteine residue of

[0019] As used herein, "peptide" refers to a targeting moiety comprising a 10-50 amino acid sequence composed of naturally occurring amino acid residues and optionally one or more non-naturally occurring amino acids. In some embodiments, R 7The peptides are peptides of 20 to 40, 20 to 30 amino acids, or 30 to 40 residues. Peptides suitable for use in the compounds of the invention are those that can insert across cell membranes via a conformational or secondary structure change in response to a change in the pH of the environment. In this way, the peptides can target acidic tissues and selectively translocate polar, cell-impermeable molecules across cell membranes in response to a decrease in extracellular pH. In some embodiments, the peptides are capable of translocating a conjugated moiety (e.g., R) across cell membranes having an acidic or hypoxic mantle with a pH below about 6.0. 8 In some embodiments, the peptide can selectively deliver the conjugated moiety (e.g., RQ-) across cell membranes having an acidic or hypoxic mantle with a pH less than about 6.5. 8 In some embodiments, the peptide can selectively deliver the conjugated moiety (e.g., RQ-) across cell membranes having an acidic or hypoxic mantle with a pH less than about 5.5. 8 In some embodiments, the peptide can selectively deliver the conjugated moiety (e.g., RQ-) across cell membranes having an acidic or hypoxic mantle with a pH of about 5.0 to 6.0. 8 Q-) can be selectively delivered.

[0020] In certain embodiments, R 7 The peptides are loaded with a payload moiety (e.g., R 8 In some embodiments, R 7 is R 7 is linked to Q via a cysteine residue in. In some embodiments, the sulfur atom of the cysteine residue can form part of the disulfide bond of the linker Q that includes a disulfide bond.

[0021] Suitable peptides that undergo conformational changes based on pH and can insert across cell membranes are described, for example, in U.S. Patent Nos. 8,076,451 and 9,289,508, each of which is incorporated herein by reference in its entirety. Other suitable peptides are described, for example, in Weerakkody, et al., PNAS 110(15), 5834-5839 (April 9, 2013), also incorporated herein by reference in its entirety.

[0022] In some embodiments, R 7 will return the following array: ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1; Pv1), AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2; Pv2), and ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3; Pv3); Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4; Pv4); and AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO: 5; Pv5), where R 7 is R 7 It is bound to Q via a cysteine residue in

[0023] In some embodiments, R 7 will return the following array: ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1; Pv1), AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2; Pv2), and A peptide comprising at least one of: ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3; Pv3); where R 7 is R 7It is bound to Q via a cysteine residue in

[0024] In some embodiments, R 7 is a peptide containing the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1; Pv1).

[0025] In some embodiments, R 7 is a peptide containing the sequence AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2; Pv2).

[0026] In some embodiments, R 7 is a peptide containing the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3; Pv3).

[0027] In some embodiments, R 7 is a peptide containing the sequence Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4; Pv4).

[0028] In some embodiments, R 7 is a peptide containing the sequence AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO: 5; Pv5).

[0029] In some embodiments, R 7 is a peptide consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1; Pv1).

[0030] In some embodiments, R 7 is a peptide consisting of the sequence AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2; Pv2).

[0031] In some embodiments, R 7is a peptide consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3; Pv3).

[0032] In some embodiments, R 7 is a peptide consisting of the sequence Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4; Pv4).

[0033] In some embodiments, R 7 is a peptide consisting of the sequence AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO: 5; Pv5).

[0034] In some embodiments, R 7 is a peptide comprising at least one sequence selected from SEQ ID NO: 6 to SEQ ID NO: 311 as shown in Table 1.

[0035] In some embodiments, R 7 is a peptide consisting of one sequence selected from SEQ ID NO: 6 to SEQ ID NO: 311 as shown in Table 1.

[0036] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0037] Any of the listed peptides useful in the present invention can be modified to contain cysteine residues by replacing a non-cysteine residue with cysteine or by adding a cysteine residue to either the N- or C-terminus.

[0038] In some embodiments, R 7 The peptides of are conformationally restricted peptides. Conformationally restricted peptides can include, for example, macrocyclic peptides and stapled peptides. Stapled peptides are peptides that are constrained by a covalent bond between two amino acid side chains to form a peptide macrocycle. Conformationally restricted peptides are described, for example, in Guerlavais et al., Annual Reports in Medicinal Chemistry 2014, 49, 331-345; Chang et al., Proceedings of the National Academy of Sciences of the United States of America (2013), 110(36), E3445-E3454; Tesauro et al., Molecules 2019, 24, 351-377; Dougherty et al., Journal of Medicinal Chemistry (2019), 62(22), 10098-10107; and Dougherty et al., Chemical Reviews (2019), 119(17), 10241-10287, each of which is incorporated herein by reference in its entirety.

[0039] The term "small molecule topoisomerase I targeting moiety" or "topoisomerase I inhibitor" refers to a chemical group that binds to topoisomerase I. A small molecule topoisomerase I targeting moiety can be a group derived from a compound that inhibits the activity of topoisomerase I. Topoisomerase inhibitors include camptothecin and its derivatives and analogs, such as opotecan, irinotecan (CPT-11), ciratecan (DB-67, AR-67), cositecan (BNP-1350), lurtotecan, gimatecan (ST1481), belotecan (CKD-602), rubitecan, topotecan, deruxtecan, and exatecan. Topoisomerase inhibitors are described, for example, in Ogitani, Bioorg. Med. Chem. Lett. 26 (2016), 5069-5072; Kumazawa, E., Cancer Chemother Pharmacol 1998, 42: 210-220; Tahara, M, Mol Cancer Ther 2014, 13 (5): 1170-1180; Nakada, T., Bioorganic & Medicinal Chemistry Letters 2016, 26: 1542-1545.

[0040] The moiety Q serves as a tether between the peptide and the topoisomerase I inhibitor that can be cleaved when the conjugate, or a portion thereof, is inside a cell. 7 and R 8 In some embodiments, Q is a chain of 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 chain atoms and 1 to 10 R q and wherein one or more chain carbon atoms of Q can be oxidized to form a carbonyl (C=O), and one or more N and S chain atoms can each be optionally oxidized to form an amine oxide, sulfoxide, or sulfonyl group, wherein Each R q are independently OH, CN, -COOH, NH2, halo, C 1-6 Haloalkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C1-6 Haloalkoxy, C 1-6 Alkylthio, phenyl, 5-6 membered heteroaryl, 4-6 membered heterocycloalkyl, C 3~6 Cycloalkyl, NH(C 1-6 alkyl), and N(C 1-6 alkyl)2, where R q C 1-6 Alkyl, phenyl, C 3-6 Cycloalkyl, 4- to 6-membered heterocycloalkyl, and 5- to 6-membered heteroaryl are each selected from halo, OH, CN, —COOH, NH, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Haloalkyl, C 1-4 Haloalkoxy, Phenyl, C 3-10 substituted with cycloalkyl, 5- or 6-membered heteroaryl, or 4- to 6-membered heterocycloalkyl, and The Two R's q The groups, together with the chain atoms to which they are attached, are selected from the group consisting of phenyl, 5- to 6-membered heteroaryl, 4- to 6-membered heterocycloalkyl, or C 3-6 A cycloalkyl ring can be formed.

[0041] In some embodiments, R q are independently OH, CN, -COOH, NH2, halo, C 1-6 Haloalkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, NH(C 1-6 alkyl) and N(C 1-6 alkyl)2.

[0042] In some embodiments, Q is [ka] [ka] [ka] [ka] [ka] is selected from.

[0043] In some embodiments, Q is [ka] is.

[0044] In some embodiments, Q is [ka] is.

[0045] In some embodiments, Q is [ka] is.

[0046] In some embodiments, Q is [ka] is.

[0047] In some embodiments, Q is [ka] is.

[0048] In some embodiments, R 1 , R 2 , R 3 , and R 4 are each independently H and C 1-4 Alkyl, Halo, CN, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)ORa1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 wherein C is selected from 1-4 Alkyl is independently selected from halo, CN, NO, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents selected from Or, R 1 and R 2 along with the carbon atoms to which they are attached, C 3-10 A cycloalkyl group or a 4- to 10-membered heterocycloalkyl group may be formed, each of which may be halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NRc1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 1 and R 3 along with the carbon atoms to which they are attached, C 3-10 A cycloalkyl group or a 4- to 10-membered heterocycloalkyl group may be formed, each of which may be halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 1 and R 4 along with the carbon atoms to which they are attached, C 3-10 A cycloalkyl group or a 4- to 10-membered heterocycloalkyl group may be formed, each of which may be halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 2 and R 3 along with the carbon atoms to which they are attached, C 3-10 A cycloalkyl group or a 4- to 10-membered heterocycloalkyl group may be formed, each of which may be halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 2 and R 4 along with the carbon atoms to which they are attached, C 3-10 A cycloalkyl group or a 4- to 10-membered heterocycloalkyl group may be formed, each of which may be halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R3 and R 4 along with the carbon atoms to which they are attached, C 3-10 A cycloalkyl group or a 4- to 10-membered heterocycloalkyl group may be formed, each of which may be halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0049] In some embodiments, R 1 , R 2 , R 3 , and R 4 are each independently H and C 1-4 alkyl, Or, R 1 and R 2 along with the carbon atoms to which they are attached, C 3-10 A cycloalkyl group or a 4- to 10-membered heterocycloalkyl group may be formed, each of which may be halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1, and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 1 and R 3 along with the carbon atoms to which they are attached, C 3-10 A cycloalkyl group or a 4- to 10-membered heterocycloalkyl group may be formed, each of which may be halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R 3 and R 4 along with the carbon atoms to which they are attached, C 3-10 A cycloalkyl group or a 4- to 10-membered heterocycloalkyl group may be formed, each of which may be halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0050] In some embodiments, R 1 and R 2 are each independently selected from H and methyl; R 3 , R 4 , R 5 , and R 6 are hydrogen atoms.

[0051] In some embodiments, R 1 , R 2 , R 3 , and R 4 are each independently selected from H and methyl; R 5 , and R 6 are hydrogen atoms.

[0052] In some embodiments, R 1 and R 2 are each independently selected from H and methyl.

[0053] In some embodiments, R 3 and R 4 are each independently selected from H and methyl.

[0054] In some embodiments, R 1 and R 2 are H respectively.

[0055] In some embodiments, R 1 and R 2 along with the carbon atoms to which they are attached, halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 Rd1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 C optionally substituted with 1, 2, or 3 substituents independently selected from 3-7 Forms a cycloalkyl group.

[0056] In some embodiments, R 1 and R 2 along with the carbon atoms to which they are attached, C 3-7 Forms a cycloalkyl group.

[0057] In some embodiments, R 1 and R 2 together with the carbon atom to which they are attached form a cyclobutyl group.

[0058] In some embodiments, R 3 and R 4 are H respectively.

[0059] In some embodiments, R 1 and R 3 along with the carbon atoms to which they are attached, halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 C optionally substituted with 1, 2, or 3 substituents independently selected from 3-7Forms a cycloalkyl group.

[0060] In some embodiments, R 1 and R 3 together with the carbon atoms to which they are attached form cyclopentyl, cyclohexyl, cycloheptyl, 1,2,3,4-tetrahydronaphthyl, tetrahydrofuranyl, or tetrahydropyranyl.

[0061] In some embodiments, R 1 and R 3 along with the carbon atoms to which they are attached, C 3-7 Forms a cycloalkyl group.

[0062] In some embodiments, R 1 and R 3 together with the carbon atom to which they are attached form a cyclohexyl group.

[0063] In some embodiments, R 2 and R 4 are H respectively.

[0064] In some embodiments, R 5 and R 6 are H respectively.

[0065] In some embodiments, R 9 , R 10 , R 11 , and R 12 are each independently selected from H and methyl.

[0066] In some embodiments, the compound of the present invention has the formula (II): [ka] or a pharmaceutically acceptable salt thereof, wherein R 7 is a peptide, R 8 is a topoisomerase I inhibitor, Ring Z is a monocyclic C 5-7 a cycloalkyl ring or a monocyclic 5- to 7-membered heterocycloalkyl ring; Each R Z independently, C 1-4 Alkyl, Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 is selected from or two adjacent R Z together with the atoms to which they are attached form a fused monocyclic C 5-7 Cycloalkyl ring, fused monocyclic 5- to 7-membered heterocycloalkyl ring, fused C 6-10 aryl ring or a fused 6- to 10-membered heteroaryl ring, each of which is C 1-4 Alkyl, Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)R b1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from R a1, R b1 , R c1 , and R d1 are independently H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 alkynyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, OH, CN, and NO2; and n is 0, 1, 2, or 3.

[0067] In some embodiments of the compound of Formula (II), R 7 is a peptide comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0068] In some embodiments of the compound of Formula (II), R 7 is Pv1, PV2, PV3, PV4, or PV5.

[0069] In some embodiments of the compound of Formula (II), R 7 is R 7 and one of the sulfur atoms of the disulfide moiety of Formula II is derived from a cysteine residue.

[0070] In some embodiments of the compound of Formula (II), R 8 are camptothecin, topotecan, irinotecan (CPT-11), ciratecan (DB-67, AR-67), cositecan (BNP-1350), lutotecan, gimatecan (ST1481), belotecan (CKD-602), rubitecan, topotecan, deruxtecan, or exatecan.

[0071] In some embodiments of the compound of Formula (II), R 8 is exatecan.

[0072] In some embodiments of the compound of Formula (II), R 8 is attached to the core via an N atom.

[0073] In some embodiments of the compound of Formula (II), ring Z is a monocyclic C 5-7 It is a cycloalkyl ring.

[0074] In some embodiments of the compound of Formula (II), ring Z is a cyclopentyl ring.

[0075] In some embodiments of the compound of Formula (II), ring Z is a cyclohexyl ring.

[0076] In some embodiments of the compound of Formula (II), ring Z is a cycloheptyl ring.

[0077] In some embodiments of the compounds of Formula (II), ring Z is a monocyclic 5- to 7-membered heterocycloalkyl ring.

[0078] In some embodiments of the compounds of Formula (II), ring Z is a 5-membered heterocycloalkyl ring.

[0079] In some embodiments of the compounds of Formula (II), ring Z is a 6-membered heterocycloalkyl ring.

[0080] In some embodiments of the compounds of Formula (II), ring Z is a 7-membered heterocycloalkyl ring.

[0081] In some embodiments of the compound of Formula (II), two adjacent R Z together with the atoms to which they are attached form a fused monocyclic C 5-7 Cycloalkyl ring, fused monocyclic 5- to 7-membered heterocycloalkyl ring, fused C 6-10 aryl ring or a fused 6- to 10-membered heteroaryl ring, each of which is C 1-4 Alkyl, Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 ,OC(O)Rb1 , OC(O)NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0082] In some embodiments of the compound of Formula (II), n is 0.

[0083] In some embodiments of the compound of Formula (II), n is 1.

[0084] In other embodiments of the compounds of Formula (II), n is 2.

[0085] In other embodiments of the compounds of Formula (II), n is 3.

[0086] In some embodiments, the compound of the present invention has Formula (III), Formula (IV), or Formula (V): [ka] or a pharmaceutically acceptable salt thereof, wherein R 7 , R 8 , R Z and n are defined as in any of the above embodiments for formula (II).

[0087] In some embodiments, the compound of formula (I) is [ka] [ka] [ka] is selected from or a pharmaceutically acceptable salt of any of the foregoing.

[0088] In some embodiments, the compound of formula (I) is [ka] [ka] [ka] [ka] [ka] [ka] is selected from.

[0089] In some embodiments, provided herein are compounds of formula (IIA): [ka] or a salt thereof, wherein Cy 1 is C 6-10 aryl or 5-10 membered heteroaryl, wherein the 5-10 membered heteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4 ring-forming heteroatoms independently selected from N, O, and S, and said C 6-10 Aryl and 5- to 10-membered heteroaryl are each C 1-4 Alkyl, Halo, OH, C 1-6 optionally substituted with 1, 2, or 3 substituents independently selected from alkoxy, CN, and NO; R 8 , ring Z, R Z , and n is as defined herein.

[0090] In some embodiments, Cy 1 is a 5-10 membered heteroaryl. In some embodiments, Cy 1 is pyridinyl. In some embodiments, Cy 1 is phenyl.

[0091] In some embodiments, the compound of Formula (IIA) has the structure: [ka] or a salt thereof.

[0092] In some embodiments, provided herein are compounds of formula (IIA) for use in preparing compounds of the invention (e.g., compounds of formula (I) or formula (II)). [ka] or a salt thereof, wherein Cy 1 , R 8 , ring Z, R Z , R a1 , R b1 , R c1 , R d1 , and n are as defined herein.

[0093] In some embodiments, provided herein are compounds of the following structure for use in preparing compounds of the invention (e.g., compounds of Formula (I) or Formula (II)): [ka] or a salt thereof.

[0094] The molecules of the present invention can be tagged with a probe, such as, for example, a fluorophore, a radioisotope, etc. In some embodiments, the probe is a fluorescent probe, such as a LICOR. The fluorescent probe can include any moiety that can re-emit light upon light excitation (e.g., a fluorophore).

[0095] Amino acids are represented by IUPAC abbreviations as follows: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0096] The term "Pv1" refers to ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1).

[0097] The term "Pv2" refers to AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2).

[0098] The term "Pv3" refers to ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3).

[0099] The term "Pv4" refers to AcAAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4).

[0100] The term "Pv5" refers to AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO: 5).

[0101] In the compounds of the present invention, peptide R 7 is linked to the disulfide moiety of the linker Q by an amino acid residue containing a sulfur atom, such as a cysteine residue. Typically, peptide R 7 The sulfur atom of the disulfide moiety of linker Q, which is the point of attachment to , is derived from an amino acid residue of the peptide, such as a cysteine residue.

[0102] The term "acidic and / or hypoxic mantle" refers to the cellular environment in the diseased tissue in question, which has a pH below 7.0, preferably below 6.5. The acidic or hypoxic mantle more preferably has a pH of about 5.5, and most preferably has a pH of about 5.0. Compounds of formula (I) intercalate across cell membranes with acidic and / or hypoxic mantles in a pH-dependent manner, resulting in R 8 Q is inserted into the cell, after which the disulfide linker is cleaved to give rise to the free R 8H Because the compounds of formula (I) are pH-dependent, they preferentially insert into cell membranes only in the presence of an acidic or hypoxic mantle surrounding the cell, and do not insert into the cell membranes of "normal" cells that do not have an acidic or hypoxic mantle. An example of a cell that has an acidic or hypoxic mantle is a cancer cell.

[0103] Peptide R 7 , or peptide R across the cell membrane 7 The terms "pH-sensitive" or "pH-dependent" as used herein to refer to the mode of insertion of the compounds of the present invention mean that the peptide has a higher affinity for cell membrane lipid bilayers with an acidic or hypoxic mantle than for membrane lipid bilayers at neutral pH. Thus, the compounds of the present invention preferentially insert through cell membranes when the cell membrane lipid bilayer has an acidic or hypoxic mantle ("diseased" cells), resulting in R 8 Insert Q into the interior of the cell (hence R as above) 8 H), but does not insert through the cell membrane if the mantle (environment of the cell membrane lipid bilayer) is not acidic or hypoxic ("normal" cells). This preferential insertion is due to the peptide R 7 is believed to be achieved as a result of forming a helical structure that facilitates membrane insertion.

[0104] It is further understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment (although it is intended that these embodiments be combined as if stated in multiple dependent forms). Conversely, for brevity, various features of the invention that are described in the context of a single embodiment can also be provided separately or in any suitable subcombination. Accordingly, it is contemplated that features described as embodiments of compounds of formula (I) can be combined in any suitable combination.

[0105] At various places in the present specification, particular features of compounds are disclosed in groups or ranges. It is specifically intended that such disclosure include each and every individual subcombination of the members of such groups and ranges. For example, "C 1-6 The term "alkyl" is specifically intended to individually (but without limitation) disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0106] The term "n-membered" refers to the number of ring-forming atoms in a moiety, where n is an integer and typically the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.

[0107] At various places in the specification, variables defining divalent linking groups may be described. It is specifically intended that each linking substituent include both the forward and reverse forms of the linking substituent. For example, -NR(CR'R") n - is -NR(CR'R") n -and-(CR'R") nBoth NR- and NR- are included, and each form is intended to be disclosed individually. When a structure requires a linking group, the Markush variable listed for that group is understood to be the linking group. For example, if a structure requires a linking group and the Markush group definition for that variable lists "alkyl" or "aryl," it is understood that "alkyl" or "aryl" represent a linking alkylene or arylene group, respectively.

[0108] The term "substituted" means that an atom or group of atoms formally replaces hydrogen as a "substituent" attached to another group. The term "substituted" refers to any level of substitution, for example, mono-, di-, tri-, tetra-, or penta-substitution, unless otherwise stated, where such substitution is permitted. Substituents are independently selected, and substitution can be at any chemically accessible position. It is understood that substitution at a given atom is limited by valence. It is understood that substitution at a given atom results in a chemically stable molecule. The phrase "optionally substituted" means unsubstituted or substituted. The term "substituted" means that a hydrogen atom has been removed and replaced with a substituent. A single divalent substituent, for example, oxo, can replace two hydrogen atoms.

[0109] "C n-m The term "" denotes a range inclusive of the endpoints, where n and m are integers and indicate the number of carbons. Examples include C 1-4 , C 1-6 Examples include:

[0110] The term "alkyl," used alone or in combination with other terms, refers to a saturated hydrocarbon group which may be straight-chained or branched. n-mThe term "alkyl" refers to an alkyl group having n to m carbon atoms. An alkyl group formally corresponds to an alkane with one C-H bond replaced at the alkyl group's point of attachment to the remainder of the compound. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, and 1,2,2-trimethylpropyl.

[0111] The term "alkenyl," used alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more double carbon-carbon bonds. An alkenyl group formally corresponds to an alkene with one C-H bond replaced at the point of attachment of the alkenyl group to the remainder of the compound. n-m The term "alkenyl" refers to an alkenyl group having n to m carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Exemplary alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

[0112] The term "alkynyl," used alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more triple carbon-carbon bonds. An alkynyl group formally corresponds to an alkyne with one C-H bond replaced at the point of attachment of the alkyl group to the rest of the compound. n-m The term "alkynyl" refers to an alkynyl group having n to m carbons. Examples of alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0113] The term "alkylene," used alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene group formally corresponds to an alkane with two C-H bonds replaced at the points of attachment of the alkylene group to the rest of the compound. n-m The term "alkylene" refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethane-1,2-diyl, ethane-1,1-diyl, propane-1,3-diyl, propane-1,2-diyl, propane-1,1-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, 2-methyl-propane-1,3-diyl, and the like.

[0114] The term "amino" refers to a group of formula -NH2.

[0115] The term "carbonyl," used alone or in combination with other terms, refers to the group --C(.dbd.O)--, which can also be written as C(O).

[0116] The term "cyano" or "nitrile" refers to a group of formula -C≡N, which can also be written -CN.

[0117] The terms "halo" or "halogen," used alone or in combination with other terms, refer to fluoro, chloro, bromo, and iodo. In some embodiments, "halo" refers to a halogen atom selected from F, Cl, or Br. In some embodiments, the halo group is F.

[0118] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced with a halogen atom. n-m The term "haloalkyl" refers to a C alkyl group having n to m carbon atoms and at least 1 to {2(n to m)+1} halogen atoms. n-mIn some embodiments, the halogen atoms are fluoro atoms. In some embodiments, the haloalkyl groups have 1 to 6 or 1 to 4 carbon atoms. Examples of haloalkyl groups include CF 3、 Examples include C2F5, CHF2, CH2F, CCl3, CHCl2, C2Cl5, etc. In some embodiments, the haloalkyl group is a fluoroalkyl group.

[0119] The term "haloalkoxy", employed alone or in combination with other terms, refers to a radical of the formula -O-haloalkyl, in which the alkyl group is as defined above. n-m The term "haloalkoxy" refers to a haloalkoxy group, where the haloalkyl group has n to m carbon atoms. Examples of haloalkoxy groups include trifluoromethoxy, and the like. In some embodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0120] The term "oxo" refers to an oxygen atom as a divalent substituent, which when attached to a carbon forms a carbonyl group, or when attached to a heteroatom forms a sulfoxide or sulfone group, or an N-oxide group. In some embodiments, heterocyclic groups can be substituted with one or two oxo (=O) substituents.

[0121] The term "oxidized" in reference to a ring-forming N atom refers to a ring-forming N-oxide.

[0122] The term "oxidized" in reference to a ring-forming S atom refers to a ring-forming sulfonyl or ring-forming sulfinyl.

[0123] The term "aromatic" refers to a carbocyclic or heterocyclic ring containing one or more polyunsaturated rings and having aromatic character (i.e., having (4n+2) delocalized π (pi) electrons, where n is an integer).

[0124] The term "aryl," used alone or in combination with other terms, refers to an aromatic hydrocarbon group that can be monocyclic or polycyclic (e.g., having two fused rings). n-m The term "aryl" refers to an aryl group having n to m ring carbon atoms. Aryl groups include, for example, phenyl, naphthyl, and the like. In some embodiments, an aryl group has 6 to about 10 carbon atoms. In some embodiments, an aryl group has 6 carbon atoms. In some embodiments, an aryl group has 10 carbon atoms. In some embodiments, an aryl group is phenyl.

[0125] The terms "heteroaryl" or "heteroaromatic," used alone or in combination with other terms, refer to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in the heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl ring has 5 to 14 ring atoms, including carbon atoms and 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl ring has 5 to 10 ring atoms, including carbon atoms and 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl ring has 5 to 6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5- or 6-membered heteroaryl ring. In other embodiments, the heteroaryl is an 8-, 9-, or 10-membered fused bicyclic heteroaryl ring.

[0126] A 5-membered heteroaryl ring is a heteroaryl group having 5 ring atoms, wherein one or more (eg, 1, 2, or 3) ring atoms are independently selected from N, O, and S.

[0127] A 6-membered heteroaryl ring is a heteroaryl group having 6 ring atoms, wherein one or more (eg, 1, 2, or 3) ring atoms are independently selected from N, O, and S.

[0128] The term "cycloalkyl," used alone or in combination with other terms, refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic, or polycyclic) including cyclized alkyl and alkenyl groups. n-m The term "cycloalkyl" refers to a cycloalkyl having n to m ring carbon atoms. Cycloalkyl groups can include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) and polycyclic groups. Cycloalkyl groups can have 3, 4, 5, 6, or 7 ring carbons (C 3-7 In some embodiments, the cycloalkyl group has 3 to 6 ring members, 3 to 5 ring members, or 3 to 4 ring members. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is monocyclic or bicyclic. In some embodiments, the cycloalkyl group is C 3-6Monocyclic cycloalkyl groups. Ring-forming carbon atoms of a cycloalkyl group can be optionally oxidized to form oxo or sulfido groups. Cycloalkyl groups also include cycloalkylidenes. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. The definition of cycloalkyl also includes moieties having one or more aromatic rings fused to (i.e., covalently bonded to) the cycloalkyl ring, such as benzo or thienyl derivatives such as cyclopentane and cyclohexane. Cycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, and the like. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0129] The term "heterocycloalkyl," used alone or in combination with other terms, refers to a non-aromatic ring or ring system, which may optionally contain one or more alkenylene groups as part of the ring structure, has at least one heteroatom ring member independently selected from nitrogen, sulfur, oxygen, and phosphorus, and has 4 to 10 ring members, 4 to 7 ring members, or 4 to 6 ring members. The term "heterocycloalkyl" includes monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups can include monocyclic or bicyclic (e.g., having two fused or bridged rings) or spirocyclic ring systems. In some embodiments, the heterocycloalkyl group is a monocyclic group having 1, 2, or 3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally oxidized to form oxo or sulfido groups, or other oxidized bonds (e.g., C(O), S(O), C(S), or S(O)2, N-oxide, etc.) or nitrogen atoms can be quaternized. A heterocycloalkyl group can be bonded through a ring-forming carbon atom or ring-forming heteroatom. In some embodiments, a heterocycloalkyl group contains zero to three double bonds. In some embodiments, a heterocycloalkyl group contains zero to two double bonds. Also included within the definition of heterocycloalkyl are moieties having one or more aromatic rings fused to (i.e., covalently bonded to) the heterocycloalkyl ring, such as benzo or thienyl derivatives such as piperidine, morpholine, and azepine. Heterocycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including a ring-forming atom of the fused aromatic ring. Examples of heterocycloalkyl groups include 2-pyrrolidinyl, morpholinyl, azetidinyl, and piperazinyl.

[0130] In certain places, definitions or embodiments refer to specific rings (e.g., azetidine ring, pyridine ring, etc.). Unless otherwise specified, these rings can be bonded to any ring member as long as the valence of the atom is not exceeded. For example, an azetidine ring can be bonded to any position on the ring, while an azetidin-3-yl ring is bonded to the 3-position.

[0131] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise specified. Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically inactive starting materials, such as by resolution of racemic mixtures or stereoselective synthesis, are known in the art. Many geometric isomers of olefins, C=N double bonds, and the like, can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can be isolated as a mixture of isomers or as separated isomeric forms.

[0132] Resolution of a racemic mixture of compounds can be carried out by any of many methods known in the art. One method involves partial recrystallization using a chiral resolving acid, which is an optically active, salt-forming organic acid. Suitable resolving agents for the partial recrystallization method are optically active acids, such as the D- and L-forms of various optically active camphorsulfonic acids, such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or β-camphorsulfonic acid. Other resolving agents suitable for the partial crystallization method include stereoisomerically pure forms (e.g., S- and R-forms, or diastereomerically pure forms) of α-methylbenzylamine, 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

[0133] Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by one skilled in the art.

[0134] In some embodiments, the compounds of the present invention have the (R) configuration. In other embodiments, the compounds have the (S) configuration. In compounds with multiple chiral centers, unless otherwise specified, each chiral center in the compound may independently be (R) or (S).

[0135] The compounds of the present invention also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions in a heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or sterically locked into one form by appropriate substitution.

[0136] The compounds herein may also include all isotopes of atoms occurring in intermediates or final compounds. Isotopes include atoms with the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds of the present invention may be replaced or substituted with an isotope of that atom at natural or non-natural abundance. In some embodiments, the compounds contain at least one deuterium atom. For example, one or more hydrogen atoms in the compounds of the present disclosure may be replaced or substituted with deuterium. In some embodiments, the compounds contain two or more deuterium atoms. In some embodiments, the compounds contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 deuterium atoms. Synthetic methods for incorporating isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, NY, Appleton-Century-Crofts, 1971); The Renaissance of H / D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in a variety of studies, such as NMR spectroscopy, metabolic experiments, and / or assays.

[0137] Substitution with heavier isotopes, such as deuterium, may confer certain therapeutic benefits, such as increased in vivo half-life or reduced dosage requirements, resulting from greater metabolic stability and, therefore, may be preferable in some cases (A. Kerekes et al. J. Med. Chem. 2011, 54, 201-210; R. Xu et al. J. Label Compd. Radiopharm. 2015, 58, 308-312).

[0138] As used herein, the term "compound" is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. The term is also meant to refer to compounds of the present invention regardless of how they are prepared, for example, synthetically, by a biological process (e.g., metabolic or enzymatic transformation), or a combination thereof.

[0139] All compounds described herein and their pharmaceutically acceptable salts may be found together with other substances, such as water and solvents (e.g., hydrates and solvates), or may be isolated. When in the solid state, the compounds described herein and their salts may occur in various forms, for example, in the form of solvates, including hydrates. Because the compounds may be in any solid form, such as polymorphs or solvates, unless otherwise specified, references herein to compounds and their salts should be understood to encompass any solid form of the compound.

[0140] In some embodiments, the compound of the present invention or a salt thereof is substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present invention. Substantial separation can include a composition containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compound of the present invention or a salt thereof.

[0141] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of safe medical judgment and commensurate with a reasonable benefit / risk ratio.

[0142] As used herein, the expressions "ambient temperature" and "room temperature" are understood in the art and generally refer to a reaction temperature that is about the same as the temperature of the room in which the reaction is carried out, e.g., a temperature of about 20°C to about 30°C.

[0143] The compounds of the present invention also include pharmaceutically acceptable salts of the compounds described herein. The term "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts of the present invention include non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (MeCN) are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 th Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19 and in Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). In some embodiments, the compounds described herein include N-oxide forms.

[0144] synthesis The compounds of the present invention, including their salts, can be prepared using known organic synthesis techniques and can be synthesized according to any of a number of possible synthetic routes, such as the following schemes.

[0145] The reaction for preparing the compounds of the present invention can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis.Suitable solvents can be substantially non-reactive with starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, i.e., temperatures that can range from the freezing temperature of the solvent to the boiling temperature of the solvent.A given reaction can be carried out in one solvent or a mixture of more than one solvent.Depending on the specific reaction step, the solvent suitable for that specific reaction step can be selected by those skilled in the art.

[0146] The preparation of compounds of the present invention may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Kocienski, Protecting Groups, (Thieme, 2007); Robertson, Protecting Group Chemistry, (Oxford University Press, 2000); Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6 th Ed. (Wiley, 2007); Peturssion et al., "Protective Groups in Carbohydrate Chemistry," J. Chem. Educ., 1997, 74(11), 1297; and Wuts et al., Protective Groups in Organic Synthesis, 4th Ed., (Wiley, 2006).

[0147] The reaction can be monitored according to any suitable method known in the art. For example, product formation can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or13 C), can be monitored by spectroscopic means such as infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0148] The following schemes provide general guidance regarding the preparation of compounds of the present invention. Those skilled in the art will understand that the preparations shown in the schemes can be modified or optimized using general knowledge of organic chemistry to prepare various compounds of the present invention.

[0149] Compounds of formula (I) can be prepared, for example, using the process as illustrated in the following scheme.

[0150] Scheme 1: Synthesis of carbonate and carbamate linked compounds [ka] Intermediate II, flanked by orthogonal leaving groups, is reacted with a nucleophilic R 8 Intermediate III can then be reacted with a thiol-containing peptide (HS-R) that participates in a disulfide exchange reaction to produce intermediate III. 7 ) to give the final compound. Suitable leaving groups are described below.

[0151] Scheme 2: Synthesis of thiopropionic acid-linked conjugates 1 [ka] Propionic acid disulfide IV with previously installed leaving groups 1 and 2 is reacted with the nucleophilic R 8 -H to produce V. This compound can then be reacted with R 7 -SH to generate the desired conjugate.

[0152] Scheme 3: Synthesis of thiopropionic acid-linked conjugates 2 [ka] The thionoester VI is a nucleophilic R 8 -H to generate propionic acid thiol VII, which can participate in a disulfide exchange reaction to provide intermediate VIII. This compound can be further reacted with R to provide the desired conjugate. 7 -Can be treated with SH.

[0153] Scheme 4: Synthesis of parabenzyl-linked conjugates 1 [ka] The alcohol group of para-aminobenzyl alcohol IX can be selectively protected to give intermediate X. This intermediate can then be reacted with intermediate II at the aniline position to give aryl carbamate XI. The protecting group can be removed to give the free alcohol XII, which can be treated with an activating agent to provide intermediate XIII, which contains an orthogonal leaving group. 8 Reaction of -H with intermediate XIII can provide intermediate XIV, followed by reaction with R 7 Treatment with -SH affords the desired parabenzyl-linked conjugate.

[0154] Scheme 5: Synthesis of parabenzyl-linked conjugates [ka] 4-Mercaptobenzyl alcohol XV can be reacted in a disulfide exchange reaction to give 4-mercaptobenzyl alcohol disulfide XVI containing leaving group 2. The remaining benzyl alcohol can be treated with an appropriate carbonyl compound to give activated compound XVII. This intermediate can be further converted to a nucleophilic R 8 -H to give intermediate XVIII, which can be reacted with R 7 -SH to give the desired conjugate.

[0155] Scheme 6: Synthesis of ortho benzyl-linked conjugates [ka] The 2-mercaptobenzyl alcohol XXIII can be reacted as previously described to give the desired conjugate.

[0156] Scheme 7: Cleavage of peptide conjugates [ka] R 8 Cleavage of the final compound to release -H can be achieved by treating the compound with excess glutathione (GSH) in buffer with incubation at 37° C. Reverse-phase HPLC analysis over the desired time course is used to follow the progress of cleavage.

[0157] Peptide R 7 can be prepared using solid-phase synthesis, first described by Merrifield in JACS, Vol. 85, pp. 2149-2154 (1963), although other methods known in the art can also be used. The Merrifield technique is well understood and is a common method for preparing peptides. Techniques useful for solid-phase peptide synthesis are described in several books, such as the 1984 text "Principles of Peptide Synthesis" by Bodanszky and Springer Verlag. This synthesis method involves the stepwise addition of protected amino acids to a growing peptide chain covalently attached to solid resin particles. This procedure eliminates the need for intermediate purification, as reagents and by-products are removed by filtration. The general concept of this method relies on the covalent attachment of the first amino acid of the chain to a solid polymer, followed by the addition of subsequent protected amino acids, one at a time, stepwise, until the desired sequence is assembled. Finally, the protected peptide is removed from the solid resin support, and the protecting groups are cleaved.

[0158] Peptide R 7 can also be produced by fermentation, for example, by modification of E. coli. Protein production in E. coli is described in detail in the R 7 The recombinant polypeptide can be controlled to produce a recombinant polypeptide having the sequence of the peptide. The production of recombinant polypeptides in E. coli is described in the following references: Zhao, Q., Xu, W., Xing, L. et al., "Recombinant production of medium- to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag." Microb Cell Fact 15, 136 (2016); de Marco, "Recombinant polypeptide production in E. coli: toward a rational approach to improve the yields of functional proteins." Microbial Cell Factories 2013, 12: 101; and Kleiner-Grote GM, Risse, JM, Friehs, K., "Secretion of recombinant proteins from E. coli." Eng. Life Sci. 2018, 18, 532-550, each of which is incorporated by reference in its entirety.

[0159] The amino acid can be attached to any suitable polymer. The polymer must be insoluble in the solvent used, have a stable physical form that allows for easy filtering, and contain functional groups to which the first protected amino acid can be securely attached by a covalent bond. A variety of polymers are suitable for this purpose, including cellulose, polyvinyl alcohol, polymethyl methacrylate, and polystyrene.

[0160] How to use Provided herein is the use of a compound of Formula (I) in the treatment of diseases such as cancer or neurodegenerative diseases. Another aspect of the present invention is the use of a compound of Formula (I) in the treatment of diseases involving acidic or hypoxic diseased tissue, such as cancer or neurodegenerative diseases. Hypoxia and acidosis are physiological markers of many disease processes, including cancer. In cancer, hypoxia is one mechanism involved in the development of an acidic environment within solid tumors. As a result, hydrogen ions must be removed from the cell (e.g., by proton pumps) to maintain a normal pH within the cell. As a result of this hydrogen ion export, cancer cells often have an increased pH gradient across the cell membrane lipid bilayer and a lower pH in the extracellular environment compared to normal cells. One approach to improving the efficacy and therapeutic index of cytotoxic agents is to exploit this physiological property to selectively deliver compounds to hypoxic cells over healthy tissue.

[0161] In the treatment methods of the present invention, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof can be administered as a single agent or in combination with other forms of treatment, such as ionizing radiation or cytotoxic agents in the case of cancer. In combination therapy, the compound of formula (I) can be administered before, simultaneously with, or after the other therapy, as will be understood by those skilled in the art. Either therapy (single agent or combination with other therapies) can be administered as a course of treatment, including multiple doses or treatment over a period of time.

[0162] Examples of cancers that can be treated using the compounds of the present disclosure include colorectal cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, uterine cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute myeloid leukemia, and ovarian cancer. These include, but are not limited to, lymphoblastic leukemia, chronic or acute leukemia including chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or urethral cancer, renal pelvic cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of these cancers.

[0163] In some embodiments, cancers treatable with the compounds of the present disclosure include bladder cancer, bone cancer, glioma, breast cancer (e.g., triple-negative breast cancer), cervical cancer, colon cancer, colorectal cancer, endometrial cancer, epithelial cancer, esophageal cancer, Ewing's sarcoma, pancreatic cancer, gallbladder cancer, gastric cancer, gastrointestinal tumors, head and neck cancer (upper aerodigestive cancer), intestinal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, adenocarcinoma), melanoma, prostate cancer, rectal cancer, renal clear cell carcinoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer.

[0164] In some embodiments, cancers treatable with the compounds of the present disclosure include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, triple-negative breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer and small cell lung cancer). Additionally, the present disclosure includes refractory or recurrent malignancies whose growth can be inhibited using the compounds of the present disclosure.

[0165] In some embodiments, cancers treatable using the compounds of the present disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, renal cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., leukemias such as lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin's lymphoma, or multiple myeloma), and combinations of these cancers.

[0166] Compounds of the invention (e.g., compounds of Formula (I)) that include a topoisomerase I targeting moiety derived from a topoisomerase I inhibitor (e.g., exatecan) can exhibit certain therapeutic advantages over topoisomerase I inhibitors themselves. For example, administration of a compound of Formula (I) can exhibit reduced toxicity (e.g., bone marrow or gastric toxicity) compared to administration of a corresponding topoisomerase I inhibitor (e.g., exatecan). In some embodiments, bone marrow toxicity is measured by total bone marrow counts from a subject's sample (e.g., total bone marrow counts in mouse femurs). In some embodiments, bone marrow toxicity is measured by PARylation in bone marrow tissue. In some embodiments, bone marrow toxicity is measured according to total nucleated bone marrow cells. In some embodiments, gastric toxicity is assessed using photographs of the stomach of a subject (e.g., a mouse) taken both in situ and ex vivo.

[0167] In certain embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof may be used in combination with a chemotherapeutic agent, a targeted cancer therapy, an immunotherapy, or a radiation therapy. The agent may be combined with the compound in a single dosage form, or the agents may be administered simultaneously or sequentially as separate dosage forms. In some embodiments, the chemotherapeutic agent, the targeted cancer therapy, the immunotherapy, or the radiation therapy, when administered together with the compound of formula (I) or a pharmaceutically acceptable salt thereof, may be combined with a corresponding topoisomerase inhibitor (e.g., R 8- H), it has been shown to be less toxic to patients, including reduced bone marrow or stomach toxicity.

[0168] Suitable chemotherapeutic or other anti-cancer agents include, for example, alkylating agents (including, but not limited to, nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, and triazenes) such as uracil mustard, chloramustine, cyclophosphamide (Cytoxan™), ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

[0169] Other drugs suitable for use in combination with the compounds of the invention include dacarbazine (DTIC), optionally with other chemotherapeutic agents such as carmustine (BCNU) and cisplatin; the "Dartmouth regimen" consisting of DTIC, BCNU, cisplatin, and tamoxifen; a combination of cisplatin, vinblastine, and DTIC; or temozolomide. The compounds according to the invention can also be combined with immunotherapeutic agents, including cytokines such as interferon alpha, interleukin 2, and tumor necrosis factor (TNF).

[0170] Suitable chemotherapeutic or other anti-cancer agents include, for example, antimetabolites (including but not limited to folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors), such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

[0171] Suitable chemotherapeutic or other anti-cancer agents further include, for example, certain natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), such as vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel (TAXOL™), mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, interferons (especially IFN-α), etoposide, and teniposide.

[0172] Other cytotoxic agents that can be administered in combination with the compounds of the invention include, for example, navelbene, CPT-11, anastrozole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, and droloxafine.

[0173] Also suitable are cytotoxic agents such as epidophyllotoxins; antitumor enzymes; topoisomerase inhibitors; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitory agents; antihormonal therapeutic agents; leucovorin; tegafur; and hematopoietic growth factors.

[0174] Other anti-cancer drug(s) include antibody therapeutics such as trastuzumab (Herceptin), antibodies against costimulatory molecules such as CTLA-4, 4-1BB, PD-1, or antibodies against cytokines (IL-10, TGF-α, etc.).

[0175] Other anti-cancer drugs also include those that block immune cell migration, such as antagonists to chemokine receptors including CCR2 and CCR4.

[0176] Other anti-cancer drugs also include those that enhance the immune system, such as adjuvants and adoptive T-cell transfer.

[0177] Anti-cancer vaccines that can be administered in combination with the compounds of the invention include, for example, dendritic cells, synthetic peptides, DNA vaccines, and recombinant viruses.

[0178] Other suitable agents for use in combination with the compounds of the invention include chemotherapy combinations such as platinum-based doublets used in lung cancer and other solid tumors (cisplatin or carboplatin plus gemcitabine; cisplatin or carboplatin plus docetaxel; cisplatin or carboplatin plus paclitaxel; cisplatin or carboplatin plus pemetrexed), or gemcitabine plus paclitaxel conjugated particles (Abraxane®).

[0179] The compounds of the present invention may be effective in combination with antihormonal agents for the treatment of breast cancer and other tumors. Suitable examples include antiestrogens, including but not limited to tamoxifen and toremifene; aromatase inhibitors, including but not limited to letrozole, anastrozole, and exemestane; adrenergic corticosteroids (e.g., prednisone); progestins (e.g., megastrol acetate); and estrogen receptor antagonists (e.g., fulvestrant). Suitable antihormonal agents used in the treatment of prostate cancer and other cancers can also be combined with the compounds of the present invention. These include antiandrogens, including but not limited to flutamide, bicalutamide, and nilutamide; luteinizing hormone-releasing hormone (LHRH) analogs, including leuprolide, goserelin, triptorelin, and histrelin; LHRH antagonists (e.g., degarelix); androgen receptor blockers (e.g., enzalutamide); and agents that inhibit androgen production (e.g., abiraterone).

[0180] The compounds of the present invention can be administered in combination with or sequentially with other agents against membrane receptor kinases, particularly for patients who have developed primary or acquired resistance to targeted therapy. These therapeutic agents include inhibitors or antibodies against EGFR, Her2, VEGFR, c-Met, Ret, IGFR1, or Flt-3, as well as inhibitors or antibodies against cancer-associated fusion protein kinases such as Bcr-Abl and EML4-Alk. EGFR inhibitors include gefitinib and erlotinib, and EGFR / Her2 inhibitors include, but are not limited to, dacomitinib, afatinib, lapitinib, and neratinib. EGFR antibodies include, but are not limited to, cetuximab, panitumumab, and necitumumab. c-Met inhibitors can be used in combination with the compounds of the present invention. These include onartumzumab, tivantinib, and INC-280. Agents directed against Abl (or Bcr-Abl) include imatinib, dasatinib, nilotinib, and ponatinib, and agents directed against Alk (or EML4-ALK) include crizotinib.

[0181] Angiogenesis inhibitors may be effective in combination with the compounds of the present invention in some tumors. These include antibodies against VEGF or VEGFR or VEGFR kinase inhibitors. Antibodies against VEGF or other therapeutic proteins include bevacizumab and aflibercept. VEGFR kinase inhibitors and other antiangiogenesis inhibitors include, but are not limited to, sunitinib, sorafenib, axitinib, cediranib, pazopanib, regorafenib, brivanib, and vandetanib.

[0182] The activation of intracellular signal transduction pathways frequently occurs in cancer, and drugs that target components of these pathways are combined with receptor targeting agents to improve efficacy and reduce resistance. Examples of drugs that can be combined with the compounds of the present invention include inhibitors of the PI3K-AKT-mTOR pathway, inhibitors of the Raf-MAPK pathway, inhibitors of the JAK-STAT pathway, and inhibitors of protein chaperones and cell cycle progression.

[0183] Drugs against PI3 kinase include, but are not limited to, pilalalisib, idelalisib, and buparlisib. mTOR inhibitors, such as rapamycin, sirolimus, temsirolimus, and everolimus, can be combined with the compounds of the present invention. Other suitable examples include, but are not limited to, vemurafenib and dabrafenib (Raf inhibitors) and trametinib, selumetinib, and GDC-0973 (MEK inhibitors). Inhibitors of one or more JAK (e.g., ruxolitinib, baricitinib, tofacitinib), Hsp90 (e.g., tanespimycin), cyclin-dependent kinase (e.g., palbociclib), HDAC (e.g., panobinostat), PARP (e.g., olaparib), and proteasome (e.g., bortezomib, carfilzomib) can also be combined with the compounds of the present invention. A further example of a PARP inhibitor that can be combined with the compounds of the invention is talazoparib.

[0184] Methods for safely and effectively administering most of these chemotherapeutic agents are known to those skilled in the art. Moreover, their administration is described in standard literature. For example, the administration of many of the chemotherapeutic agents is described in the "Physicians' Desk Reference" (PDR, e.g., 1996 edition, Medical Economics Company, Montvale, NJ), the disclosure of which is incorporated herein by reference as if set forth in its entirety.

[0185] The phrase "therapeutically effective amount" of a compound (therapeutic agent, active ingredient, drug, etc.) refers to the amount of compound administered to a subject in need of therapy or treatment that alleviates symptoms, improves the condition, or delays the onset of the disease state according to clinically acceptable criteria for the disorder or condition being treated. For example, a therapeutically effective amount can be an amount that has been demonstrated to have the desired therapeutic effect in in vitro assays, in vivo animal assays, or clinical trials. A therapeutically effective amount can vary based on the particular dosage form, method of administration, treatment protocol, the particular disease or condition being treated, the benefit / risk ratio, etc., among many other factors.

[0186] The therapeutically effective amount can be obtained from clinical trials, animal models, or in vitro cell culture assays. It is known in the art that the effective amount suitable for human use can be calculated from the effective amount determined from animal models or in vitro cell culture assays. For example, as reported by Reagan-Shaw et al., FASEB J. 2008:22(3)659-61, "μg / ml" (effective amount based on in vitro cell culture assay) is equivalent to "mg / kg body weight / day" (effective amount in mice). Furthermore, based on the fact that the metabolic rate of mice is six times faster than that of humans, the effective amount in humans can be calculated from the effective amount in mice.

[0187] As an example of the treatment using the compound of formula (I) in combination with a cytotoxic agent, a therapeutically effective amount of the compound of formula (I) can be administered to a patient suffering from cancer as part of a treatment plan that also includes a therapeutically effective amount of ionizing radiation or a cytotoxic agent.In the context of this treatment plan, the term "therapeutically effective" amount should be understood to mean effective in combination therapy.Those skilled in the art of cancer treatment will understand how to adjust the dosage to achieve optimal therapeutic results.

[0188] Similarly, appropriate dosages of the compounds of the invention for treating non-cancerous diseases or conditions (such as cardiovascular disease) can be readily determined by medical technicians.

[0189] As used herein, the term "treatment" includes the administration of a compound or composition to reduce the frequency, delay the onset, or reduce the progression of diseases involving acidic or hypoxic diseased tissue, such as cancer, stroke, myocardial infarction, or long-term neurodegenerative diseases, in a subject compared to subjects not receiving the compound or composition. This includes reversing, alleviating, or preventing the symptoms, clinical signs, or underlying pathology of a condition in a manner that improves or stabilizes the subject's condition (e.g., regression of tumor growth in the case of cancer, or reduction or amelioration of myocardial ischemia-reperfusion injury in infarction, stroke, or similar cardiovascular diseases). The terms "inhibit" or "reduce" are used in cancer to refer to methods of inhibiting or reducing tumor growth (e.g., reducing tumor size) in a population compared to an untreated control population.

[0190] All publications (including patents) mentioned herein are incorporated by reference for the purpose of describing and disclosing, for example, the structures and procedure described in the publications, which might be used in connection with the disclosure set forth herein. The publications discussed throughout the text are provided solely for their disclosure prior to the filing date of the present application.

[0191] Disclosed herein are several types of ranges. When any type of range is disclosed or claimed, the intention is to individually disclose or claim each possible number that such range can reasonably encompass, including the endpoints of the range and any subranges and combinations of subranges encompassed therein. For example, when a range of a therapeutically effective amount of an active ingredient is disclosed or claimed, the intention is to individually disclose or claim every possible number that such range can encompass, consistent with the disclosure herein. For example, it is disclosed or claimed by disclosing that a therapeutically effective amount of a compound can be in the range of about 1 mg / kg to about 50 mg / kg (of control body weight).

[0192] Formulation, Dosage Forms, and Administration To prepare the pharmaceutical compositions of the present invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is combined as an active ingredient intimately mixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. This carrier can take a variety of forms, e.g., oral or parenteral, depending on the form of preparation desired for administration. When preparing compositions in oral dosage form, any of the usual pharmaceutical media, such as water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents, can be used for oral liquid preparations such as suspensions, elixirs, and solutions; or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used for oral solid preparations such as powders, capsules, and tablets. Because of their ease of administration, tablets and capsules are the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are obviously used. If desired, tablets can be sugar-coated or enteric-coated by standard techniques. For parenteral preparations, the carrier usually comprises sterile water, although other ingredients, such as those that aid solubility or preservatives, may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, etc. may be employed. Those skilled in the pharmaceutical and medical arts will be able to readily determine the dosage of the pharmaceutical compositions of the present invention which will be suitable for the particular disease or condition being treated. [Example]

[0193] As used herein, all abbreviations, symbols, and conventions are consistent with those used in the contemporary scientific literature. See, e.g., Janet S. Dodd, ed., The ACS Style Guide: A Manual for Authors and Editors, 2nd Ed., Washington, DC: American Chemical Society, 1997. The following definitions explain the terms and abbreviations used herein.

[0194] Brine: A saturated solution of NaCl in water DCM: dichloromethane TFA: Trifluoroacetic acid DIPEA: Diisopropylethylamine DMA: Dimethylacetamide DME: Dimethoxyethane DMF: dimethylformamide DMSO: methyl sulfoxide DTT: dithiothreitol MSD: Mass spectrometry detector Et2O: Ethyl ether EtOAc: ethyl acetate EtOH: Ethyl alcohol HATU: O-(7-aza-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HOBt: 1-hydroxybenzotriazole RP: Reverse phase HPLC: High-performance liquid chromatography IPA: Isopropanol LAH: Lithium aluminum hydride N-BuLi: n-butyllithium LC-MS: Liquid chromatography mass spectrometry LDA: lithium diisopropyl ethyl amide Me: Methyl MeOH: Methanol MTBE: Methyl t-butyl ether NMP: N-methylpyrrolidine Ph: Phenyl PNPC: para-nitrophenyl chloroformate RT or rt: room temperature SFC: Supercritical Fluid Chromatography TBAI: Tetrabutylammonium iodide TBME: tert-butyl methyl ether tBu: tertiary butyl THF: tetrahydrofuran TEA: Triethylamine TMEDA: Tetramethylethylenediamine GSH: glutathione GS: sulfur-bound glutathione LiOH: Lithium hydroxide DPPA: Diphenylphosphoryl azide Sn(Bu)2(lauric acid)2: Dibutyltin dilaurate PBS: phosphate buffered saline ACN: acetonitrile AcOH: acetic acid EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline DMAP: 4-dimethylaminopyridine EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0195] The sources of the starting materials used in the examples are shown in the table below.

[0196] [Table 2-1] [Table 2-2] [Table 2-3]

[0197] [Table 3-1] [Table 3-2]

[0198] The HPLC method used is shown below.

[0199] HPLC method A: Sunfire C18 150 x 4.6 mm; H2O / acetonitrile w / TFA modifier (0.05%); flow rate: 1 ml / min; wavelength = 217 nM.

[0200] B: Ace Equivalence 250x4.6mm; H2O / acetonitrile w / TFA modifier (0.05%); flow rate: 1 ml / min; wavelength = 217 nM.

[0201] C: Sunfire C18 150 x 30 mm; H2O / acetonitrile w / TFA modifier (0.05%); flow rate: 30 ml / min; wavelength = 217 nM.

[0202] mass spectrometry Matrix-assisted laser desorption / ionization-time of flight (Maldi-TOF) mass spectrometry was performed on an Applied Biosystems Voyager System 6268. Samples were prepared in a matrix of α-cyanohydroxycinnamic acid on an AB Science plate (part number V700666).

[0203] ESI (electrospray ionization) mass spectrometry was measured on either an Agilent 1100 series LC-MS equipped with a 1946 MSD or a Waters Xevo Qtof high-resolution MS, both of which provide mass / charge species (m / z=3).

[0204] Synthesis of cis-S-(3-hydroxybutan-2-yl)ethanethioate (L-4 and L-5) [ka] To a stirred solution of trans-2,3-dimethyloxirane (5.0 g, 69.3 mmol) in water (50 mL) was added thioacetic acid (5.8 mL, 76.2 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (200 mL). The organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to give cis-S-(3-hydroxybutan-2-yl)ethanethioate (4.0 g, crude) as an oily compound. MS m / z 149.0 [M+H] + .

[0205] Synthesis of cis-3-mercaptobutan-2-ol [ka] To a stirred solution of S-(3-hydroxybutan-2-yl)ethanethioate (4 g, 26.9 mmol) in THF (40 mL) was added lithium aluminum hydride (1 M solution in THF) (27 mL, 26.9 mmol) dropwise at 0 °C. The reaction mixture was gradually warmed to room temperature and stirred for 3 h. The reaction mixture was slowly quenched with 1 N HCl at 0 °C and the pH was adjusted to 2-3. The reaction mixture was extracted with ethyl acetate (50 mL), and the organic layer was dried over anhydrous sodium sulfate and evaporated to give cis-3-mercaptobutan-2-ol as a crude oily compound.

[0206] Synthesis of trans-S-(3-hydroxybutan-2-yl)ethanethioate (L-6 and L-7) [ka] To a stirred solution of cis-2,3-dimethyloxirane (1.0 g, 13.9 mmol) in water (15 mL) was added thioacetic acid (1.1 mL, 15.6 mmol) at room temperature and stirred for 16 hours. The reaction mixture was quenched with sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (20 mL). The organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to give trans-S-(3-hydroxybutan-2-yl)ethanethioate (0.7 g, crude) as a yellow oil.

[0207] Synthesis of trans-3-mercaptobutan-2-ol [ka] To a stirred solution of trans-S-(3-hydroxybutan-2-yl)ethanethioate (700 mg, 4.72 mmol) in THF (10 mL), lithium aluminum hydride (1 M solution in THF) (4.8 mL, 4.72 mmol) was added dropwise at 0 °C and stirred at the same temperature for 3 h. The reaction mixture was quenched with 1 N HCl at 0 °C to adjust the pH to 2-3. The reaction mixture was extracted with CHCl (10 mL). The organic layer was dried over anhydrous sodium sulfate and used directly in the next step.

[0208] Synthesis of trans-S-(2-hydroxycyclohexyl)ethanethioate (L-8 and L-9) [ka] To a stirred solution of 7-oxabicyclo[4.1.0]heptane (5.0 g, 51.0 mmol) in water (50.0 mL) was added thioacetic acid (4.92 mL, 61.0 mmol). The reaction mixture was stirred at room temperature for 16 hours. The reaction progress was monitored by TLC (20% EtOAc / hexane). After completion of the reaction, the reaction mixture was diluted with diethyl ether. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give trans-S-(2-hydroxycyclohexyl)ethanethioate (3.8 g, crude) as a brown liquid.

[0209] Synthesis of trans-2-mercaptocyclohexan-1-ol [ka] To a stirred solution of trans-S-(2-hydroxycyclohexyl)ethanethioate (3.8 g, 21.8 mmol) in THF (20.0 mL) was added 1 M LiAH in THF (21.8 mL, 21.8 mmol) at 0 °C. The reaction mixture was gradually warmed to room temperature and stirred for 1 h. The reaction progress was monitored by TLC (20% EtOAc / hexane). Upon completion of the reaction, the reaction mixture was cooled to room temperature and quenched with 1.0 N HCl (30 mL). The reaction mixture was extracted with CHCl (300.0 mL). The organic layer was washed with brine solution (30.0 mL), concentrated, and the crude trans-2-sulfanylcyclohexanol was used in the next step (2.88 g, crude).

[0210] Synthesis of intermediate I from L [ka]

[0211] [Table 4]

[0212] Intermediate I-1: Synthesis of (2R)-2-(2-pyridyldisulfanyl)propan-1-ol [ka] To 2-(2-pyridyldisulfanyl)pyridine (5.00 g, 22.7 mmol) in 40 mL of N2-degassed MeOH was added (2R)-2-sulfanylpropan-1-ol (0.75 g, 8.14 mmol) dropwise. The mixture was stirred under N2 for 2 h. The mixture was concentrated to dryness and loaded directly onto a SiO2 flash column, eluting with 0-50% EtOAc / hexane to give 1.17 g, 71% of (2R)-2-(2-pyridyldisulfanyl)propan-1-ol. MS m / z 202.1 [M+H] + .

[0213] Intermediate I-2 was prepared in a similar manner from L-2.

[0214] Intermediate I-3: Synthesis of [1-[(5-nitro-2-pyridyl)disulfanyl]cyclobutyl]methanol [ka] To 5-nitro-2-[(5-nitro-2-pyridyl)disulfanyl]pyridine (17.4 g, 56.0 mmol) in degassed (N)MeOH (100 mL) was added (1-mercaptocyclobutyl)methanol (8.3 mL, 70.0 mmol) (degassed with N) dropwise and stirred at room temperature under a N atmosphere for 16 hours. The reaction mixture was concentrated to dryness in vacuo. The resulting crude product was purified by column chromatography using 30% EtOAc / hexane to give [1-[(5-nitro-2-pyridyl)disulfanyl]cyclobutyl]methanol as a yellow liquid (9.0 g, 46% yield). MS m / z 272.9 [M+H] + .

[0215] I-4 and I-5: Synthesis of 3-(pyridin-2-yldisulfanyl)butan-2-ol isomer 1 and isomer 2 [ka] A stirred solution of 2,2-dipyridyl disulfide (520 mg, 2.35 mmol) in MeOH (15 mL) was purged with nitrogen gas for 5 minutes. A nitrogen-purged solution of cis-3-mercaptobutan-2-ol (500 mg) in CHCl (10 mL) was added at 0°C. The reaction mixture was allowed to warm gradually to room temperature and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure to give the crude material, which was purified by column chromatography using 30–40% EA / hexane. The racemic product was separated by ChiralPrep HPLC (CHIRALPAK IG; 100 mm × 4.6 mm × 3 microns; mobile phase: n-hexane:ethanol 80:20 with 0.1% DEA; flow rate: 1.0 mL / min), and the individual enantiomers were separated. The solvent was removed to give (2S,3S)-3-(2-pyridyldisulfanyl)butan-2-ol* (140 mg, isomer-1) MS m / z 216.1 [M+H] + and (2R,3R)-3-(2-pyridyldisulfanyl)butan-2-ol (140 mg, isomer-2). MS m / z 216.1 [M+H] + .

[0216] I-6 and I-7: Synthesis of 3-(pyridin-2-yldisulfanyl)butan-2-ol isomer 1 and isomer 2 [ka] A stirred solution of 2,2-dipyridyl disulfide (520 mg, 2.35 mmol) in MeOH (15 mL) was purged with nitrogen gas for 5 minutes. A nitrogen-purged solution of cis-3-mercaptobutan-2-ol (500 mg) in CHCl (10 mL) was added at 0°C. The reaction mixture was allowed to warm gradually to room temperature and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure to give the crude material, which was purified by column chromatography using 30–40% EA / hexane. The racemic product was separated by ChiralPrep HPLC (Column: CHIRALPAK IG (100 mm × 4.6 mm × 3 micrometers), Mobile phase: n-hexane:ethanol with 0.1% DEA (80:20); Flow rate: 1.0 mL / min) to separate the individual enantiomers. The solvent was removed to give (2R,3S)-3-(2-pyridyldisulfanyl)butan-2-ol* (0.6 g, Isomer-I) MS m / z 215.9 [M+H] + and (2S,3R)-3-(2-pyridyldisulfanyl)butan-2-ol* (0.6 g, Isomer-II) MS m / z 216.2 [M+H] + was obtained as an oily compound.

[0217] Intermediate I-6: Synthesis of trans-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol [ka] To a solution of 1,2-di(pyridin-2-yl)disulfane (2.41 g, 10.9 mmol) in MeOH (degassed with N) (30 mL), trans-2-sulfanylcyclohexanol (2.88 g, 21.0 mmol) (degassed with N) was added dropwise and stirred at room temperature under a N atmosphere for 16 hours. The reaction mixture was concentrated to dryness in vacuo. The resulting crude product was purified by column chromatography using 30% EtOAc / hexane to give trans-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol as a yellow liquid.

[0218] [ka] Chiral separation was performed using a chiralpak IG (100 mm × 4.6 mm × 3 micrometers) using n-hexane:IPA (80:20) containing 0.1% diethylamine to give (1R,2R)-2-(2-pyridyldisulfanyl)cyclohexanol* isomer-1 (350 mg) and (1S,2S)-2-(2-pyridyldisulfanyl)cyclohexanol* isomer-2 (400 mg).

[0219] Intermediate XV from XXI [ka] [Table 5]

[0220] Intermediate XV-1: Synthesis of [4-(2-pyridyldisulfanyl)phenyl]methanol [ka] A stirred solution of 1,2-di(pyridin-2-yl)disulfane (2.68 g, 12.1 mmol) in a mixture of AcOH:ethanol (5 mL, 1:10) solvent was degassed under N. Following this, 4-mercaptophenyl)methanol (0.74 g, 5.2 mmol) was added dropwise over 20 min to the AcOH / ethanol (5 mL) solvent mixture and stirred at room temperature under a N atmosphere for 12 h. The reaction was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (SiO, 60–70% EtOAc / hexanes) to give [4-(2-pyridyldisulfanyl)phenyl]methanol as a colorless liquid (800 mg, 61% yield).

[0221] Carbonate leaving group intermediate II from intermediate I [ka] [Table 6]

[0222] II-1: Synthesis of (4-nitrophenyl)[(2R)-2-(2-pyridyldisulfanyl)propyl]carbonate [ka] To (2R)-2-(2-pyridyldisulfanyl)propan-1-ol (0.39 g, 1.94 mmol) in THF under N2 was added pyridine (0.16 mL, 1.94 mmol) and (4-nitrophenyl)carboxynochloridate (0.59 g, 2.91 mmol). The mixture was stirred under N2 for 16 h. The mixture was diluted with EtOAc and quenched with 20 mL of saturated NH4Cl. The mixture was washed with water and brine, and the organic layer was concentrated. The crude mixture was purified by column chromatography (SiO2, 0-50% EtOAc / hexanes) to afford 0.59 g, 83% of (4-nitrophenyl)[(2R)-2-(2-pyridyldisulfanyl)propyl]carbonate. MS m / z found: 367.1 [M+H] + .

[0223] Intermediates II-2 and II-3 were synthesized similarly to II-1.

[0224] II-4: Synthesis of 4-nitrophenyl ((2R,3R)-3-(pyridin-2-yldisulfanyl)butan-2-yl) carbonate [ka] To a stirred solution of (2R,3R)-3-(pyridin-2-yldisulfanyl)butan-2-ol (140 mg, 0.651 mmol) in CHCl (2.0 mL) was added pyridine (0.11 mL, 1.43 mmol), 4-nitrophenyl carbonochloridate (150 mg, 0.781 mmol), and a catalytic amount of 4-dimethylaminopyridine at room temperature. The reaction vessel was sealed and stirred at room temperature for 48 h. The reaction mixture was diluted with CHCl (10 mL) and then washed with water (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product, which was purified by column chromatography using 30–40% ethyl acetate / hexanes. Fractions were concentrated to give the crude product, which was further purified on a C18 reverse-phase column. The pure fractions were concentrated to give 4-nitrophenyl ((2R,3R)-3-(pyridin-2-yldisulfanayl)butan-2-yl) carbonate (70 mg, 28%) as an oily compound. MS m / z 381.0 [M+H] + .

[0225] II-5: Synthesis of 4-nitrophenyl ((2S,3S)-3-(pyridin-2-yldisulfanyl)butan-2-yl) carbonate [ka] To a stirred solution of (2R,3R)-3-(pyridin-2-yldisulfanyl)butan-2-ol (80 mg, 0.372 mmol) in CHCl (1.0 mL) was added pyridine (0.066 mL, 0.818 mmol), 4-nitrophenyl carbonochloridate (89 mg, 0.446 mmol), and a catalytic amount of 4-dimethylaminopyridine at room temperature. The reaction vessel was sealed and stirred at room temperature for 48 h. The reaction mixture was diluted with CHCl (5 mL) and then washed with water (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude titrated product, which was purified by column chromatography using 30–40% ethyl acetate / hexanes. Fractions were concentrated to give the crude product, which was further purified on a C18 reverse-phase column. The pure fractions were concentrated to give 4-nitrophenyl ((2S,3S)-3-(pyridin-2-yldisulfanayl)butan-2-yl) carbonate (140 mg, 58%) as an oily compound. MS m / z 381.0 [M+H] + .

[0226] II-6: Synthesis of 4-nitrophenyl ((2R,3S)-3-(pyridin-2-yldisulfanyl)butan-2-yl) carbonate [ka] To a stirred solution of (2R,3S)-3-(pyridin-2-yldisulfanyl)butan-2-ol (0.4 g, 1.86 mmol) in CHCl (10 mL) was added pyridine (0.36 mL, 4.09 mmol), 4-nitrophenyl carbonochloridate (0.44 g, 2.32 mmol), and a catalytic amount of 4-dimethylaminopyridine at 0 °C. The reaction vessel was sealed and stirred at room temperature for 48 h. The reaction mixture was diluted with CHCl (20 mL) and washed with water (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product, which was purified by silica gel flash column chromatography using 30–40% ethyl acetate / hexanes. The compound eluted as a mixture in 30% EtOAc:hexanes. Fractions were concentrated to give the crude product, which was further purified on a C18 reverse-phase column. Evaporation of pure fractions gave 4-nitrophenyl ((2R,3S)-3-(pyridin-2-yldisulfanyl)butan-2-yl) carbonate (0.17 g, 24.2%) as an oily compound. MS m / z 381.0 [M+H] + .

[0227] II-7: Synthesis of 4-nitrophenyl ((2S,3R)-3-(pyridin-2-yldisulfanyl)butan-2-yl) carbonate [ka] To a stirred solution of (2S,3R)-3-(pyridin-2-yldisulfanyl)butan-2-ol (0.4 g, 1.86 mmol) in CHCl (10 mL) was added pyridine (0.36 mL, 4.09 mmol), 4-nitrophenyl carbonochloridate (0.44 g, 2.32 mmol), and a catalytic amount of 4-dimethylaminopyridine at 0 °C. The reaction vessel was sealed and stirred at room temperature for 48 h. The reaction mixture was diluted with CHCl (20 mL) and washed with water (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product, which was purified by silica gel flash column chromatography using 30–40% ethyl acetate / hexane. The compound eluted as a mixture in 30% EtOAc:hexane. Fractions were concentrated to give the crude product, which was further purified on a C18 reverse-phase column. The pure fractions were concentrated to give 4-nitrophenyl ((2S,3R)-3-(pyridin-2-yldisulfanyl)butan-2-yl) carbonate (0.18 g, 26%) as an oily compound. MS m / z 381.0 [M+H] + .

[0228] II-8: Synthesis of (4-nitrophenyl)[(1R,2R)-2-(2-pyridyldisulfanyl)cyclohexyl]carbonate [ka] To a solution of (1R,2R)-2-(2-pyridyldisulfanyl)cyclohexanol* (130.0 mg, 0.5 mmol) in THF (3.0 mL) was added potassium carbonate (0.20 g, 1.5 mmol), a catalytic amount of DMAP, and 4-nitrophenyl chloroformate (0.21 g, 0.10 mmol) at room temperature. The reaction vessel was sealed and stirred at room temperature for 48 hours. The progress of the reaction was monitored by TLC (20% EtOAc / hexane). After completion of the reaction, the reaction mixture was quenched with water (20.0 mL) and extracted with EtOAc (20.0 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by column chromatography using EtOAc / hexane to give 4-nitrophenyl (4-nitrophenyl) [(1R,2R)-2-(2-pyridyldisulfanyl)cyclohexyl] carbonate as a white solid (89 mg, 40% yield). MS m / z 407.0 [M+H] + .

[0229] II-9: Synthesis of 4-nitrophenyl)[(1S,2S)-2-(2-pyridyldisulfanyl)cyclohexyl]carbonate [ka] To a solution of (1S,2S)-2-(2-pyridyldisulfanyl)cyclohexanol* (0.42 g, 1.7 mmol) in THF (10.0 mL) was added potassium carbonate (0.70 g, 5.1 mmol), a catalytic amount of DMAP, and 4-nitrophenyl chloroformate (0.69 g, 3.4 mmol) at room temperature. The reaction vessel was sealed and stirred at room temperature for 48 hours. The progress of the reaction was monitored by TLC (20% EtOAc / hexane). After completion of the reaction, the reaction mixture was quenched with water (20.0 mL) and extracted with EtOAc (20.0 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by column chromatography using EtOAc / hexane to give 4-nitrophenyl (4-nitrophenyl) [(1R,2R)-2-(2-pyridyldisulfanyl)cyclohexyl] carbonate* as a white solid (250 mg, 35% yield). MS m / z 406.7 [M+H] + .

[0230] Carbonate leaving group intermediate XV from XIV [ka] [Table 7]

[0231] XV-1: Synthesis of (4-nitrophenyl)[4-(2-pyridyldisulfanyl)phenyl]methyl carbonate [ka] To a stirred solution of (4-(pyridin-2-yldisulfanyl)phenyl)methanol (0.40 g, 1.60 mmol) in CHCl (10 mL) was added 4-nitrophenyl chloroformate (0.65 g, 3.2 mmol), pyridine (0.25 mL, 3.20 mmol), and a catalytic amount of DMAP (0.005 g) at room temperature. The mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with 1.5 N HCl solution. The organic layer was separated, washed with brine, dried over anhydrous NaSO, and concentrated. The crude product was purified by column chromatography (SiO, 20–30% EtOAc / hexane) to give (4-nitrophenyl)[4-(2-pyridyldisulfanyl)phenyl]methyl carbonate as a colorless liquid (600 mg, 91% yield); MS m / z 415.0 [M+H]. + .

[0232] Carbonate and carbamate linked intermediates III [ka] [Table 8]

[0233] III-1: Synthesis of [(2S)-2-(2-pyridyldisulfanyl)propyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate [ka]

[0234] 1-Hydroxybenzotriazole hydrate (8.64 mg, 0.0564 mmol) in 2 mL of anhydrous DMF, finely ground molecular sieves 4 Å (50 mg) of (10S,23S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19-methyl-8-oxa-4,15-diazahexacyclo[14.7.1.02, 14.04, 13.06, 11.020, 24] To a mixture of tetracosa-1,6(11),12,14,16(24),17,19-heptaene-5,9-dione, methanesulfonic acid (25.0 mg, 0.0470 mmol), and pyridine (0.0190 mL, 0.235 mmol) was added (4-nitrophenyl)[(2S)-2-(2-pyridyldisulfanyl)propyl]carbonate (19.0 mg, 0.0517 mmol). After stirring at room temperature for 16 hours, the mixture was filtered and the solution was concentrated. The residue was purified by column chromatography (0-5% MeOH / DCM) to give [(2S)-2-(2-pyridyldisulfanyl)propyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (29.0 mg, 93.0% yield). MS m / z 663.0 [M+H] + .

[0235] Intermediates III-2 to III-9 are prepared from II-2 to II-9 in the same manner as III-1.

[0236] Carbonate and carbamate linked intermediates XVI [ka] [Table 9]

[0237] Intermediate XVI-1 is prepared from XV-1 in the same manner as III-1.

[0238] Synthesis of 4-nitrophenyl (trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-yl) carbonate [ka]

[0239] Step 1: Synthesis of racemic trans-(4-hydroxytetrahydrofuran-3-yl)ethanethioate [ka]

[0240] To a stirred solution of 3,6-dioxabicyclo[3.1.0]hexane (5.0 g, 0.051 mol) in water (40.0 mL) was added thioacetic acid (4.98 mL, 0.069 mol), and the resulting reaction mixture was stirred at room temperature for 16 hours. The progress of the reaction was monitored by TLC (20% EtOAc / hexane). Upon completion of the reaction, the reaction mixture was diluted with diethyl ether and washed with 10% sodium bicarbonate solution. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography using 20% EtOAc:n-hexane to give the title product as a brown liquid (4.0 g, 42% yield). 1 HNMR (400 MHz, CDCl3): δ 4.35-4.28 (m, 2H), 4.02-3.98 (m, 1H), 3.81-3.73 (m, 2H), 3.69-3.62 (m, 1H), 2.37 (s, 3H).

[0241] Step 2: Synthesis of racemic trans-4-mercaptotetrahydrofuran-3-ol [ka]

[0242] To a stirred solution of racemic trans-(4-hydroxytetrahydrofuran-3-yl)ethanethioate (4.0 g, 24.7 mmol) in dry THF (20.0 mL) under a nitrogen atmosphere, LAH (1 M in THF) (27.1 mL, 27.1 mmol) was added dropwise at 0 °C. The reaction mixture was gradually warmed to room temperature and stirred for 2 h. The reaction progress was monitored by TLC (20% EtOAc:n-hexane). Upon completion of the reaction, the reaction mixture was cooled to room temperature and quenched with 1.0 N HCl (50 mL). The reaction mixture was extracted into DCM (3 × 20 mL), and the organic layer was washed with brine solution (20 mL), dried over anhydrous sodium sulfate, filtered, partially distilled, and directly carried on to the next step. (2.9 g, crude).

[0243] Step 3: Synthesis of trans-(4RS,3RS)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol and trans-(4SR,3SR)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol [ka]

[0244] To a solution of 2-(pyridin-2-yldisulfanyl)pyridine (0.9 g, 21.7 mmol) in MeOH (degassed with N) (10 mL), 4-sulfanyloxolan-3-ol (2.9 g, 24.1 mmol) (degassed with N) was added dropwise and stirred at room temperature under a nitrogen atmosphere for 16 hours. The reaction mixture was concentrated to dryness in vacuo. The resulting crude product was purified by flash column chromatography using 30% EtOAc:n-hexane to afford the title compound 4-(pyridin-2-yldisulfanyl)oxolan-3-ol (racemic mixture) as a yellow oil. The isomers were separated by chiral preparative HPLC.

[0245] Chiral preparative HPLC conditions: Column: Chiralpak IA (250 mm x 20 mm x 5 mic) Mobile phase: EtOH (90:10) containing 0.1% DEA Flow rate: 19ml / min The separated fractions of the separated isomers were collected from the chiral preparative HPLC and evaporated under reduced pressure to give the title compounds as Isomer 1 (600 mg) and Isomer 2 (620 mg).

[0246] Isomer 1: (trans-(4RS,3RS)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol): LC-MS m / z C9H 11 Calculated for NO2S2: 229; Found: 230 [M+H] + . 1 H-NMR (400 MHz, CDCl3): δ 8.53-8.52 (m, 1H), 7.67-7.63 (m, 1H), 7.56 (d, J = 8.0Hz, 1H), 7.23-7.19 (m, 1H), 4.45-4.48 (m, 1H), 4.25 (t, J = 8.8Hz,1H), 4.12 (t, J = 6.8Hz,1H), 3.74-3.67(m, 2H), 3.48-3.41 (m, 1H).

[0247] Isomer 2: (trans-(4SR,3SR)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol): LC-MS m / z C9H 11 Calculated for NO2S2: 229; Found: 230 [M+H] + . 1 H-NMR (400 MHz, CDCl3): δ 8.54-8.53 (m, 1H), 7.68-7.64 (m, 1H), 7.56 (d, J = 8.0Hz, 1H), 7.23-7.20 (m, 1H), 4.49-4.45 (m, 1H), 4.25 (t, J = 7.6Hz,1H), 4.12 - 4.10 (m,1H), 3.74-3.67 (m, 2H), 3.47 - 3.44 (m, 1H).

[0248] The absolute stereochemistry of the isomers was arbitrarily assigned.

[0249] Step 4: Synthesis of 4-nitrophenyl (trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-yl) carbonate [ka]

[0250] To a stirred solution of trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol (0.61 g, 2.69 mmol) in DMF (10 mL) under a nitrogen atmosphere, DIPEA (1.45 mL, 8.08 mmol) and bis(4-nitrophenyl)carbonate (1.64 g, 5.38 mmol) were added at room temperature. The reaction vessel was sealed and stirred at room temperature for 12 hours. The progress of the reaction was monitored by TLC (20% EtOAc:n-hexane). Upon completion of the reaction, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 × 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by flash column chromatography using 20-30% EtOAc:n-hexane to give 4-nitrophenyl (trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-yl) carbonate as an off-white solid (790 mg, 77% yield). 1 HNMR (400 MHz, CDCl3): δ 8.50 (d, J = 4.4 Hz, 1H), 8.27 (d, J = 8.8 Hz, 2H), 7.67 - 7.59 (m, 2H), 7.36 (d, J = 8.8 Hz, 2H), 7.15 (t, J = 5.2 Hz, 1H), 5.44 - 5.43 (m, 1H), 4.40 - 4.25 (m, 2H), 4.03 (d, J = 11.2 Hz, 1H), 3.92 - 3.86 (m, 1H), 3.85 - 3.79 (m, 1H);LC-MS m / z C 16 H 14 Calculated for N2O6S2: 394; Found: 395 [M+H] +.

[0251] Synthesis of 4-nitrophenyl (trans-(trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-yl) carbonate [ka]

[0252] To a stirred solution of trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol (550 mg, 2.46 mmol) in DMF (10.0 mL) under nitrogen, DIPEA (1.32 mL, 7.38 mmol) and bis(4-nitrophenyl)carbonate (1.5 g, 4.92 mmol) were added at room temperature. The reaction vessel was sealed and stirred at room temperature for 12 hours. The progress of the reaction was monitored by TLC (20% EtOAc:n-hexane). Upon completion of the reaction, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and filtered. The mixture was stirred for 1 hour at rt and concentrated under reduced pressure to give the crude product. The crude product was purified by flash column chromatography using 20-30% EtOAc. The crude product was purified by flash column chromatography using 20-30% EtOAc:n-hexane to give 4-nitrophenyl (trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-yl) carbonate as an off-white solid (0.6 g, 70% yield). 1HNMR (400 MHz, CDCl3): δ 8.85 (d, J = 4.4 Hz, 1H), 8.26 (d, J = 8.8 Hz, 2H), 7.68 - 7.59 (m, 2H), 7.35 (d, J = 8.8 Hz, 2H), 7.14 (t, J = 5.2 Hz, 1H), 5.44 - 5.43 (m, 1H), 4.40 - 4.25 (m, 2H), 4.03 (d, J = 11.2 Hz, 1H), 3.92 - 3.86 (m, 1H), 3.85 - 3.79 (m, 1H);LC-MS m / z C 16 H 14 Calculated for N2O6S2: 394; Found: 395 [M+H] + .

[0253] Synthesis of 4-nitrophenyl(trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cyclopentyl) carbonate [ka]

[0254] Step 1: Synthesis of racemic trans-(5-hydroxycyclopentan-1-yl)ethanethioate [ka]

[0255] To a stirred solution of 6-oxabicyclo[3.1.0]hexane (3.0 g, mmol) in water (30 mL), thioacetic acid (3 mL, 39.2 mmol) was added at room temperature and stirred for 16 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 x 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give racemic trans-(5-hydroxycyclopentan-1-yl)ethanethioate as an oily compound (2.6 g, crude). LC-MS m / z C7H 12 Calculated for O2S: 160.2; Found: 143.3 [M+H - 17]+ .

[0256] Step 2: Synthesis of racemic trans-2-mercaptocyclopentan-1-ol [ka]

[0257] To a stirred solution of racemic trans-(5-hydroxycyclopentan-1-yl)ethanethioate (2.6 g, 16.2 mmol) in THF (20 mL) at 0 °C under a nitrogen atmosphere, LAH (1 M in THF) (24 mL, 24.3 mmol) was added dropwise. The reaction mixture was gradually warmed to room temperature and stirred for 2 h. The reaction progress was monitored by TLC (20% EtOAc:n-hexane). Upon completion of the reaction, the reaction mixture was cooled to room temperature, quenched with 1 N HCl, and extracted into DCM. The organic layer was dried over anhydrous sodium sulfate, filtered, and partially evaporated, and the crude racemic trans-2-mercaptocyclopentan-1-ol was used in the next step (1.9 g, crude).

[0258] Step 3: Synthesis of trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol and trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol [ka]

[0259] To a stirred solution of 2-(pyridin-2-yldisulfanyl)pyridine (2.1 g, 9.65 mmol) in MeOH (10 mL) under a nitrogen atmosphere, racemic trans-2-mercaptocyclopentan-1-ol (1.9 g, 16.1 mmol) was added dropwise at 0 °C. The reaction mixture was gradually warmed to room temperature and stirred for 16 h. After completion of the reaction, the reaction mixture was concentrated to dryness under vacuum. The resulting crude product was purified by silica gel flash column chromatography. The compound was eluted with 15% EtOAc:n-hexane. Fractions containing the desired product were combined and evaporated under reduced pressure to give the title compound (racemic mixture) as a yellow liquid. The isomers were separated by chiral preparative HPLC.

[0260] Chiral preparative HPLC conditions: Column: Chiralpak IA (250 mm x 20 mm x 5 mic) Mobile phase: EtOH (70:30) containing 0.1% DEA Flow rate: 19ml / min The separated fractions of the separated isomers were collected from the chiral preparative HPLC and evaporated under reduced pressure to give the title compounds as Isomer 1 (300 mg) and Isomer 2 (300 mg) as colorless oils.

[0261] Isomer 1 (trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol): LC-MS m / z calculated for C10H13NOS2: 227.34; found: 228.1 [M+H] + . 1 HNMR (400 MHz, CDCl3): δ 8.51 - 8.50 (m, 1H), 7.61 - 7.57 (m, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.17 - 7.14 (m, 1H), 4.03 - 3.97 (m, 1H), 3.0 - 2.87 (m,1H), 2.11 - 2.02 (m, 3H), 1.75 -1.65 (m, 4H).

[0262] Isomer 2 (trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol): LC-MS m / z calculated for C10H13NOS2: 227.34; found: 228.1 [M+H] + . 1 HNMR (400 MHz, CDCl3): δ 8.51 - 8.50 (m, 1H), 7.61 - 7.57 (m, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.17 - 7.14 (m, 1H), 4.03 - 3.97 (m, 1H), 3.0 - 2.87 (m,1H), 2.11 - 2.02 (m, 3H), 1.75 -1.65 (m, 4H).

[0263] The absolute stereochemistry of the isomers was arbitrarily assigned.

[0264] Step 4: Synthesis of 4-nitrophenyl ((1R,2R)-2-(pyridin-2-yldisulfanyl)cyclopentyl) carbonate [ka]

[0265] To a stirred solution of trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol (0.3 g, 1.34 mmol) in DMF (10 mL) under a nitrogen atmosphere, DIPEA (0.65 mL, 3.96 mmol) and bis(4-nitrophenyl)carbonate (0.8 g, 2.64 mmol) were added at room temperature. The reaction vessel was sealed and stirred at room temperature for 16 h. The reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 × 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel flash column chromatography. The compound was eluted as a mixture in 10% EtOAc:n-hexane. Evaporation of fractions gave the crude compound, which was purified by reverse-phase column chromatography. The product-containing fractions were evaporated under reduced pressure to give 4-nitrophenyl (trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cyclopentyl)carbonate as a colorless oil (305 mg, 59%).

[0266] 1 HNMR (400 MHz, CDCl3): δ 8.46 (d, J = 4.1Hz, 1H), 8.25 (d, J = 6.8 Hz, 2H), 7.66 - 7.62 (m, 2H), 7.34 (d, J = 6.4 Hz, 2H), 7.10 - 7.08 (m, LC-MS m / z C 17 H 16 Calculated for N2O5S2: 392.44; Found: 393.0 [M+H] + .

[0267] Synthesis of 4-nitrophenyl(trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cyclopentyl) carbonate [ka]

[0268] To a stirred solution of (1SR,2SR)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol (0.26 g, 1.14 mmol) in DMF (10.0 mL) under a nitrogen atmosphere, DIPEA (0.57 mL, 3.43 mmol) and bis(4-nitrophenyl)carbonate (0.7 g, 2.29 mmol) were added at room temperature. The reaction vessel was sealed and stirred at room temperature for 16 h. The reaction mixture was quenched with water (20.0 mL) and extracted with EtOAc (3 × 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel flash column chromatography. The compound was eluted as a mixture in 10% EtOAc:n-hexane. Fractions were evaporated to give the crude compound, which was purified by reverse-phase column chromatography. The product-containing fractions were evaporated under reduced pressure to give 4-nitrophenyl(trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cyclopentyl)carbonate (330 mg, 73.5%) as a colorless oil. 1 HNMR (400 MHz, CDCl3): δ 8.46 (d, J = 4Hz, 1H), 8.25 (d, J = 6.8 Hz, 2H), 7.66 - 7.62 (m, 2H), 7.34 (d, J = 6.4 Hz, 2H), 7.10 - 7.08 (m, 1H), 5.29 - 5.10 (m, 1H), 3.52 - 3.45 (m, 1H), 2.32 - 2.28 (m, 2H), 1.9 -1.76 (m, 4H). LC-MS m / z C 17 H 16 Calculated for N2O5S2: 392.44; Found: 393.0 [M+H] + .

[0269] Synthesis of 4-nitrophenyl (trans-(2RS,3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl) carbonate [ka]

[0270] Step 1: Synthesis of 1aH,2H,7H,7aH-naphtho[2,3-b]oxirene [ka]

[0271] To a stirred solution of 1,4-dihydronaphthalene (100 mg, 768 μmol) in dichloromethane (2.00 mL) at 0° C. under a nitrogen atmosphere, 3-chlorobenzene-1-carboperoxoic acid (199 mg, 1.5 equiv., 1.15 mmol) was added in lots and stirred at room temperature for 16 hours. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mass was filtered, extracted with dichloromethane, washed with sodium bicarbonate solution, followed by water and brine. The two layers were separated, and the combined organic layer was dried over sodium sulfate, filtered, and evaporated to give the crude product, which was purified by silica gel flash column chromatography. The product was eluted with 10% EtOAc and n-hexane (the product is UV inactive), and the fractions were collected and dried under vacuum to give 1aH,2H,7H,7aH-naphtho[2,3-b]oxirene (85.0 mg, 581 μmol) as an oily compound.

[0272] 1 HNMR (400 MHz, CDCl3): δ 7.14 (t, J = 3.2 Hz, 2H), 7.05 (t, J = 3.2 Hz, 2H), 3.48 (s, 2H), 3.32 (d, J = 17.6 Hz, 2H), 3.19 (d, J = 17.6 Hz, 2H).

[0273] Step 2: Synthesis of racemic [trans-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)sulfanyl](phenyl)methanone [ka]

[0274] To a stirred solution of 1aH,2H,7H,7aH-naphtho[2,3-b]oxirane (100 mg, 684 μmol) in ethoxyethane (4.00 mL) under a nitrogen atmosphere, aluminum oxide (1.00 g) (acidic) was added. The solution was cooled to 0 °C. Next, thiobenzoic acid (482 mg, 5.1 equiv., 3.49 mmol) was added to the reaction mixture, which was then stirred at room temperature for 24 h. After completion of the reaction (reaction progress was monitored by TLC), the reaction mixture was filtered and washed with sodium bicarbonate solution, followed by water and brine solution to obtain the crude product. The crude product was purified by silica gel flash column chromatography. The product was eluted with 20% EtOAc:n-hexane to give racemic [trans-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)sulfanyl](phenyl)methanone (125 mg, 440 μmol) as a colorless liquid.

[0275] 1 HNMR (400 MHz, DMSO): δ 7.89 (d, J = 7.2 Hz, 2H), 7.66 (t, 1H), 7.53 (d, J = 7.2 Hz, 2H), 7.09 (m, 4H), 5.39 (s, 1H), 4.00 (s, 2H), 3.42 (d, J = 17.6 Hz, 1H), 3.12 (t, J = 16 Hz, 1H), 2.81 (t, J = 18.4 Hz, 2H).

[0276] Step 3: Synthesis of racemic trans-3-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol [ka]

[0277] To a stirred solution of [(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)sulfanyl](phenyl)methanone (115 mg, 404 μmol) in methanol (3.00 mL), K2CO3 (113 mg, 2 equiv., 809 μmol) was added, and the reaction mixture was stirred at room temperature for 0.5 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mass was concentrated (to remove methanol) and then acidified with 1 N HCl solution until the pH reached 2-3 to give racemic trans-3-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol (70.0 mg, 388 μmol), which was used directly in the next step.

[0278] Step 4: Synthesis of trans-(2RS,3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol and trans-(2SR,3SR)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol [ka]

[0279] To a stirred solution of racemic trans-3-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol (350 mg, 1.94 mmol) in methanol (2.50 ml) under a nitrogen atmosphere, 2-(pyridin-2-yldisulfanyl)pyridine (428 mg, 1 equiv., 1.94 mmol) was added and stirred at room temperature for 16 hours. The reaction progress was monitored by TLC and LC-MS. After completion of the reaction, the reaction mass was concentrated, then diluted with DCM, washed with water, then brine, and dried over sodium sulfate. The resulting crude product was purified by silica gel flash column chromatography. The desired product was eluted with 20% EtOAc:hexane. The product was repurified by reverse-phase column chromatography (10-20% 0.1% formic acid in water / acetonitrile). Fractions containing the desired product were collected and evaporated in vacuo to give 3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (350 mg, 1.21 mmol) as a yellow solid. The isomers were separated by chiral preparative HPLC.

[0280] 1 HNMR (400 MHz, DMSO): δ 8.44 (d, J = 4.4 Hz, 1H), 7.79 (d, J = 3.2 Hz, 2H), 7.26 - 7.24 (m, 1H), 7.06 (s, 4H), 5.61 (s, 1H), 3.91 - 3.80 (m, 1H), 3.31 - 3.19 (m, 2H), 3.13 - 3.07 (m, 1H), 2.92 - 2.84 (m, 1H), 2.75 - 2.65 (m, 1H).

[0281] Fractionation conditions: Column: Chiralpak IA (250 mm x 20 mm x 5 mic) Mobile phase: n-hexane:ethanol containing 0.1% DEA (50:50) Flow rate: 19ml / min

[0282] The isomers were separated and each fraction was collected from the chiral prep. Combined HPLC and evaporation afforded the individual isomers. Isomer 1 was collected first and assigned as trans-(2RS,3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol. Isomer 2 was collected second and assigned as trans-(2SR,3SR)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol. The absolute stereochemistry of the isomers was arbitrarily assigned.

[0283] Step 5: Synthesis of 4-nitrophenyl (trans-(2RS,3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl) carbonate To a stirred solution of trans-(2RS,3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (150 mg, 518 μmol) in dimethylformamide (3.00 mL, 38.7 mmol), bis(4-nitrophenyl)carbonate (315 mg, 2 equiv., 1.04 mmol) was added, followed by N,N-diisopropylethylamine (271 μL, 3 equiv., 1.55 mmol). The reaction mixture was stirred at room temperature for 12 hours. After completion of the reaction, the reaction mass was quenched with water and extracted with DCM (3 × 5). The combined organic phases were dried over sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product. The crude product was purified by silica gel flash column chromatography (0–40% EtOAc:n-hexane) and repurified by reverse-phase column chromatography (10–50% 0.1% formic acid in water:ACN) to give (trans-(2RS,3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl)carbonate (133 mg, 293 μmol) as an off-white solid.

[0284] 1HNMR (400 MHz, DMSO): δ 8.44 (d, 1H), 8.30 (d, J = 9.2 Hz, 2H), 7.80 - 7.76 (m, 2H), 7.54 (d, J = 9.2 Hz, 2H), 7.26 - 7.24 (m, 1H), 7.14 - 7.06 (m, 4H), 5.21 - 5.19 (m, 1H), 3.78 - 3.77 (m, 1H), 3.45 - 3.25 (m, 2H), 3.10 - 3.01 (m, 2H).

[0285] Synthesis of 4-nitrophenyl (trans-(2SR,3SR)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl) carbonate [ka]

[0286] To a stirred solution of trans-(2SR,3SR)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (130 mg, 449 μmol) in dimethylformamide (2.60 mL, 33.6 mmol), bis(4-nitrophenyl)carbonate (273 mg, 2 equiv., 898 μmol) was added, followed by diisopropylethylamine (13.0 mL, 3 equiv., 74.6 mmol). The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction (reaction progress was monitored by TLC), the reaction mass was quenched with water and extracted with DCM (3 × 5). The combined organic phases were dried over sodium sulfate, filtered, and evaporated under reduced pressure to obtain the crude product. This crude product was purified by flash column chromatography (0–40% EtOAc:n-hexane). The product was repurified by reverse-phase column chromatography (10-50% 0.1% formic acid in water:ACN) to give 4-nitrophenyl (trans-(2SR,3SR)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl)carbonate (30.0 mg, 66.0 μmol) as an off-white solid.

[0287] 1 HNMR (400 MHz, DMSO): δ 8.44 (d, 1H), 8.30 (d, J = 9.2 Hz, 2H), 7.80 - 7.76 (m, 2H), 7.54 (d, J = 9.2 Hz, 2H), 7.26 - 7.24 (m, 1H), 7.14 - 7.06 (m, 4H), 5.21 - 5.19 (m, 1H), 3.78 - 3.77 (m, 1H), 3.45 - 3.25 (m, 2H), 3.10 - 3.01 (m, 2H).

[0288] Synthesis of 4-nitrophenyl (trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)oxan-3-yl) carbonate [ka]

[0289] Step 1: Synthesis of 3,7-dioxabicyclo[4.1.0]heptane [ka]

[0290] To a stirred solution of 3,6-dihydro-2H-pyran (2.0 g, 23.8 mmol) in dichloromethane (20.0 mL) at 0 °C, 3-chlorobenzene-1-carboperoxoic acid (4.92 g, 1.2 equiv., 28.5 mmol) was slowly added in portions and stirred at room temperature for 16 hours. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mass was quenched with saturated sodium bicarbonate solution, and the organic layer was separated, washed with water followed by brine solution, dried over anhydrous sodium sulfate, filtered, and evaporated to give the title compound dioxabicyclo[4.1.0]heptane (1.00 g, 9.99 mmol) as a colorless oil. 1H NMR (400 MHz, CDCl3): 4.03 - 3.94 (m, 2H), 3.55 - 3.49 (m, 1H), 3.46 - 3.41 (m, 1H), 3.35 (m, 1H), 3.18 (m, 1H), 2.00 (m, 2H).

[0291] Step 2: Synthesis of racemic [trans-(3-hydroxytetrahydropyran-4-yl)sulfanyl](phenyl)methanone [ka]

[0292] To a stirred solution of 3,7-dioxabicyclo[4.1.0]heptane (1.00 g, 9.99 mmol) in ethoxyethane (40 mL) at room temperature, benzenecarbothioic acid S-acid (5.88 mL, 5 equiv., 49.9 mmol) was added, followed by silanedione (3.00 g, 5 equiv., 49.9 mmol), and the reaction mixture was stirred at room temperature for 12 h. The reaction progress was monitored by TLC, and upon completion of the reaction of the starting material, the reaction mass was quenched with saturated sodium bicarbonate solution and then extracted with ethyl acetate (2 × 10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and then evaporated under reduced pressure to give the crude product, which was purified by flash column chromatography (0–30% EtOAc:n-hexane). The compound was eluted with 20% EtOAc:n-hexane. The pure fractions were collected and evaporated to give racemic [trans-(3-hydroxytetrahydropyran-4-yl)sulfanyl](phenyl)methanone (2.0 g, 8.39 mmol).

[0293] LC-MS m / z C 12 H 14 Calculated for O3S: 238.3, Found: 239.1 [M+H] + ; 1H-NMR (400 MHz, CDCl3): δ 7.43 (d, J = 7.6 Hz, 2H), 7.09 (t, J = 17.2 Hz, 1H), 6.93 (t, J = 8.0 Hz, 2H), 3.55 (dd, J = 4.0 Hz, 4.0 Hz, 1H), 3.37 (d, J = 11.2 Hz, 1H), 3.28 - 3.24 (m, 1H), 3.22 - 3.18 (m, 1H), 3.01 (t, J = 10.8 Hz, 1H), 2.83 (t, J = 12.4 Hz, 1H), 1.36 -1.27 (m, 1H), 1.27 (s, 2H).

[0294] Step 3: Synthesis of racemic trans-4-sulfaniloxan-3-ol [ka]

[0295] To a stirred solution of racemic [trans-3-hydroxytetrahydropyran-4-yl)sulfanyl](phenyl)methanone (2.50 g, 10.5 mmol) in dichloromethane (25 mL) at room temperature, hydrazine hydrate (5.15 mL, 10 equiv., 105 mmol) was slowly added and the reaction mixture was stirred for 1 h. The reaction progress was monitored by TLC, and immediately upon completion of the reaction, the reaction mass was quenched with 1 N HCl to adjust the pH to 2-3. The two layers were separated, and the organic layer was dried over sodium sulfate, filtered, and partially evaporated. The crude product, racemic trans-4-sulfanyltetrahydropyran-3-ol, was used in the next step.

[0296] Step 4: Synthesis of trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-ol and trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-ol [ka]

[0297] To a stirred solution of 2-(pyridin-2-yldisulfanyl)pyridine (1.85 g, 0.8 equiv., 8.41 mmol) in methanol (40 mL) was added racemic trans-4-sulfanyltetrahydropyran-3-ol (1.41 g, 10.5 mmol) in DCM at 0° C., and the reaction mixture was then stirred at room temperature for 12 hours. Upon completion of the reaction, the completed reaction mass was evaporated under reduced pressure to give the crude product, which was purified by flash column chromatography. The product was eluted with 20% EtOAc:n-hexane, and the pure fractions were collected and evaporated to give the title product, 4-(pyridin-2-yldisulfanyl)oxan-3-ol (racemic mixture). The isomers were separated by chiral preparative HPLC.

[0298] Chiral preparative HPLC conditions: Column: Chiralpak IA (250 mm x 20 mm x 5 mic) Mobile phase: n-hexane:IPA containing 0.1% DEA (90:10) Flow rate: 19ml / min The isomers were separated and each fraction was collected from the chiral preparative HPLC. The fractions were combined and evaporated to give each isomer.

[0299] (isomer 1-350mg, isomer 2-350mg) LC-MS m / z C 10 H 13 Calculated for NO2S2: 243.34, Found: 244 [M+H] + .

[0300] Isomer 1 (trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-ol): 1H-NMR (400 MHz, DMSO): δ 8.53 (s, 1H), 7.60 (t, J = 6.40 Hz, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.23 (t, J = 20.8 Hz, 1H), 4.28 - 4.06 (m, 1H), 3.94 (d, J = 12 Hz, 1H), 3.54 - 3.40 (m, 3H), 3.33 - 3.21 (m, 1H), 3.07 - 2.74 (m, 1H), 2.04 - 1.94 (m, 2H).

[0301] Isomer 2 (trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-ol) 1 H-NMR (400 MHz, DMSO): δ 8.52 (d, J = 2.8 Hz, 1H), 7.61 (t, J = 6.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.18 (t, J = 5.2 Hz, 1H), 4.12 - 4.09 (m, 1H), 3.94 (d, J = 12 Hz, 1H), 3.53 - 3.47 (m, 1H), 3.47 - 3.37 (m, 1H), 3.25 (t, J = 10.4 Hz, 1H), 2.80 - 2.73 (m, 1H), 1.96 -1.42 (m, 1H), 1.20 (d, J = 6.0 Hz, 2H).

[0302] The absolute stereochemistry of the isomers was arbitrarily assigned.

[0303] Step 5: Synthesis of 4-nitrophenyl (trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-yl) carbonate DMF To a stirred solution of (trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-ol) (300 mg, 1.23 mmol) in 8 mL of hexane was added bis(4-nitrophenyl)carbonate (750 mg, 2 equiv., 2.47 mmol), followed by diisopropylethylamine (644 μL, 3 equiv., 3.70 mmol) at room temperature for 12 h. Immediately after completion of the reaction, the reaction mass was partitioned between water and DCM. The organic layer was separated, washed with brine solution, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product, which was purified by flash column chromatography. The desired compound was eluted in 25% EtOAc:n-hexane as a mixture. The mixture was purified by reverse-phase column chromatography (10–60% 0.1% formic acid / ACN in water). Fractions containing the desired product were combined and evaporated to give 4-nitrophenyl (trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-yl) carbonate (270 mg, 0.66 mmol). LC-MS m / z C 17 H 16 Calculated for N2O6S2: 408.4, Found: 409.1 [M+H] + ; 1 H-NMR (400 MHz, CDCl3): δ 8.46 (d, 1H), 8.28 (d, J = 8.8 Hz, 2H), 7.64 - 7.52 (m, 2H), 7.41 (d, J = 8.8 Hz, 2H), 7.09 (s, 1H), 4.87 (d, J = 2.8 Hz, 1H), 4.25 - 4.18 (m, 1H), 3.91 (d, J = 11.6 Hz, 1H), 3.52 - 3.42 (m, 1H), 3.20 (d, J = 2.8 Hz, 1H), 2.21 (d, J = 12.4 Hz, 1H), 1.98 (d, J = 7.6 Hz, 1H), 1.25 (s, 1H).

[0304] Synthesis of 4-nitrophenyl (trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-yl) carbonate [ka]

[0305] To a stirred solution of (trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-ol) (340 mg, 1.40 mmol) in DMF (8 mL), bis(4-nitrophenyl)carbonate (850 mg, 2 equiv., 2.79 mmol) was added, followed by diisopropylethylamine (730 μL, 3 equiv., 4.19 mmol) at room temperature for 12 h. Immediately after completion of the reaction of the starting material, the reaction mixture was partitioned between water and DCM. The organic layer was separated, washed with brine solution, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product, which was purified by flash column chromatography (0–40% EtOAc:n-hexane). The desired product eluted as a mixture and was then repurified by reverse-phase column chromatography (10–50% 0.1% formic acid / ACN in water). Fractions containing the desired product were combined and evaporated to give 4-nitrophenyl (trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-yl) carbonate (300 mg, 735 μmol). LC-MS m / z C 17 H 16 Calculated for N2O6S2: 408.4, Found: 409.1 [M+H] + ; 1 H-NMR (400 MHz, DMSO): δ 8.46 (d, 1H), 8.28 (d, J = 8.0 Hz, 2H), 7.66 - 7.58 (m, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 4.87 (d, J = 3.6 Hz, 1H), 4.25 - 4.22 (m, 1H), 3.91 (d, J = 11.6 Hz, 1H), 3.52 - 3.42 (m, 2H), 3.20 (d, J = 3.6 Hz, 1H), 2.21 (d, J = 12.0 Hz, 1H), 1.98 - 1.95 (m, 1H).

[0306] Synthesis of 4-nitrophenyl(trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cycloheptyl) carbonate [ka]

[0307] Step 1: Synthesis of 8-oxabicyclo[5.1.0]octane [ka]

[0308] To a stirred solution of cycloheptene (1.0 g, 10.4 mmol) in dichloromethane (10 mL) at 0 °C was added 3-chlorobenzene-1-carboperoxoic acid (2.15 g, 1.2 equiv., 12.5 mmol). The reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 16 h. The reaction progress was monitored by TLC. Upon completion of the reaction, the reaction mixture was slowly quenched with saturated aqueous sodium bicarbonate solution and the mixture was vigorously stirred for approximately 30 min. The two layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give the desired product as a colorless liquid (700 mg, 6.24 mmol). 1 H-NMR (400 MHz, CDCl3): δ 3.07 (s, 2H), 1.93 - 1.86 (m, 4H), 1.60 - 1.43 (m, 4H), 1.21 - 1.17(m, 2H).

[0309] Step 2: Synthesis of racemic [trans-(2-hydroxycycloheptyl)sulfanyl](phenyl)methanone [ka]

[0310] To a stirred solution of 8-oxabicyclo[5.1.0]octane (3.00 g, 26.7 mmol) in toluene (60 mL) under a nitrogen atmosphere at room temperature, benzenecarbothioic acid S-acid (4.72 mL, 1.5 equiv., 40.1 mmol) was added, followed by 2-methylpropan-2-aminium chloride (293 mg, 0.1 equiv., 2.67 mmol). The reaction mixture was stirred at 50 °C for 16 h (reaction progress was monitored by TLC). Upon completion of the reaction, the reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product, which was purified by flash column chromatography. The desired product was eluted with 20% EtOAc:n-hexane, and the pure fractions were collected and evaporated to give the title compound racemic [trans-(2-hydroxycycloheptyl)sulfanyl](phenyl)methanone (3.0 g, 12.0 mmol). LC-MS m / z C 14 H 18 Calculated value for O2S: 250.4, 1 H-NMR (400 MHz, CDCl3): δ 7.96 (d, J = 8.0 Hz, 2H), 7.57 (t, J = 6.8 Hz, 1H), 7.4 (t, J = 7.6 Hz, 2H), 3.88 - 3.85 (m, 1H), 3.81 - 3.77 (m, 1H), 2.09 - 2.05 (m, 1H), 2.04 - 1.62 (m, 8H), 1.55 - 1.53 (m, 2H).

[0311] Step 3: Synthesis of racemic trans-4-sulfanylcycloheptan-3-ol [ka]

[0312] Racemic [trans-(2-hydroxycycloheptyl)sulfanyl](phenyl)methanone (2.80 g, 11.2 mmol) in dichloromethane (25 mL) was added at room temperature under a nitrogen atmosphere to 1,4-disulfanylbutane-2,3-diol (173 mg, 0.1 equiv., 1.12 mmol), followed by hydrazine hydrate (1.37 mL, 2.5 equiv., 28.0 mmol). The reaction mixture was stirred at room temperature for 3 hours (reaction progress was monitored by TLC). Upon completion of the reaction, the reaction mixture was quenched with 1N HCl and extracted with DCM (2 × 30 mL). The organic layers were combined, dried over sodium sulfate, filtered, and the organic layer was partially evaporated. The crude racemic trans-4-sulfanylcycloheptan-3-ol was used directly in the next step.

[0313] Step 4: Synthesis of trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol and trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol [ka]

[0314] To a stirred solution of 2-(pyridin-2-yldisulfanyl)pyridine (1.73 g, 0.7 equiv., 7.85 mmol) in methanol (25 mL) at 0 °C under a nitrogen atmosphere, racemic trans-4-sulfanylcycloheptan-3-ol (1.64 g, 11.2 mmol) in DCM was added, and the reaction mixture was stirred at room temperature for 12 h. The reaction progress was monitored by TLC and LCMS, and the reaction mass was evaporated under reduced pressure. The crude product was purified by flash column chromatography, and the desired product was eluted with 20% EtOAc:n-hexane. Once the product was collected as a mixture, it was repurified by reverse-phase column chromatography (10–50% 0.1% formic acid in water:acetonitrile) to give racemic trans-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol (1.5 g, 52%) (racemic mixture). The isomers were separated by chiral preparative HPLC.

[0315] (Isomer-1: 550mg, Isomer-2: 550mg).

[0316] Chiral preparative HPLC conditions: Column: Chiralpak IA (250 mm x 20 mm x 5 mic) Mobile phase: n-hexane:IPA containing 0.1% DEA (90:10) Flow rate: 19ml / min The isomers were separated and each fraction was collected from chiral preparative HPLC. The fractions were evaporated separately to give each isomer.

[0317] Isomer 1 (trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol): LC-MS m / z C 12 H 17 Calculated for NOS2: 255.4, Found: 256.2 [M+H] + ; 1 H-NMR (400 MHz, CDCl3): δ 8.49 (s, 1H), 7.56 (d, J = 6.8 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 6.17 (s, 1H), 3.51 (m, 1H), 2.75 - 2.73 (m, 1H), 2.08 - 1.95 (m, 2H), 1.82 - 1.67 (m, 4H), 1.57 - 1.25 (m, 4H).

[0318] Isomer 2 (trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol): LC-MS m / z C 12 H 17 Calculated for NOS2: 255.4, Found: 256.2 [M+H] + ; 1H-NMR (400 MHz, CDCl3): δ 8.50 (d, J = 4.40 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 8.40 Hz, 1H), 7.13 (t, J = 6.4 Hz, 1H), 6.18 (s, 1H), 3.53 - 3.49 (m, 1H), 2.77 - 2.72 (m, 1H), 2.11 - 2.08 (m, 1H), 2.00 - 1.96 (m, 1H), 1.84 - 1.67 (m, 4H), 1.59 - 1.45 (m, 4H).

[0319] The absolute stereochemistry of the isomers was arbitrarily assigned.

[0320] Step 5: Synthesis of 4-nitrophenyl (trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cycloheptyl) carbonate To a stirred solution of trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol (500 mg, 1.96 mmol) in DMF (10 mL) under a nitrogen atmosphere, bis(4-nitrophenyl)carbonate (1.49 g, 2.5 equiv., 4.89 mmol) was added, followed by diisopropylethylamine (1.02 mL, 3 equiv., 5.87 mmol) at room temperature. The reaction mixture was stirred for 12 h. Upon completion of the reaction, the reaction mixture was partitioned between water and DCM. The two layers were separated, and the organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product, which was purified by flash column chromatography. The desired product was eluted with a 23% EtOAc:hexane mixture. The mixture was re-purified by reverse-phase column chromatography (10-60% 0.1% formic acid / ACN in water) to give the title product 4-nitrophenyl(trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cycloheptyl)carbonate (450 mg, 1.07 mmol). LC-MS m / z C 19 H 20Calculated for N2O5S2: 420.5, Found: 421.3 [M+H] + ; 1 H-NMR (400 MHz, CDCl3): δ 8.45 (s, 1H), 8.27 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 7.6 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.09 (m, 1H), 5.04 - 5.03 (m, 1H), 3.22 (m, 1H), 2.15 - 2.00 (m, 3H), 1.87 - 1.79 (m, 2H), 1.72 - 1.63 (m, 4H), 1.54 - 1.49 (m, 2H).

[0321] Synthesis of 4-nitrophenyl(trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cycloheptyl) carbonate [ka]

[0322] To a stirred solution of trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol (580 mg, 2.27 mmol) in DMF (10 mL) under a nitrogen atmosphere, bis(4-nitrophenyl)carbonate (1.73 g, 2.5 equiv., 5.68 mmol) was added, followed by diisopropylethylamine (1.38 mL, 3.5 equiv., 7.95 mmol). The reaction mixture was stirred at room temperature for 12 h. Upon completion of the reaction, as monitored by TLC, the reaction mixture was partitioned between water and DCM. The two layers were separated, and the combined organic layer was washed with brine solution, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude product was purified by flash column chromatography. The desired product was eluted with a 23-25% EtOAc:n-hexane mixture. The product was re-purified by reverse-phase column chromatography (10-60% 0.1% formic acid / ACN in water) to give the title compound 4-nitrophenyl(trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cycloheptyl)carbonate (450 mg, 1.07 mmol). LC-MS m / z C 19 H 20 Calculated for N2O5S2: 420.5, Found: 421.3 [M+H] + ;1H-NMR (400 MHz, CDCl3): δ 8.46 (s, 1H), 8.27 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.12 (m, 1H), 5.04 - 5.03 (m, 1H), 3.23 (m, 1H), 2.12 - 2.00 (m, 2H), 1.87 - 1.79 (m, 3H),1.63 - 1.49 (m, 6H).

[0323] Synthesis of 4-nitrophenyl (trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl) carbonate [ka]

[0324] Step 1: Synthesis of 1aH,2H,3H,7bH-naphtho[1,2-b]oxirene [ka]

[0325] To a stirred solution of 1,2-dihydronaphthalene (2.0 g, 15.4 mmol) in dichloromethane (75 mL) was added a saturated solution of sodium bicarbonate (75 mL). The mixture was then cooled to 0 °C. To this mixture was added 3-chlorobenzene-1-carboperoxoic acid (5.30 g, 2 equivalents, 30.7 mmol) in portions over 30 minutes. After the addition, the reaction mass was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the two layers were separated, and the organic layer was dried over sodium sulfate and concentrated under reduced pressure to give 1aH,2H,3H,7bH-naphtho[1,2-b]oxirene (2.77 g). The crude product was used in the next step without further purification.

[0326] Step 2: Synthesis of racemic [trans-(2-hydroxy-1,2,3,4-tetrahydronaphthalen-1-yl)sulfanyl](phenyl)methanone [ka]

[0327] To a stirred solution of 1aH,2H,3H,7bH-naphtho[1,2-b]oxirene (2.25 g, 15.4 mmol) in ethoxyethane (20 mL) was added dropwise silanedione (4.50 g, 74.9 mmol) and benzenecarbothioic acid S-acid (9.06 mL, 5 equiv., 77.0 mmol). The mixture was stirred at room temperature for 16 h. The reaction progress was monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with saturated sodium carbonate solution (25 mL) and extracted with ethyl acetate (2 × 20 mL). The combined organic layers were washed with water, brine, dried over sodium sulfate and concentrated under reduced pressure to give the crude product, which was purified by column chromatography to give racemic trans-(2-hydroxy-1,2,3,4-tetrahydronaphthalen-1-yl)sulfanyl](phenyl)methanone (1.57 mg, 35.87%) as a yellow liquid.

[0328] Step 3: Synthesis of racemic trans-1-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol [ka]

[0329] To a stirred solution of racemic trans-[(2-hydroxy-1,2,3,4-tetrahydronaphthalen-1-yl)sulfanyl](phenyl)methanone (1.40 g, 4.92 mmol) in dichloromethane (25.0 mL) was added (2R,3R)-1,4-disulfanylbutane-2,3-diol (144 mg, 0.19 equiv., 935 μmol) and hydrazine hydrate (60.4 μL, 0.25 equiv., 1.23 mmol). The reaction mass was stirred at room temperature for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched with HCl solution (pH = 1-2). The DCM layer was separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give racemic trans-1-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol, which was used in the next step.

[0330] Step 4: Synthesis of trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol and trans-(1SR,2SR)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol [ka]

[0331] To a stirred solution of 2-(pyridin-2-yldisulfanyl)pyridine (867 mg, 0.8 equiv., 3.94 mmol) in methanol (5 mL) at 0° C., dropwise addition of racemic trans-1-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol in DCM obtained in the previous step was performed. The reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by LCMS and TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure to obtain the crude product. This crude product was purified by column chromatography to obtain racemic 1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol as a yellow oil, which was further purified by reverse-phase column chromatography to obtain a colorless oil (380 mg, 26.69%). The resulting racemic product was separated by chiral chromatography to obtain Isomer-1: 130 mg and Isomer-2: 190 mg.

[0332] Fractionation conditions: Column: Chiralpak IA (250 mm x 20 mm x 5 mic) Mobile phase: n-hexane:ethanol containing 0.1% DEA (50:50) Flow rate: 19ml / min Isomer-1 (trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol): LC-MS m / z C 15 H 15 Calculated for NOS2: 289.4, Found: 290.1 [M+H] +;1H-NMR (400 MHz, CDCl3): δ 8.07(d, J = 7.2 Hz, 2H), 7.61 - 7.57(m, 1H), 7.48 - 7.44 (m, 2H), 7.36 - 7.34 (m, 1H), 7.18-7.13 (m, 3H), 4.98 (d, J=4.4 Hz, 1H), 4.24 (m, 1H), 3.07-2.99 (m, 1H), 2.91-2.80 (m, 1H).

[0333] Isomer-2 (trans-(1SR,2SR)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol) LC-MS m / z C 15 H 15 Calculated for NOS2: 289.4, Found: 290.1 [M+H] + ;1H-NMR (400 MHz, CDCl3): δ 8.55 (d, J=4.0 Hz, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.58 (t, J=7.6 Hz, 1H), 7.37(d, J=8.4 Hz, 1H), 7.26-7.17 (m, 3H), 7.08 (d, J=7.2 Hz, 1H), 4.15 (d, J=8.0 Hz, 1H), 3.97-3.93 (m, 1H), 2.89 (d, J=4.8 Hz, 2H), 2.32-2.28 (m, 1H), 1.97-1.87 (m, 2H).

[0334] The absolute stereochemistry of the isomers was arbitrarily assigned.

[0335] Step 5: Synthesis of 4-nitrophenyl (trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl) carbonate To a stirred solution of trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (170 mg, 587 μmol) in N,N-dimethylformamide (2.50 mL), bis(4-nitrophenyl)carbonate (447 mg, 2.5 equiv., 1.47 mmol) was added dropwise at room temperature, followed by diisopropylethylamine (307 μL, 3 equiv., 1.76 mmol). The reaction mixture was stirred in a sealed tube at room temperature for 12 hours. The reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mass was partitioned between water (5 mL) and DCM (5 mL). The organic layer was dried over sodium sulfate and evaporated under reduced pressure to give the crude product, which was purified by flash column chromatography (0-40% EA in hexanes) and repurified by reverse-phase column chromatography (10-70% 0.1% formic acid / ACN in water) to give 4-nitrophenyl (trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl) carbonate (70.0 mg, 154 μmol) as a colorless sticky solid (70 mg, 26.22%). LC-MS m / z C 22 H 18 Calculated for N2O4S2: 454.5 Found: 455.3 [M+H] + ;1H-NMR (400 MHz, CDCl3): δ 8.73(d, J=20.4 Hz, 1H), 8.22(d, J=8.4 Hz, 2H), 7.67(s, 2H), 7.50(m, 1H), 7.32(d, J=8.4 Hz, 2H), 7.25-7.16(m, 4H), 5.51(s, 1H), 4.52(s, 1H), 3.01-2.85(m, 2H), 2.63(m, 1H), 2.26-2.22(m, 1H).

[0336] Synthesis of 4-nitrophenyl (trans-(1SR,2SR)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl) carbonate [ka]

[0337] To a stirred solution of trans-(1SR,2SR)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (120 mg, 415 μmol) in N,N-dimethylformamide (1.50 mL) was added bis(4-nitrophenyl)carbonate (315 mg, 2.5 equiv., 1.04 mmol) followed by diisopropylethylamine (217 μL, 3 equiv., 1.24 mmol) dropwise at room temperature. The reaction mixture was stirred in a sealed tube at room temperature for 12 hours. The progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mass was partitioned between water (5 mL) and DCM (5 mL), the organic layer was dried over sodium sulfate and evaporated under reduced pressure to give the crude product, which was purified by flash column chromatography (0-40% EA in hexanes) and re-purified by reverse phase column chromatography (10-70% 0.1% formic acid / ACN in water) to give 4-nitrophenyl (trans-(1SR,2SR)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl)carbonate (65.0 mg, 143 μmol)) as a colorless sticky solid (65 mg, 34.49%).

[0338] LC-MS m / z C 22 H 18 Calculated for N2O4S2: 454.5 Found: 455.3 [M+H] + ;1H-NMR (400 MHz, CDCl3): δ 8.55(m, 1H), 8.22(d, J=7.6 Hz, 2H), 7.69(s, 2H), 7.51(m, 1H), 7.32(d, J=7.6 Hz, 2H), 7.25-7.16(m, 4H), 5.51(s, 1H), 4.52(s, 1H), 3.01-2.86(m, 2H), 2.62(m, 1H), 2.26(m, 1H).

[0339] Synthesis of 4-nitrophenyl(trans-4-(pyridin-2-yldisulfanyl)cyclohexyl) carbonate [ka]

[0340] Step 1: Synthesis of trans-4-mercaptocyclohexan-1-ol [ka]

[0341] To a stirred solution of 7-oxabicyclo[2.2.1]heptane (1.00 g, 10.2 mmol) in ethanol (10 mL), 4-methylbenzene-1-sulfonic acid (2.63 g, 1.5 eq, 15.3 mmol), thiourea (1.16 g, 1.5 eq, 15.3 mmol) were added and the reaction mass was heated to 80°C for 24 hours. Then, the reaction mass was cooled to room temperature, and 50% aqueous sodium hydroxide solution (1.30 g, 3.2 eq, 32.6 mmol) was added to the reaction mass and heated at 100°C for 2 hours. After completion of the reaction, the reaction mass was cooled to room temperature, concentrated under reduced pressure, and acidified with 10% HSO solution. The reaction mass was then extracted with DCM and used directly in the next step.

[0342] Step 2: Synthesis of trans-4-(pyridin-2-yldisulfanyl)cyclohexan-1-ol [ka]

[0343] To a stirred solution of 2-(pyridin-2-yldisulfanyl)pyridine (1.60 g, 0.8 eq, 7.26 mmol) in methanol (10.0 mL) at 0 °C, the organic layer from (Step 1) 4-sulfanylcyclohexan-1-ol (1.20 g, 9.08 mmol) was added. After completion of the addition, the reaction mass was stirred at room temperature for 16 hours. After completion of the reaction, the reaction mass was concentrated, and the crude product was purified by column chromatography (using 0-40% EtOAc:n-hexane) to give the desired product. The product was repurified by reverse-phase column chromatography using 0.1% formic acid and ACN. The fractions containing the desired product were collected and concentrated under reduced pressure to give the title product as a yellow oil (1.60 g, 73% yield). LC-MS m / z C 11 H 15 Calculated for NOS2: 241; Found: 242 [M+H]+.

[0344] Step 3: Synthesis of 4-nitrophenyl (trans-4-(pyridin-2-yldisulfanyl)cyclohexyl) carbonate Under a nitrogen atmosphere, to a stirred solution of trans-4-(pyridin-2-yldisulfanyl)cyclohexan-1-ol (400 mg, 1.66 mmol) in N,N-dimethylformamide (3 mL), bis(4-nitrophenyl)carbonate (907 mg, 1.8 equiv., 2.98 mmol) and ethyl bis(propan-2-yl)amine (892 μL, 3 equiv., 4.97 mmol) were added and stirred at room temperature for 16 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mass was quenched with water (15 mL) and extracted with DCM (3 × 10 mL). The two layers were separated, and the combined organic layer was washed with water followed by brine solution, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography (0–30% EtOAc:n-hexane). The product was repurified by reverse-phase column chromatography using 0.1% formic acid and ACN. Fractions containing the desired product were collected and concentrated under reduced pressure to give 4-nitrophenyl (trans-4-(pyridin-2-yldisulfanyl)cyclohexyl) carbonate as a yellow oil (0.3 g, 73% yield). LC-MS m / z C 18 H 18 Calculated for N2O5S2: 407; Found: 407 [M+H] +; 1 HNMR (400 MHz, CDCl3): δ 8.49 - 8.42 (m, 1H), 8.26 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.0 Hz, 1H), 7.65 - 7.60 (m, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.12 - 7.05 (m, 1H), 4.75 - 4.65 (m, 1H), 2.98 - 2.87 (m, 1H), 2.28 - 2.18 (m, 4H), 1.68 - 1.50 (m, 4H).

[0345] Synthesis of (2R)-3-methyl-2-(pyridin-2-yldisulfanyl)butyl 4-nitrophenyl carbonate [ka]

[0346] Step 1: Synthesis of cesium benzoylsulfanide [ka]

[0347] To a stirred solution of benzenecarbothioic acid S-acid (5.00 g, 36.2 mmol) in methanol (40.0 mL) under a nitrogen atmosphere, cesium carbonate (7.72 g, 1.1 equiv., 39.8 mmol) was added portionwise over 10–15 min. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (as determined by TLC), the reaction mixture was concentrated under reduced pressure. The solid residue was diluted with 10 mL of acetone, and the white solid (CsHCO3) was filtered off. This process was repeated twice to ensure all CsHCO3 was removed. The acetone was then concentrated to afford cesium benzoylsulfanide (9.50 g, 35.2 mmol) as a colorless solid. 1 HNMR (400 MHz, CD3OD): δ 8.08 (d, J = 6.8 Hz, 2H), 7.37 - 7.27 (m, 3H).

[0348] Step 2: Synthesis of (2R)-2-(benzoylsulfanyl)-3-methylbutanoic acid [ka]

[0349] To a stirred solution of (2S)-2-bromo-3-methylbutanoic acid (2.00 g, 11.0 mmol) in N,N-dimethylformamide (14.0 mL) was added cesium benzoylsulfanide (2.98 g, 11.0 mmol). The reaction mixture was stirred at room temperature for 20 hours. The progress of the reaction was monitored by TLC. Upon completion of the reaction, the reaction mixture was diluted with diethyl ether (3 × 15 mL) and washed with water (3 × 15 mL). The ether layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was recrystallized from n-hexane to give (2R)-2-(benzoylsulfanyl)-3-methylbutanoic acid (2.50 g, 10.5 mmol) as an oily compound. 1 HNMR (400 MHz, DMSO-d6): δ 12.93 (s, 1H), 7.92 (d, J = 7.2 Hz, 2H), 7.69 (t, J = 7.6 Hz, 1H), 7.55 (t, J = 7.2 Hz, 2H), 4.14 (d, J = 6.8 Hz, 1H), 2.30 - 2.22 (m, 1H), 1.01 - 0.89 (m, 6H).

[0350] Step 3: Synthesis of (2R)-3-methyl-2-sulfanylbutan-1-ol [ka]

[0351] To a stirred solution of (2R)-2-(benzoylsulfanyl)-3-methylbutanoic acid (2.50 g, 10.5 mmol) in ethoxyethane (50.0 mL) at 0 °C, lithium aluminum hydride (52.5 mL, 5 equiv., 52.5 mmol) was added dropwise under a nitrogen atmosphere. After the addition was complete, the ice bath was removed, and the reaction mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After completion of the reaction of the starting material, the reaction mixture was cooled in an ice bath and quenched with 1.0 N HCl (30 mL) at 0 °C. The reaction mixture was extracted with DCM (20 mL), and the remaining gel-like material from the LAH reduction was washed with diethyl ether (10 mL). The combined organic layers were dried over sodium sulfate, filtered, and used further in the next step.

[0352] Step 4: Synthesis of (2R)-3-methyl-2-(pyridin-2-yldisulfanyl)butan-1-ol [ka]

[0353] To a stirred solution of (2R)-3-methyl-2-sulfanylbutan-1-ol (1.20 g, 9.98 mmol) in MeOH (5 mL) was added 2-(pyridin-2-yldisulfanyl)pyridine (1.76 g, 0.8 equiv., 7.99 mmol) under nitrogen atmosphere and stirred at room temperature for 16 h. The reaction progress was monitored by TLC and LC-MS. After completion of the reaction, the reaction mass was concentrated and extracted with DCM. The two layers were separated, and the combined organic layer was washed with water followed by brine, dried over sodium sulfate, filtered, and evaporated. The crude product was purified by silica gel flash column chromatography (using a 12 g column) eluted with 50% EtOAc:n-hexane and repurified by reverse-phase column chromatography (10–20% 0.1% formic acid in water / acetonitrile). The product-containing fractions were collected and evaporated under vacuum to give the title product. The product was repurified by preparative HPLC.

[0354] Preparative HPLC conditions: Column: X-Bridge C-18 (250 mm x 4.6 mm x 5 mic) Mobile phase (A): 0.1% ammonia in water Mobile phase (B): Acetonitrile Flow rate: 19ml / min Gradient B: 0 / 10, 12 / 60, 22 / 95, 25 / 95, 27 / 10, 30 / 10 The fractions collected from the preparative HPLC were combined and evaporated to give the title product 3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (350 mg, 1.21 mmol) as a yellow solid. 1 HNMR (400 MHz, CDCl3): δ 8.49 (d, J = 4 Hz,1H), 7.55 - 7.54 (m, 1H), 7.36 (d, J = 8.4 Hz,1H), 7.13 (t, J = 6.4Hz, 1H), 3.82 (dd, J = 12.4 Hz, 1H), 3.66 - 3.60 (m, 1H), 2.75 - 2.70 (m, 1H), 2.01 - 1.92 (m, 1H), 1.10 - 1.01 (m, 7H).

[0355] Step 5: Synthesis of (2R)-3-methyl-2-(pyridin-2-yldisulfanyl)butyl 4-nitrophenyl carbonate To a stirred solution of (2R)-3-methyl-2-(pyridin-2-yldisulfanyl)butan-1-ol (800 mg, 3.49 mmol) in N,N-dimethylformamide (2.50 mL) was added bis(4-nitrophenyl)carbonate (2.12 g, 2 equiv., 6.98 mmol), followed by diisopropylethylamine (1.82 mL, 3 equiv., 10.5 mmol) at room temperature. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the reaction mass was partitioned between water and DCM. The two layers were separated, and the organic layer was dried over sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product, which was purified by flash column chromatography (0-40% EtOAc:n-hexane). The product was repurified by reverse-phase chromatography (10–70% 0.1% formic acid / ACN in water) to give the title product (2R)-3-methyl-2-(pyridin-2-yldisulfanyl)butyl 4-nitrophenyl carbonate (600 mg, 1.52 mmol) as a colorless gum. 1 HNMR (400 MHz, CDCl3): δ 8.45 (d, J = 4.0 Hz, 1H), 8.26 (d, J = 9.2 Hz, 2H), 7.72 (d, J = 8.4 Hz,1H), 7.63 (t, J = 7.2 Hz, 1H), 7.35 (d, J = 9.2 Hz, 2H), 7.08 (t, J = 6.8 Hz, 1H), 4.59 - 4.48 (m, 2H), 3.08 (q, J = 6.0 Hz, 1H), 2.21 - 2.13 (m, 1H), 1.14 - 1.06 (m, 6H).

[0356] Following the same procedure from step 2, (2S)-3-methyl-2-(pyridin-2-yldisulfanyl)butyl 4-nitrophenyl carbonate was synthesized using (2R)-2-bromo-3-methylbutanoic acid.

[0357] Synthesis of the compound of Example 2 from intermediate III-2 [ka]

[0358] To a vial containing Pv2 (25.0 mg, 0.061 mmol; as a free-flowing solid) and [(2S)-2-(2-pyridyldisulfanyl)propyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (6.03 mg, 0.091 mmol), 1 mL of CH3CN and 0.5 mL of water were added. N-methylmorpholine (22.7 mg, 0.224 mmol) was added. The mixture was stirred at room temperature overnight. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20–85% CH3CN / H2O + 0.05% TFA, 15 min) to give the desired product (13.0 mg, yield: 47.0%).

[0359] The compounds of Examples 1 and 3 to 9 (see Table 4 below) were synthesized in the same manner as the compound of Example 2 from intermediates III-1 and III-3 to III-9, respectively.

[0360] Synthesis of Compound of Example 10 from Intermediate XVI-1 [ka]

[0361] The DMF and PBS were degassed for 30 min using a stream of N. In a separate vial, Pv2 (25.0 mg, 0.061 mmol; as a free-flowing solid), [4-(2-pyridyldisulfanyl)phenyl]methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (6.5 mg, 0.09 mmol), 1.5 mL of DMF, and 0.5 mL of PBS were added. To this was added CH3CO2H (0.0347 mL, 0.606 mmol). The mixture was stirred at room temperature overnight. LC-MS indicated a complete reaction. The reaction mixture was purified by reverse-phase HPLC (PrepSlope_4 min, 30–100% CH3CN / H2O + 0.05% TFA, 18 min) to give the desired product (3.0 mg, yield: 10.7%).

[0362] The compounds of the present invention and analytical data are shown below. [Table 10-1] [Table 10-2] [Table 10-3]

[0363] Example 11: Synthesis of Compound 11 [ka]

[0364] Step 1: Synthesis of 2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol [ka]

[0365] To a solution of 1,2-di(pyridin-2-yl)disulfane (15.2 g, 68.9 mmol) in MeOH (degassed with N) (30 mL) was added (1-mercaptocyclobutyl)methanol (11.4 g, 86.2 mmol) (degassed with N) dropwise and stirred at room temperature under a N atmosphere for 16 hours. The reaction mixture was concentrated to dryness in vacuo. The resulting crude material was purified by column chromatography using 30% EtOAc / hexane to give the title compound as a yellow liquid. 1 HNMR (400 MHz, CDCl3): δ 8.54-8.53 (m, 1H), 7.60-7.56 (m, 1H), 7.40-7.38 (m, 1H), 7.17-7.14 (m, 1H), 3.38-3.34 (m, 1H), 2.62-2.57 (m, 1H), 2.11-2.02 (m, 1H), 1.75-1.74 (m, 2H), 1.61-1.60 (m, 1H), 1.42-1.24 (m, 4H).

[0366] The title compound was subjected to chiral preparative HPLC conditions (Chiralpak IG: 250 mm x 20 mm x 5 mm; n-hexane:IPA with 0.1% diethylamine (80:20); 19 mL / min; 25 °C (room temperature). (1R,2R)-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol (4.5 g, 18.6 mmol) eluted first (retention time: 3.9 min), followed by (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol (retention time: 11.3 min). The absolute stereochemistry was confirmed by comparing the product of Step 2 with chiral material with the reported absolute stereochemistry (see Monaco, M.R.; J. Am. Chem. Soc. 2014, 136, 49, 16982-16985).

[0367] Step 2: Synthesis of 4-nitrophenyl ((1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl) carbonate [ka]

[0368] To a solution of (1R,2R)-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol (4.5 g, 18.6 mmol) in DMF (90.0 mL) was added DIPEA (10.3 mL, 56.0 mmol) and bis(4-nitrophenyl)carbonate (11.35 g, 27.3 mmol) at room temperature. The reaction vessel was sealed and stirred at room temperature for 12 h. The reaction progress was monitored by TLC (20% EtOAc / hexane). After completion of the reaction, the reaction mixture was quenched with water (20.0 mL) and extracted with EtOAc (20.0 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by column chromatography using 20–30% EtOAc / hexane to give the title product as an off-white solid (5.0 g, 66% yield). 1 HNMR (400 MHz, CDCl3): δ 8.44 (d, J = 4 Hz, 1H), 8.28 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.61-7.57 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 9.6 Hz, 2H), 7.08-7.05 (t, J = 5.2 Hz, 1H), 4.85-4.74 (m, 1H), 3.03-2.92 (m, 1H), 2.28 (d, J = 9.6 Hz, 1H), 2.20-2.12 (m, 1H), 1.85-1.62 (m, 3H), 1.45-1.25 (m, 3H). LC-MS calculated for m / z: 406.7; found: 407.4 [M+H] + .

[0369] Step 3: Synthesis of [(1S,2S)-2-(2-pyridyldisulfanyl)cyclohexyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate [ka]

[0370] To (10S,23S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19-methyl-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptene-5,9-dione methanesulfonic acid (250 mg, 0.470 mmol) in 10 mL of dry DMF was added (1R,2R)-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol (from step 2; 191 mg, 0.470 mmol), N,N-diisopropylethylamine (122 mg, 0.941 mmol), and DMAP (115 mg, 0.941 mmol). The mixture was stirred at room temperature overnight. LC-MS showed that the desired coupling product had formed. The reaction mixture was then diluted with EtOAc and washed with saturated aqueous NH4Cl, HO, and brine. The mixture was dried over sodium sulfate, filtered, and concentrated. The crude residue was purified by column chromatography using 0-5% MeOH / dichloromethane to give 240 mg of the desired product in 72.6% yield (240 mg).

[0371] Step 4: Coupling with Pv1 (compound 11) To a vial was added Pv1 (275 mg, 0.0811 mmol), the compound from Step 3 (74.1 mg, 0.105 mmol), acetonitrile (10 mL), and water (5 mL), and n-methylmorpholine (0.303 g, 0.0030 mol) was added to the mixture. The mixture was stirred at room temperature overnight. LC-MS showed that the desired coupling product had formed.

[0372] The reaction mixture was directly purified by reverse-phase HPLC (20-85% acetonitrile / water, 0.5% acetic acid, retention time: 7.022 min on a Sunfire Prep C18 column (10 μm, 50 × 150 mm)) to give the desired product in 68% yield (213 mg). ESI (M+3H / 3) 3+ : 1291.6

[0373] Example 12: Synthesis of Compound 12 [ka]

[0374] Step 1: Synthesis of [(1R,2R)-1-methyl-2-(2-pyridyldisulfanyl)propyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate 1-Hydroxybenzotriazole hydrate (8.64 mg, 0.0564 mmol), finely ground molecular sieves 4 Å (50 mg), (10S,23S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19-methyl-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaethene in 2 mL of anhydrous DMF To a mixture of 4-nitrophenyl-5,9-dione, methanesulfonic acid (25.0 mg, 0.0470 mmol), and pyridine (0.0190 mL, 0.235 mmol) was added [(1R,2R)-1-methyl-2-(2-pyridyldisulfanyl)propyl](4-nitrophenyl)carbonate (19.7 mg, 0.470 mmol) (see II-4: Synthesis of 4-nitrophenyl((2R,3R)-3-(pyridin-2-yldisulfanyl)butan-2-yl)carbonate). The mixture was stirred at room temperature for 16 hours, then filtered, and the solution was concentrated.

[0375] The residue was then purified by column chromatography (0-5% MeOH / DCM) to give the title compound (35.0 mg, 0.0517 mmol, yield: 110%).

[0376] Step 2: Coupling with peptide Pv1 (compound 12) A vial was charged with peptide Pv1 (50.0 mg, 14.7 e-5 mol), [(1R,2R)-1-methyl-2-(2-pyridyldisulfanyl)propyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.013 g, 1.92 e-5 mol), 2 mL of ACN, and 1 mL of water. N-methylmorpholine (0.060 mL, 0.000545 mol) was added. The mixture was stirred overnight at room temperature. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-85% ACN / HO + 0.05% TFA, 13 min; retention time: 6.95 min) to give compound 12 (0.0350 g, 9.10 e-6 mol, yield: 61.8%). ESI (M+3H / 3) 3+ : 1281.9

[0377] Example 13: Synthesis of Compound 13 [ka]

[0378] Compound 13 was prepared in a manner similar to compound 11, replacing ((1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl)carbonate with ((1R,2R)-2-(pyridin-2-yldisulfanyl)cyclohexyl)carbonate in step 2. Sunfire Prep C18 column (10 μm, 50 x 150 mm) (20-85% acetonitrile / water, 0.5% acetic acid); retention time: 6.609 min. ESI (M+3H / 3) 3+ : 1290.3

[0379] Example 14: Synthesis of Compound 14 [ka]

[0380] Step 1: Synthesis of (4-nitrophenyl)[trans-(1RS,2RS)-2-(2-pyridyldisulfanyl)cyclopentyl]carbonate The title compound was prepared using the first stereoisomer eluted from chiral chromatographic separation of racemic trans-2-(2-pyridyldisulfanyl)cyclopentyl, assigned as trans-(1RS,2RS)-2-(2-pyridyldisulfanyl)cyclopentan-1-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0381] Step 2: Synthesis of [(1RS,2RS)-2-(2-pyridyldisulfanyl)cyclopentyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol), DMAP (23.0 mg, 0.188 mmol), and (4-nitrophenyl)[trans-(1RS,2RS)-2-(2-pyridyldisulfanyl)cyclopentyl]carbonate (40.6 mg, 0.103 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 μL, 0.188 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-5% MeOH / DCM) to give the title compound (33.0 mg, 0.0479 mmol, 50.9% yield).

[0382] Step 2: Coupling with peptide Pv1 (compound 14) A vial was charged with peptide Pv1 (50.0 mg, 1.47 e-5 mol), [trans-(1RS,2RS)-1-methyl-2-(2-pyridyldisulfanyl)cyclopentyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.0124 g, 1.80 e-5 mol), 2 mL of ACN, and 1 mL of water. N-methylmorpholine (0.060 mL, 0.000545 mol) was added. The mixture was stirred overnight at room temperature. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-90% ACN / HO + 0.05% TFA, 16 min; retention time: 6.761 min) to give compound 14 (0.0360 g, 9.34 e-6 mol, yield: 63.3%). ESI (M+3H / 3) 3+ : 1286.3.

[0383] Example 15: Synthesis of Compound 15 [ka]

[0384] Step 1: Synthesis of (4-nitrophenyl)[trans-(1SR,2SR)-2-(2-pyridyldisulfanyl)cyclopentyl]carbonate The title compound was synthesized from the second stereoisomer eluted from a chiral chromatographic separation of racemic trans-2-(2-pyridyldisulfanyl)cyclopentyl, assigned as trans-(1SR,2SR)-2-(2-pyridyldisulfanyl)cyclopentan-1-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0385] Step 2: Synthesis of [(1SR,2SR)-2-(2-pyridyldisulfanyl)cyclopentyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol), DMAP (23.0 mg, 0.188 mmol), and (4-nitrophenyl)[trans-(1SR,2SR)-2-(2-pyridyldisulfanyl)cyclopentyl]carbonate (38.2 mg, 0.0974 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 μL, 0.188 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-5% MeOH / DCM) to give the title compound (29.0 mg, 0.0421 mmol, yield: 44.8%).

[0386] Step 3: Coupling with peptide Pv1 (compound 15) A vial was charged with peptide Pv1 (50.0 mg, 1.47 e-5 mol), trans-(1SR,2SR)-1-methyl-2-(2-pyridyldisulfanyl)cyclopentyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.0124 g, 1.80 e-5 mol), 2 mL of ACN, and 1 mL of water. N-methylmorpholine (0.060 mL, 0.000545 mol) was added. The mixture was stirred overnight at room temperature. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-90% ACN / HO + 0.05% TFA, 16 min; retention time: 6.883 min) to give compound 15 (0.0280 g, 7.26 e-6 mol, yield: 49.3%). ESI (M+3H / 3) 3+ : 1285.9.

[0387] Example 16: Synthesis of Compound 16 [ka]

[0388] Step 1: Synthesis of (4-nitrophenyl)[trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]carbonate The title compound was synthesized from the first stereoisomer eluted from chiral chromatographic separation of racemic trans-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-ol, assigned as trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0389] Step 2: Synthesis of [(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol), DMAP (23.0 mg, 0.188 mmol), and (4-nitrophenyl)[trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]carbonate (38.2 mg, 0.0969 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 μL, 0.188 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-5% MeOH / DCM) to give the title compound (40.0 mg, 0.0579 mmol, 61.6% yield).

[0390] Step 3: Coupling with peptide Pv1 (compound 16) A vial was charged with peptide Pv1 (50.0 mg, 1.47 e-5 mol), [trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.0124 g, 1.80 e-5 mol), 2 mL of ACN, and 1 mL of water. N-methylmorpholine (0.060 mL, 0.000545 mol) was added. The mixture was stirred overnight at room temperature. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-80% ACN / HO + 0.05% TFA, 15 min; retention time: 6.633 min) to give compound 16 (0.0290 g, 7.52 e-6 mol, yield: 51.0%). ESI (M+3H / 3) 3+ : 1286.4.

[0391] Example 17: Synthesis of Compound 17 [ka]

[0392] Step 1: Synthesis of (4-nitrophenyl)[trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]carbonate The title compound was synthesized from the second stereoisomer eluted from chiral chromatographic separation of racemic trans-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-ol, assigned as trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0393] Step 2: Synthesis of [(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol), DMAP (23.0 mg, 0.188 mmol), and (4-nitrophenyl)[trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]carbonate (38.2 mg, 0.0969 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 μL, 0.188 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-5% MeOH / DCM) to give [trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamate (31.0 mg, 0.0449 mmol, yield: 47.7%).

[0394] Step 3: Coupling with peptide Pv1 (compound 17) A vial was charged with peptide Pv1 (50.0 mg, 1.47 e-5 mol), [trans-(3SRS,4SR)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.0124 g, 1.80 e-5 mol), 2 mL of ACN, and 1 mL of water. To this was added N-methylmorpholine (0.060 mL, 0.000545 mol). The mixture was stirred at room temperature overnight. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-85% ACN / HO + 0.05% TFA, 13 min; retention time: 6.670 min) to give compound 17 (0.0170 g, 4.41 e-6 mol, yield: 29.9%). ESI (M+3H / 3) 3+ : 1286.7.

[0395] Example 18: Synthesis of Compound 18 [ka]

[0396] Step 1: Synthesis of (4-nitrophenyl)[trans-(2RS,3RS)-3-(2-pyridyldisulfanyl)tetralin-2-yl]carbonate The title compound was synthesized from the first stereoisomer eluted from chiral chromatographic separation of racemic trans-3-(2-pyridyldisulfanyl)tetralin-2-ol, assigned as trans-(2RS,3RS)-3-(2-pyridyldisulfanyl)tetralin-2-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0397] Step 2: Synthesis of [(2RS,3RS)-3-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(2RS,3RS)-3-(2-pyridyldisulfanyl)tetralin-2-yl]carbonate (32.1 mg, 0.0705 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 μL, 0.941 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give the title compound (26.0 mg, 0.0346 mmol, yield: 73.6%).

[0398] Step 3: Coupling with peptide Pv1 (compound 18) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6 mol), [trans-(2RS,3RS)-3-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.00719 g, 9.58 e-6 mol), 1 mL of ACN, and 0.5 mL of water. To this was added N-methylmorpholine (0.030 mL, 0.000273 mol). The mixture was stirred at room temperature for 65 h. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN / HO + 0.05% TFA, 20 min; retention time: 6.851 min) to give compound 18 (0.0080 g, 2.04 e-6 mol, yield: 27.7%). ESI (M+3H / 3) 3+ : 1307.4.

[0399] Example 19: Synthesis of Compound 19 [ka]

[0400] Step 1: Synthesis of (4-nitrophenyl)[trans-(2SR,3SR)-3-(2-pyridyldisulfanyl)tetralin-2-yl]carbonate The title compound was synthesized from the second stereoisomer eluted from chiral chromatographic separation of racemic trans-3-(2-pyridyldisulfanyl)tetralin-2-ol, assigned as trans-(2SR,3SR)-3-(2-pyridyldisulfanyl)tetralin-2-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0401] Step 2: Synthesis of [(2SR,3SR)-3-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(2SR,3SR)-3-(2-pyridyldisulfanyl)tetralin-2-yl]carbonate (32.1 mg, 0.0705 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 μL, 0.941 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give [trans-(2SR,3SR)-3-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamate (10.0 mg, 0.0133 mmol, yield: 28.3%).

[0402] Step 3: Coupling with peptide Pv1 (compound 19) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6 mol), [trans-(2SR,3SR)-3-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.010 g, 1.33 e-5 mol), 1 mL of ACN, and 0.5 mL of water. N-methylmorpholine (0.030 mL, 0.000273 mol) was added. The mixture was stirred at room temperature for 65 hours. LC-MS showed a complete reaction.

[0403] The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN / HO + 0.05% TFA, 20 min; retention time: 6.855 min) to give compound 19 (0.0060 g, 1.33 e-5 mol, yield: 20.8%). ESI (M+3H / 3) 3+ : 1307.6.

[0404] Example 20: Synthesis of Compound 20 [ka]

[0405] Step 1: Synthesis of (4-nitrophenyl)[trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]carbonate The title compound was synthesized from the first stereoisomer eluted from chiral chromatographic separation of racemic trans-4-(2-pyridyldisulfanyl)tetrahydropyran-3-ol, assigned as trans-(2RS,3RS)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0406] Step 2: Synthesis of [(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]carbonate (23.1 mg, 0.0564 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 μL, 0.941 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give [trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamate (30.0 mg, 0.0426 mmol, yield: 90.5%).

[0407] Step 3: Coupling with peptide Pv1 (compound 20) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6 mol), [trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.00779 g, 1.11 e-5 mol), 1 mL of ACN, and 0.5 mL of water. To this was added N-methylmorpholine (0.030 mL, 0.000273 mol). The mixture was stirred at room temperature for 65 h. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 30-85% ACN / HO + 0.05% TFA, 13 min; retention time: 6.380 min) to give compound 20 (0.0060 g, 1.55 e-6 mol, yield: 21.0%). ESI (M+3H / 3) 3+ : 1292.3.

[0408] Example 21: Synthesis of Compound 21 [ka]

[0409] Step 1: Synthesis of (4-nitrophenyl)[trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]carbonate The title compound was synthesized from the second stereoisomer eluted from chiral chromatographic separation of racemic trans-4-(2-pyridyldisulfanyl)tetrahydropyran-3-ol, assigned as trans-(2SR,3SR)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0410] Step 2: Synthesis of [(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]carbonate (23.1 mg, 0.0564 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 μL, 0.941 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give [trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamate (25.0 mg, 0.0355 mmol, yield: 75.4%).

[0411] Step 3: Coupling with peptide Pv1 (compound 21) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6), [trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.00779 g, 1.11 e-5 mol), 1 mL of ACN, and 0.5 mL of water. To this was added N-methylmorpholine (0.030 mL, 0.000273 mol). The mixture was stirred at room temperature for 65 hours. LC-MS showed a complete reaction.

[0412] The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-70% ACN / HO + 0.05% TFA, 17 min; retention time: 6.765 min) to give compound 21 (0.021 g, 5.42 e-6 mol, yield: 73.6%). ESI (M+3H / 3) 3+ : 1291.1.

[0413] Example 22: Synthesis of Compound 22 [ka]

[0414] Step 1: Synthesis of (4-nitrophenyl)[trans-(1RS,2RS)-2-(2-pyridyldisulfanyl)cycloheptyl]carbonate The title compound was synthesized from the first stereoisomer eluted from the chiral chromatographic separation of racemic trans-2-(2-pyridyldisulfanyl)cycloheptane, assigned as trans-(1RS,2RS)-2-(2-pyridyldisulfanyl)cycloheptan-1-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0415] Step 2: Synthesis of [(1RS,2RS)-2-(2-pyridyldisulfanyl)cycloheptyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(1RS,2RS)-2-(2-pyridyldisulfanyl)cycloheptyl]carbonate (23.7 mg, 0.0564 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 μL, 0.941 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give the title compound (29.0 mg, 0.0405 mmol, 86.0% yield).

[0416] Step 3: Coupling with peptide Pv1 (compound 22) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6), [trans-(1RS,2RS)-2-(2-pyridyldisulfanyl)cycloheptyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.00792 g, 1.11 e-5 mol), 1 mL of ACN, and 0.5 mL of water. N-methylmorpholine (0.030 mL, 0.000273 mol) was added. The mixture was stirred at room temperature for 65 h. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-70% ACN / HO + 0.05% TFA, 17 min; retention time: 6.868 min) to give compound 22 (0.020 g, 5.15 e-6 mol, yield: 69.9%). ESI (M+3H / 3) 3+ : 1296.3.

[0417] Example 23: Synthesis of Compound 23 [ka]

[0418] Step 1: Synthesis of (4-nitrophenyl)[trans-(1SR,2SR)-2-(2-pyridyldisulfanyl)cycloheptyl]carbonate The title compound was synthesized from the second stereoisomer eluted from a chiral chromatographic separation of racemic trans-2-(2-pyridyldisulfanyl)cycloheptane, assigned as trans-(1SR,2SR)-2-(2-pyridyldisulfanyl)cycloheptan-1-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0419] Step 2: Synthesis of [(1SR,2SR)-2-(2-pyridyldisulfanyl)cycloheptyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(1SR,2SR)-2-(2-pyridyldisulfanyl)cycloheptyl]carbonate (23.7 mg, 0.0564 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 μL, 0.941 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give the title compound (31.0 mg, 0.0432 mmol, yield: 91.9%).

[0420] Step 3: Coupling with peptide Pv1 (compound 23) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6), [trans-(1SR,2SR)-2-(2-pyridyldisulfanyl)cycloheptyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.00792 g, 1.11 e-5 mol), 1 mL of ACN, and 0.5 mL of water. N-methylmorpholine (0.030 mL, 0.000273 mol) was added. The mixture was stirred at room temperature for 65 h. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-88% ACN / HO + 0.05% TFA, 17 min; retention time: 7.178 min) to give compound 23 (0.020 g, 5.15 e-6 mol, yield: 69.9%). ESI (M+3H / 3) 3+ : 1296.0.

[0421] Example 24: Synthesis of Compound 24 [ka]

[0422] Step 1: Synthesis of (4-nitrophenyl)[trans-1-(1RS,2RS)-1-(2-pyridyldisulfanyl)tetralin-2-yl]carbonate The title compound was synthesized from the first stereoisomer eluted from chiral chromatographic separation of racemic trans-1-(2-pyridyldisulfanyl)tetralin-2-ol, assigned as trans-(1RS,2RS)-1-(2-pyridyldisulfanyl)tetralin-2-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0423] Step 2: Synthesis of [trans-(1RS,2RS)-1-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-1-(1RS,2RS)-2-pyridyldisulfanyl)tetralin-2-yl]carbonate (32.1 mg, 0.0705 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 μL, 0.941 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give the title compound (20.0 mg, 0.0266 mmol, yield: 56.6%).

[0424] Step 3: Coupling with peptide Pv1 (Example 24) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6), [trans-1-(1RS,2RS)-1-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.0083 g, 1.11 e-5 mol), 1 mL of ACN, and 0.5 mL of water. To this was added N-methylmorpholine (0.030 mL, 0.000273 mol). The mixture was stirred at room temperature for 65 h. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN / HO + 0.05% TFA, 20 min; retention time: 6.968 min) to give compound 24 (0.012 g, 3.06 e-6 mol, yield: 41.6%). ESI (M+3H / 3) 3+ : 1307.2.

[0425] Example 25: Synthesis of Compound 25 [ka]

[0426] Step 1: Synthesis of (4-nitrophenyl)[trans-(1SR,2SR)-3-(2-pyridyldisulfanyl)tetralin-2-yl]carbonate The title compound was synthesized from the second stereoisomer eluted from chiral chromatographic separation of racemic trans-1-(2-pyridyldisulfanyl)tetralin-2-ol, assigned as trans-(1SR,2SR)-1-(2-pyridyldisulfanyl)tetralin-2-ol, following a synthetic method similar to that described for the synthesis of compound 11.

[0427] Step 2: Synthesis of [trans-(1SR,2SR)-1-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(1SR,2SR)-1-(2-pyridyldisulfanyl)tetralin-2-yl]carbonate (32.1 mg, 0.0705 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 μL, 0.941 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give the title compound (22.0 mg, 0.0293 mmol, yield: 62.3%).

[0428] Step 3: Coupling with peptide Pv1 (compound 25) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e 6), [trans-(1SR,2SR)-1-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.0083 g, 1.11 e 5 mol), 1 mL of ACN, and 0.5 mL of water. N-methylmorpholine (0.030 mL, 0.000273 mol) was added. The mixture was stirred at room temperature for 65 hours. LC-MS showed a complete reaction.

[0429] The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN / HO + 0.05% TFA, 20 min; retention time: 6.944 min) to give compound 25 (0.013 g, 3.32 e-6 mol, yield: 45.0%). ESI (M+3H / 3) 3+ : 1307.0.

[0430] Example 26: Synthesis of Compound 26 [ka]

[0431] Step 1: Synthesis of [trans-4-(2-pyridyldisulfanyl)cyclohexyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol) and (4-nitrophenyl)[4-(2-pyridyldisulfanyl)cyclohexyl]carbonate (synthesized from commercially available trans-4-mercaptocyclohexan-1-ol) (42.1 mg, 0.103 mmol) in 2 mL of anhydrous DMF, N,N-diisopropylethylamine (35 μL, 0.188 mmol) was added. After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give the title compound (45.0 mg, 0.0640 mmol, yield: 68.1%).

[0432] Step 2: Coupling with peptide Pv1 (compound 26) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6), [trans-4-(2-pyridyldisulfanyl)cyclohexyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.00777 g, 1.11 e-5 mol), 1 mL of ACN, and 0.5 mL of water. N-Methylmorpholine (0.030 mL, 0.000273 mol) was added. The mixture was stirred at room temperature for 65 h. LC-MS showed complete reaction.

[0433] The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN / HO + 0.05% TFA, 20 min; retention time: 6.593 min) to give compound 26 (0.028 g, 7.23 e-6 mol, yield: 98.2%). ESI (M+3H / 3) 3+ : 1291.0.

[0434] Example 27: Synthesis of Compound 27 [ka]

[0435] Step 1: Synthesis of [(2S)-3-methyl-2-(2-pyridyldisulfanyl)butyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol) and [(2S)-3-methyl-2-(2-pyridyldisulfanyl)butyl](4-nitrophenyl)carbonate (synthesized from L-valine, cf. J. Org. Chem. 1990, 55, 2286-2288) (40.8 mg, 0.103 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 μL, 0.188 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give the title compound (48.0 mg, 0.0695 mmol, yield: 73.9%).

[0436] Step 2: Coupling with peptide Pv1 (compound 27) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6 mol), [(2S)-3-methyl-2-(2-pyridyldisulfanyl)butyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.00764 g, 1.11 e-5 mol), 1 mL of ACN, and 0.5 mL of water. N-methylmorpholine (0.030 mL, 0.000273 mol) was added. The mixture was stirred at room temperature for 65 h. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN / HO + 0.05% TFA, 20 min; retention time: 6.773 min) to give compound 27 (0.024 g, 6.22 e-6 mol, yield: 84.4%). ESI (M+3H / 3) 3+ : 1286.8.

[0437] Example 28: Synthesis of Compound 28 [ka]

[0438] Step 1: Synthesis of [(2R)-3-methyl-2-(2-pyridyldisulfanyl)butyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol) and (4-nitrophenyl)(2R)-3-methyl-2-(2-pyridyldisulfanyl)butyl]carbonate (synthesized from D-valine, cf. J. Org. Chem. 1990, 55, 2286-2288) (40.8 mg, 0.103 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 μL, 0.188 mmol). After stirring at room temperature for 16 h, the mixture was diluted with EtOAc (50 mL) and washed with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated, and the residue was purified by column chromatography (0-3% MeOH / DCM) to give the title compound (41.0 mg, 0.0594 mmol, yield: 63.1%).

[0439] Step 2: Coupling with peptide Pv1 (compound 28) A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6), [(2R)-3-methyl-2-(2-pyridyldisulfanyl)butyl]N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate (0.00764 g, 1.11 e-5 mol), 1 mL of ACN, and 0.5 mL of water. N-methylmorpholine (0.030 mL, 0.000273 mol) was added. The mixture was stirred at room temperature for 65 h. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse-phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN / HO + 0.05% TFA, 20 min; retention time: 6.708 min) to give compound 28 (0.012 g, 3.08 e-6 mol, yield: 41.8%). ESI (M+3H / 3) 3+ : 1287.8.

[0440] Example 29: Synthesis of Compound 29 [ka]

[0441] Analytical Method: Chromatographic purity was measured on an Agilent 1200 Series, 1100 Series, or 6130 Series LC / MS system using a Merck Chromolith RP-18e analytical HPLC column (monolithic, 50 x 2 mm) and the following analytical HPLC method: injection volume 5 μL; flow rate 1 mL / min; 5 to 95% acetonitrile in water with 0.05% AcOH (Method A) or 0.05% TFA (Method B) over 5 min; Agilent diode array detector at I = 254, 220, or 195 nm; room temperature.

[0442] Step 1: Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-(pyridin-2-yldisulfanayl)propanamide Solid exatecan mesylate [CAS: 169869-90-3] (80 mg, 0.150 mmol) was added to a solution of 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanayl)propanoate (180 mg, 0.576 mmol) in DMF (4 mL), followed by aqueous PBS buffer (4 mL, pH = 7.4, 50 mM) and sonication for approximately 5 minutes. The cloudy mixture was stirred at ambient temperature for 2 hours, at which point the reaction was determined to be approximately 25% complete. Ammonium acetate (11 mg, 0.143 mmol) was added to an additional 2 mL of DMF, and the resulting mixture was stirred at ambient temperature for 18 hours. The mixture was acidified with TFA (80 mL, 0.98 mmol) and divided into two equal portions. Each portion was then purified by Redi-SepC. 18Purification was performed on a 50 g cartridge and eluted with a gradient (5% to 95%) of acetonitrile in water containing TFA (0.05% v / v). The combined fractions were frozen and lyophilized to give the title compound as a pale yellow solid (42 mg, 44%). HPLC purity at 254 nm: 97%. Retention time: 2.50 min (Method A). MS data, 633.2 (M+H). + .

[0443] Step 2: Coupling with peptide Pv1 (compound 29) Solid peptide Pv1 (168.4 mg, 0.0480 mmol) was added to solid N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-(pyridin-2-yldisulfanayl)propanamide (30.5 mg, 0.0482 mmol) and dissolved in DMF (2 mL) by sonication (approximately 1 min) and flushing with nitrogen. 4-Methylmorpholine (20 mL, 0.182 mmol) was added, and the solution was maintained at ambient temperature for 18 h. The solution was acidified with acetic acid (17 mL, 0.296 mmol) and purified using Biotage™ C. 18 The product was applied to a 300A 25 g reverse-phase column and eluted with a gradient (25% to 95%) of acetonitrile in water containing TFA (0.05% v / v). The combined fractions were frozen and lyophilized to give a pale yellow solid. The product was dissolved in DMSO (3 mL) and a 1 mL portion of the solution was purified by Biotage™ chromatography. 18 Separate purifications were performed on a 300A 25 g reverse-phase column and eluted with a gradient (25% to 95%) of 10 mM ammonium acetate in water (acetonitrile / water / 2-propanol, 3 / 2 / 1). The combined fractions were frozen and lyophilized to give a pale yellow solid, which was dissolved in 0.4% TFA in 2 / 1 water / acetonitrile, transferred to a tared vial, and lyophilized to give solid compound 29 (128 mg, 66%). HPLC purity at 254 nm: >95%. Retention time: 3.19 min (Method B). MS data: 1900.6 (M+2H / 2). 2+ , 1267.3 (M+3H / 3)3+ .

[0444] Example A: Growth Retardation Assay Cells were plated in 96-well black-walled, clear-bottom plates (Griener) at 2500 DLD-1 WT cells per well, 5000 FaDu and HeLa cells per well, and 3000 HCT116 cells per well in growth medium containing 10% FBS. Cells were allowed to adhere for 60 minutes at room temperature and then returned to a 37°C, 5% CO2 incubator. After 24 hours, the medium was removed and replaced with fresh growth medium containing various drug concentrations. Each drug concentration was added in triplicate. Drug-untreated controls contained growth medium only. Cells were returned to the incubator. 96 hours after drug addition, cells were fixed with 4% paraformaldehyde for 20 minutes and stained with 1 μg / mL Hoechst. Plates were imaged using a Cytation5 autoimager (BioTek), and cells were counted using CellProfiler (http: / / cellprofiler.org). Percent cell growth delay was calculated and data were plotted using GraphPadPrism. [Table 11] [Table 12]

[0445] Example B: Plasma Pharmacokinetics of Compound 11 in a Rat Model Animal medication Male Sprague-Dawley rats underwent jugular vein cannulation and vascular access button (VAB, Instech Labs catalog number VABR1B / 22) insertion at Envigo Labs prior to shipping. A magnetic aluminum cap (Instech Labs catalog number VABRC) was used to protect the jugular vein catheter access port, and animals were housed two per cage on corncob bedding for 4-5 days prior to the study. Rats received a single intravenous dose of 5 mg / kg of compound 11 prepared in a vehicle of 5% mannitol in citrate buffer. Blood (250 μL) was collected from fed rats into K2EDTA-filled microtainers at 1, 2, 4, 8, 24, and 30 hours after compound administration. Plasma was separated by centrifugation, and 100 μL aliquots were transferred to a 96-well polypropylene plate on dry ice. Samples were stored at −80°C until processed for quantification of total peptides by ELISA and exatecan release by LC-MS / MS.

[0446] ELISA measurement of total peptide plasma concentrations A 96-well plate was coated with 0.1 μM BSA-labeled peptide prepared in 0.2 M carbonate-bicarbonate buffer, pH 9.4, at 100 μL / well and incubated overnight at 4°C. The plate was washed four times with ELISA wash buffer (PBS + 0.05% Tween 20), incubated with blocking buffer (PBS + 5% dry milk + 0.05% Tween 20) (300 μL / well) at room temperature for 2 hours, and washed four times again with ELISA wash buffer. Simultaneously, 2x Compound 11 standards in control plasma and study plasma samples were preincubated with 1–10 ng / mL primary antibody specific for the Pv1 peptide for 30 minutes at room temperature. The preincubated samples were added to the precoated, preblocked assay plate at 100 μL / well and incubated at room temperature for 1 hour. Plates were washed four times with ELISA wash buffer and incubated with 100 μL / well of secondary goat anti-mouse IgG HRP antibody (1:5,000 in antibody diluent) for 1 hour at room temperature. Plates were washed four times with ELISA wash buffer and incubated with 100 μL / well of SuperSignal substrate for 1 minute at room temperature with gentle shaking. Plates were read for luminescence using a BioTek Cytation5 plate reader.

[0447] LC-MS / MS measurement of exatecan plasma concentrations To quantify exatecan, 20 μL of plasma samples were added to polypropylene autosampler vials. 20 μL of PPT-IS (ACN:H2O (50:50) + 0.5% FA containing 1000 ng / mL internal standard) and 20 μL of diluent (ACN:H2O (50:50) + 0.5% FA) were added to each sample. 120 μL of ACN + 5% FA was then added. The vials were capped and vortexed for 2 minutes. Samples were centrifuged at 3700 rpm for 5–10 minutes and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0448] Figure 1 shows plots of the plasma concentration of compound 11 and released exatecan after a single intravenous dose of 5 mg / kg of compound 11 in rats (data are expressed as mean ± SEM). As shown in Figure 1, less than 0.002% of the exatecan payload was released after 30 hours of circulation. Figure 1 demonstrates that compound 11 is stable in plasma for at least 30 hours.

[0449] Example C: Tumor and bone marrow pharmacokinetics of compound 11 in a mouse model Animal medication Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog number NCRNU-F) and housed five per cage on AlphaDri bedding in a disposable cage system (Innovive). Human HCT116 cancer cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and placed at 2.5 x 10 cells per 100 µL. 6 Cells were implanted subcutaneously into the left flank of each mouse at a density of 1000 xenografts with a minimum volume of 300 mm. 3 When the tumor reached 100 mg / kg, mice were administered a single intraperitoneal injection of 10 mg / kg of compound 11 prepared in a vehicle of 5% mannitol in citrate. Tumor and bone marrow samples were collected from fed, anesthetized mice 1, 2, 4, 8, 16, 24, 32, and 48 hours after compound administration. Total peptide concentrations in tumor and bone marrow were determined via ELISA.

[0450] ELISA measurement of total peptide tissue concentrations A 96-well plate was coated with 0.1 μM BSA-labeled peptide prepared in 0.2 M carbonate-bicarbonate buffer, pH 9.4, at 100 μL / well and incubated overnight at 4 °C. The plate was washed four times with ELISA wash buffer (PBS + 0.05% Tween 20), incubated with blocking buffer (PBS + 5% dry milk + 0.05% Tween 20) (300 μL / well) for 2 h at room temperature, and washed four times again with ELISA wash buffer. Simultaneously, 2x Compound 11 standards (in their respective tissue matrices) or sample tumor homogenates or bone marrow samples diluted in diluent (PBS + 2% dry milk + 0.05% Tween 20) were preincubated with 1–10 ng / mL primary antibody specific for the Pv1 peptide for 30 min at room temperature. The preincubated samples were added to the precoated, preblocked assay plate at 100 μL / well and incubated for 1 h at room temperature. Plates were washed four times with ELISA wash buffer and incubated with 100 μL / well of secondary goat anti-mouse IgG HRP antibody (1:5,000 in antibody diluent) for 1 hour at room temperature. Plates were washed four times with ELISA wash buffer and incubated with 100 μL / well of SuperSignal substrate for 1 minute at room temperature with gentle shaking. Plates were read for luminescence using a BioTek Cytation5 plate reader.

[0451] Figure 2 shows plots of peptide concentrations in tumors and bone marrow after a single intraperitoneal administration of 10 mg / kg of compound 11 in mice (data are expressed as mean ± SEM), demonstrating that compound 11 effectively targets tumors.

[0452] Example D: Bone marrow toxicity studies in mouse models Animal medication Six-week-old female athymic nude Fox nuMice were obtained from Taconic Labs (catalog number NCRNU-F) and housed five per cage on AlphaDri bedding in a disposable cage system (Innovive). Human HCT116 cancer cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and placed at 2.5 x 10 cells per 100 µL. 6 Cells were implanted subcutaneously into the left flank of each mouse at a density of 200 mm xenografts. 3 When the minimum volume of 100 μg / kg of exatecan was reached, mice were administered intraperitoneal doses of vehicle, or unconjugated exatecan (equivalent to 1.15 or 2.3 mg / kg exatecan, respectively) or Compound 11 (equivalent to 10 or 20 mg / kg Compound 11, respectively) at 2.6 or 5.2 μmol / kg. Compounds were administered once daily for 4 days.

[0453] Bone marrow collection Tumor-bearing mice were euthanized by cervical dislocation 6 hours after the last dose. The femurs were removed, and bone marrow was extruded into a 50 mL conical tube by flushing the bone with a 23-gauge needle attached to a 5 cc syringe containing PBS + 2% fetal bovine serum. The bone marrow was homogenized by gentle pipetting, filtered through a 100 μm nylon mesh filter, and the cells were pelleted by centrifugation at 1200 rpm for 5 minutes at 4°C. Red blood cells were lysed with 3 mL of lysis buffer for 2 minutes at room temperature. PBS was added to a volume of 25 mL, and the cells were repelleted by centrifugation as described above. The cell pellet was suspended in 5 mL of PBS, and cell counts were assessed by trypan blue exclusion. Cell counts from four independent studies were averaged and plotted.

[0454] Figure 3 shows a graph of total bone marrow counts from femurs of tumor-bearing nude mice after 4 days of once-daily administration of 2.6 and 5.2 μmol / kg of either compound 11 (equivalent to 10 and 20 mg / kg conjugate) or free exatecan (equivalent to 1.15 and 2.3 mg / kg exatecan) (data are expressed as mean ± SEM). Compound 11 did not exhibit the myelotoxicity that limits the clinical usefulness of exatecan.

[0455] Example E: Gastric Toxicity Studies in a Mouse Model Animal dosing and gastric imaging Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog number NCRNU-F) and housed five per cage on AlphaDri bedding in a disposable caging system (Innovive). Human HCT116 cells derived from colorectal cancer were diluted 1:1 in phenol red-free Matrigel and cultured at 2.5 x 10 cells per 100 µL. 6 Cells were implanted subcutaneously into the left flank of each mouse at a density of 300 mm xenografts. 3 When the stomach reached a minimum volume of 1000 mg / kg, mice were administered an intraperitoneal dose of vehicle, or 5.2 μmol / kg of unconjugated exatecan (equivalent to 2.3 mg / kg exatecan) or Compound 11 (equivalent to 20 mg / kg Compound 11). Compounds were administered once daily for 4 days. Six hours after the last administration, mice were euthanized by cervical dislocation and gross necropsies were performed. Photographs of the stomach were taken both in situ and ex vivo.

[0456] Figure 4A shows the excised stomach of a tumor-bearing nude mouse after four days of daily administration of 5.2 μmol / kg of either vehicle, compound 11 (equivalent to 20 mg / kg conjugate), or free exatecan (equivalent to 2.3 mg / kg exatecan). Figure 4B shows the in situ stomach of a tumor-bearing nude mouse after four days of daily administration of 5.2 μmol / kg of either compound 11 (equivalent to 20 mg / kg conjugate) or free exatecan (equivalent to 2.3 mg / kg exatecan). Compound 11 did not exhibit the gastric toxicity that limits the clinical usefulness of exatecan.

[0457] Example F: Efficacy of Compound 11 in the HCT116 colorectal cancer model Six-week-old female athymic nude Fox nuMice were obtained from Taconic Labs (catalog number NCRNU-F) and housed five per cage on AlphaDri bedding in a disposable cage system. Human HCT116 cells derived from colorectal cancer were diluted 1:1 in phenol red-free Matrigel at 2.5 x 10 cells per 100 µL. 6 Cells were subcutaneously implanted into the left flank of each mouse at a density of 100–200 mm. 3 When the tumor reached an average volume of 1000 mg / kg, mice were randomly divided into groups and treated as detailed in the table below. Mice received intraperitoneal (IP) doses of vehicle or unconjugated exatecan (equivalent to 1.15 or 2.3 mg / kg exatecan, respectively) or Compound 11 (equivalent to 10 or 20 mg / kg Compound 11, respectively) at 2.6 or 5.2 mol / kg. Doses were prepared by diluting a 0.1 mg / μL DMSO stock with 5% mannitol in citrate buffer and administered once daily, four times a week, for 3 weeks in a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured with calipers, and the volume was calculated using the formula for ellipsoid volume: volume = π / 6 x (length) x (width). 2 The animals died and tumor size reached 2000 mm 3 Patients were excluded from the study if their weight loss exceeded 100g or their weight loss exceeded 20%. The table below shows the dosing schedules for the various treatment groups. [Table 13]

[0458] Figure 5A shows plots of the mean tumor volumes resulting from the administration of equimolar amounts of either free exatecan or compound 11 in nude mice bearing HCT116 colorectal flank tumors. Animals were parenterally dosed once daily, four times per week, for three weeks. Figure 5B shows Kaplan-Meier survival curves for the administration of equimolar amounts of either free exatecan or compound 11 in nude mice bearing HCT116 colorectal flank tumors. Data are expressed as mean ± SEM. These data demonstrate that compound 11 exhibits potent antitumor activity in preclinical colorectal cancer models.

[0459] Example G: Efficacy of Compound 11 in the MKN45 HER2-negative gastric cancer model Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog number NCRNU-F) and housed five per cage on AlphaDri bedding in a disposable cage system. Human MKN45 cells derived from gastric cancer were diluted 1:1 in phenol red-free Matrigel and cultured at 2 x 10 cells per 100 µL. 6 Cells were subcutaneously implanted into the left flank of each mouse at a density of 100–200 mm. 3 When the tumor reached an average volume of 1000 mg / kg, mice were randomly divided into groups and treated as detailed in the table below. Mice received intraperitoneal (IP) doses of vehicle or unconjugated exatecan (equivalent to 1.15 or 2.3 mg / kg exatecan, respectively) or Compound 11 (equivalent to 10 or 20 mg / kg Compound 11, respectively) at 2.6 or 5.2 mol / kg. Doses were prepared by diluting a 0.1 mg / μL DMSO stock with 5% mannitol in citrate buffer and administered once daily, four times a week, for two weeks in a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured with calipers, and the volume was calculated using the formula for ellipsoid volume: volume = π / 6 x (length) x (width). 2 The animals died and tumor size reached 2000 mm 3 Patients were excluded from the study if their weight loss exceeded 100g or their weight loss exceeded 20%. The table below shows the dosing schedules for the various treatment groups. [Table 14]

[0460] Figure 6A shows the single-agent efficacy of compound 11 in nude mice bearing MKN45 HER2-negative gastric cancer flank tumors. Animals were parenterally dosed once daily, four times a week, for two weeks. Figure 6B shows Kaplan-Meier survival curves for nude mice bearing MKN45 HER2-negative gastric cancer flank tumors administered equimolar doses of either free exatecan or compound 11. Data are expressed as mean ± SEM. These data demonstrate that compound 11 exhibits potent antitumor activity in preclinical gastric cancer models.

[0461] In Figure 6B, Kaplan-Meier analysis was used to assess survival based on death or exclusion from the study.

[0462] Example H: Efficacy of Compound 11 in the JIMT-1 HER2-Intermediate Breast Cancer Model Five- to six-week-old female NOD.SCID mice were obtained from Beijing Anikeeper Biotech Co., Ltd. (Beijing, China). Human J1MT-1 cells derived from breast cancer were diluted 1:1 with phenol red-free Matrigel and cultured at 5 x 10 cells / 100 μL. 6 Cells were implanted subcutaneously into the left flank of each mouse at a density of 100 mm xenografts. 3 When the tumor reached an average volume of 1000 mg / kg, mice were randomly divided into groups and treated as detailed in the table below. Mice received an intraperitoneal (IP) dose of vehicle or 2.6 or 5.2 μmol / kg of compound 11 (equivalent to 10 or 20 mg / kg of compound 11, respectively). Doses were prepared by diluting a 0.1 mg / μL DMSO stock with 5% mannitol in citrate buffer and administered once daily, four times a week, for three weeks in a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured with calipers, and the volume was calculated using the formula for ellipsoid volume: volume = π / 6 x (length) x (width). 2 The body weight of the animals was measured at the same time as the tumor volume assessment. Animals were killed when the tumor size reached 2000 mm 3 Patients were excluded from the study if their weight loss exceeded 100g or their weight loss exceeded 20%. The table below shows the dosing schedules for the various treatment groups. [Table 15]

[0463] Figure 7A shows a plot of the mean tumor volume resulting from administration of compound 11 in SCID mice bearing JIMT-1 HER2 intermediate breast cancer flank tumors. Animals were administered parenterally once daily, four times per week, for three weeks. Figure 7B shows a plot of the percent change in body weight in SCID mice bearing JIMT-1 HER2 intermediate breast cancer flank tumors administered compound 11. Data are expressed as mean ± SEM. These data demonstrate that compound 11 exhibits potent antitumor activity in preclinical breast cancer models.

[0464] Example I: Efficacy of Compound 11 in the MDA-MB-231 triple-negative breast cancer model 3-4 week old female athymic nude Fox nu Mice were obtained from Envigo Lab. Human MDA-MB-231 cells, derived from breast adenocarcinoma, were diluted 1:1 in phenol red-free Matrigel and cultured at 2 x 10 cells per 100 μL. 6 Cells were subcutaneously implanted into the left flank of each mouse at a density of 50–100 mm. 3 When the tumor reached an average volume of 1000 mg / kg, mice were randomly divided into groups and treated as detailed in the table below. Mice were administered intraperitoneal (IP) doses of vehicle or 5, 10, or 20 mg / kg of compound 11. Doses were prepared by diluting a 0.1 mg / μL DMSO stock with 5% mannitol in citrate buffer and administered once daily, four times a week, for three weeks in a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured with calipers, and the volume was calculated using the formula for ellipsoid volume: volume = π / 6 x (length) x (width). 2 The body weight of the animals was measured at the same time as the tumor volume assessment. Animals were killed when the tumor size reached 2000 mm 3 Patients were excluded from the study if their weight loss exceeded 100g or their weight loss exceeded 20%. The table below shows the dosing schedules for the various treatment groups.

[0465] [Table 16]

[0466] Figure 8A shows a plot of mean tumor volume in nude mice bearing MDA-MB-231 triple-negative breast cancer flank tumors treated with compound 11. Animals were dosed parenterally, once daily, four times per week for three weeks. Figure 8B shows a plot of percent change in body weight relative to day 0 in nude mice bearing MDA-MB-231 triple-negative breast cancer flank tumors treated with compound 11. Data are expressed as mean ± SEM. These data demonstrate that compound 11 exhibits potent antitumor activity in preclinical breast cancer models.

[0467] Example J: Effect of the combination of Compound 11 and talazoparib in the MDA-MB-231 triple-negative breast cancer model 3-4 week old female athymic nude Fox nu Mice were obtained from Envigo Lab. Human MDA-MB-231 cells, derived from breast adenocarcinoma, were diluted 1:1 in phenol red-free Matrigel and plated at 2 x 10 cells per 100 μL. 6 Cells were subcutaneously implanted into the left flank of each mouse at a density of 50–100 mm. 3 When the tumor reached an average volume of 1000 mg / kg, mice were randomly divided into groups and treated as detailed in the table below. Mice were administered an intraperitoneal (IP) dose of vehicle or 5 mg / kg of Compound 11 alone or in combination with an oral (PO) dose of 0.33 mg / kg of talazoparib. Doses were prepared by diluting a 0.1 mg / μL DMSO stock with 5% mannitol in citrate buffer. Compound 11 was administered once daily, four times a week, for three weeks at a volume of 12 mL / kg (300 μL per 25 g mouse), and talazoparib was administered once daily for 15 days. Xenograft tumors were measured with calipers, and the volume was calculated using the formula for ellipsoid volume: volume = π / 6 x (length) x (width). 2 The body weight of the animals was measured at the same time as the tumor volume assessment. Animals were killed when the tumor size reached 2000 mm 3Patients were excluded from the study if their weight loss exceeded 100g or their body weight loss exceeded 20%. The table below shows the dosing schedules for the various treatment groups.

[0468] [Table 17]

[0469] Figure 9A shows a plot of the mean tumor volume in nude mice bearing MDA-MB-231 triple-negative breast cancer flank tumors treated with Compound 11 and talazoparib. Animals were administered Compound 11 parenterally once daily, four times a week, for three weeks, and talazoparib orally once daily for 18 days. Figure 9B shows a plot of the percent change in body weight relative to day 0 in nude mice bearing MDA-MB-231 triple-negative breast cancer flank tumors treated with Compound 11 and talazoparib.

[0470] Example K: Glutathione Cleavage Studies A 20 mM stock of the conjugate was prepared in 100% DMSO. The stock was then diluted with 100 mM Tris, pH 7.5, to generate a 500 μM intermediate dilution, followed by a further 1:5 dilution in 100 mM Tris, pH 7.5, to a final concentration of 100 μM conjugate. 100 mM glutathione was prepared immediately before use in HO and diluted 1:10 in the challenge sample to a final glutathione challenge concentration of 10 mM. Samples were mixed by inversion and incubated at 37°C for up to 24 hours. 50 μL samples were aliquoted into siliconized microfuge tubes at 0, 4, and 24 hours and immediately frozen at -80°C.

[0471] Samples were thawed and extracted as follows: 8 μL of 25% phosphoric acid, followed by 117 μL of 100% acetonitrile / 0.1% TFA, was added to each sample, mixed, and centrifuged at 13,000 x G for 10 minutes. The supernatant was pipetted into 0.2 mL HPLC vials and loaded onto a Perkin Elmer Flexar HPLC autosampler. The following table summarizes the HPLC conditions.

[0472] [Table 18]

[0473] The data was analyzed by calculating the compound reduction rate (area of the retention time peak of the cleaved conjugate / area of the retention time peak of the conjugate at time 0)×100.

[0474] Figure 10 shows a graph of the degradation of Compound 11 and Compound 29 resulting from treatment with 10 mM glutathione over 16 hours. As shown in Figure 10, Compound 29 was released much faster than Compound 11 under similar glutathione exposure.

[0475] The following table summarizes the degradation data for Compounds 11 through 29 measured at 4 and 24 hours for the 10 mM glutathione exposure conditions described above.

[0476] [Table 19]

[0477] Example L: Plasma Stability Study A 20 mM stock of the conjugate was prepared in 100% DMSO. The stock was subsequently diluted with 100 mM Tris, pH 7.5, to generate a 500 μM intermediate dilution, which was then diluted 1:5 directly with rat plasma to generate a final concentration of 100 μM conjugate. Samples were mixed by inversion and incubated at 37°C for up to 24 hours. 50 μL samples were aliquoted into siliconized microfuge tubes at 0, 4, and 24 hours and immediately frozen at -80°C.

[0478] Samples were thawed and extracted as follows: 8 μL of 25% phosphoric acid, followed by 117 μL of 100% acetonitrile / 0.1% TFA, was added to each sample, mixed, and centrifuged at 13,000 x G for 10 minutes. The supernatant was pipetted into 0.2 mL HPLC vials and loaded onto a Perkin Elmer Flexar HPLC autosampler. The following table summarizes the HPLC conditions.

[0479] [Table 20]

[0480] The data was analyzed by calculating the compound reduction rate (area of the retention time peak of the incubated conjugate / area of the retention time peak of the conjugate at time 0) X 100. The results of the study are shown in the table below.

[0481] [Table 21]

[0482] Various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including but not limited to all patents, patent applications, and publications cited in this application, is hereby incorporated by reference in its entirety.

Claims

1. Formula (I) 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein R ７ is a peptide, R ８ teeth, 【Chemistry 2-1】 【Chemistry 2-2】 【Chemistry 2-3】 【Chemistry 2-4】 【Chemistry 2-5】 【Chemistry 2-6】 【Chemistry 2-7】 is selected from the group consisting of Q is, 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 【Chemistry 3-4】 【Chemistry 3-5】 is selected from the group consisting of R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ９ , R １０ , R １１ , and R １２ are each independently H, C １－４ Alkyl, C １－４ Alkenyl, C ６－１０ Aryl, C ３－１０ Cycloalkyl, 5- to 10-membered heteroaryl, 4- to 10-membered heterocycloalkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ wherein said C １－４ Alkyl, C １－４ Alkenyl, C ６－１０ Aryl, C ３－１０ Cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R １ and R ２ together with the carbon atoms to which they are attached, C ３－１４ cycloalkyl group or 4- to 14-membered heterocycloalkyl group, each of which is １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R １ and R ３ together with the carbon atoms to which they are attached, C ３－１４ cycloalkyl group or 4- to 14-membered heterocycloalkyl group, each of which is １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R ２ and R ３ together with the carbon atoms to which they are attached, C ３－１４ cycloalkyl group or 4- to 14-membered heterocycloalkyl group, each of which is １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R ３ and R ４ together with the carbon atoms to which they are attached, C ３－１４ cycloalkyl group or 4- to 14-membered heterocycloalkyl group, each of which is １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R ５ and R ６ together with the carbon atoms to which they are attached, C ３－１４ cycloalkyl group or 4- to 14-membered heterocycloalkyl group, each of which is １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ and optionally substituted with 1, 2, or 3 substituents independently selected from R １３ is H or C １－６ is alkyl, A is H or C １－４ is alkyl, R ａ１ , R ｂ１ , R ｃ１ , and R ｄ１ are each independently H, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Haloalkyl, OH, CN, NO ２ , and CO ２ CH ３ wherein said C １－６ Alkyl and C ２－６ Alkenyl is OH, CN, NO ２ , or CO ２ CH ３ may be substituted with 【Chemistry 4】 is C ６－１０ aryl or 5- to 10-membered heteroaryl, wherein said 5- to 10-membered heteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4 ring-forming heteroatoms independently selected from N, O, and S; Ring G is C ３－１４ a cycloalkyl group or a 4- to 14-membered heterocycloalkyl group, each of which is C １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ and optionally substituted with 1, 2, or 3 substituents independently selected from [N,O,S] is NH, O, or S; [N,O] is NH or O; [C,N,O] is CR Ｘ R Ｙ , NH, or O, and Each R Ｘ and R Ｙ is H and C １－４ or a pharmaceutically acceptable salt thereof, wherein each of the following is independently selected from alkyl:

2. R ７ is a peptide, R ８ teeth, 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】 【Chemistry 5-5】 【Chemistry 5-6】 is selected from the group consisting of Q is, 【Chemistry 6-1】 【Chemistry 6-2】 【Chemistry 6-3】 【Chemistry 6-4】 【Chemistry 6-5】 is selected from the group consisting of R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ９ , R １０ , R １１ , and R １２ are each independently H, C １－４ Alkyl, C １－４ Alkenyl, C ６－１０ Aryl, 5-10 membered heteroaryl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ wherein said C １－４ Alkyl, C １－４ Alkenyl, C ６－１０ Aryl and 5- to 10-membered heteroaryl are each halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R １ and R ２ together with the carbon atom to which they are attached, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ C optionally substituted with 1, 2, or 3 substituents independently selected from ３－７ forming a cycloalkyl group, Or, R １ and R ３ together with the carbon atom to which they are attached, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ C optionally substituted with 1, 2, or 3 substituents independently selected from ３－７ forming a cycloalkyl group, Or, R ２ and R ３ together with the carbon atom to which they are attached, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ C optionally substituted with 1, 2, or 3 substituents independently selected from ３－７ forming a cycloalkyl group, Or, R ３ and R ４ together with the carbon atom to which they are attached, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ C optionally substituted with 1, 2, or 3 substituents independently selected from ３－７ forming a cycloalkyl group, Or, R ５ and R ６ together with the carbon atom to which they are attached, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ C optionally substituted with 1, 2, or 3 substituents independently selected from ３－７ forming a cycloalkyl group, R １３ is H or C １－６ is alkyl, A is H or C １－４ is alkyl, R ａ１ , R ｂ１ , R ｃ１ , and R ｄ１ are H and C, respectively. １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Haloalkyl, OH, CN, NO ２ , and CO ２ CH ３ wherein said C １－６ Alkyl and C ２－６ Alkenyl is OH, CN, NO ２ , or CO ２ optionally substituted with CH; 【Chemistry 7】 is C ６－１０ aryl or 5- to 10-membered heteroaryl, wherein the 5- to 10-membered heteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4 ring-forming heteroatoms independently selected from N, O, and S; [N,O,S] is NH, O, or S; [N,O] is NH or O; [C,N,O] is CR Ｘ R Ｙ , NH, or O, and Each R Ｘ and R Ｙ is H and C １－４ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R is independently selected from alkyl;

3. The R ７ R across cell membranes with acidic or hypoxic mantles having a pH below about 6.0 ８ 3. The compound according to claim 1 or 2, which is a peptide capable of selectively delivering Q-, or a pharmaceutically acceptable salt thereof.

4. The R ７ But the following array ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1; Pv1), AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2; Pv2), ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3; Pv3), Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4; Pv4), and AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO: 5; Pv5), Here, the R ７ But, R ７ 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein:

5. The R ７ But the following array ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1; Pv1), AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2; Pv2), and ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3; Pv3), Here, the R ７ But, R ７ 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein:

6. The R ７ 3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein is a peptide comprising the sequence: ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1, Pv1).

7. The R ７ 3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein is a peptide comprising the sequence: AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2, Pv2).

8. The Q is 【Chemistry 8】 8. The compound according to any one of claims 1 to 7, wherein:

9. The R １ , R ２ , R ３ , and R ４ are each independently H, C １－４ Alkyl, C １－４ Alkenyl, C ６－１０ Aryl, 5-10 membered heteroaryl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ wherein said C １－４ Alkyl, C １－４ Alkenyl, C ６－１０ Aryl and 5- to 10-membered heteroaryl are each halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ 9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, optionally substituted with 1, 2, or 3 substituents independently selected from:

10. The R １ and R ３ together with the carbon atoms to which they are attached, C ３－１４ cycloalkyl group or 4- to 14-membered heterocycloalkyl group, each of which is １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ 9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, optionally substituted with 1, 2, or 3 substituents independently selected from:

11. The R １ and R ３ together with the carbon atoms to which they are attached form cyclopentyl, cyclohexyl, cycloheptyl, 1,2,3,4-tetrahydronaphthyl, tetrahydrofuranyl, or tetrahydropyranyl, or a pharmaceutically acceptable salt thereof.

12. The R １ and R ３ together with the carbon atoms to which they are attached, C ３－７ The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, which forms a cycloalkyl group.

13. The R １ and R ３ 9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein: together with the carbon atom to which they are attached, form a cyclohexyl group.

14. The Q is 【Chemistry 9】 9. The compound according to any one of claims 1 to 8, wherein:

15. The Q is 【Chemistry 10】 9. The compound according to any one of claims 1 to 8, wherein:

16. The R １ and R ２ are each independently selected from H and methyl; ３ , R ４ , R ５ , and R ６ The compound of any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, wherein each is H.

17. The R １ and R ２ are each independently selected from H and methyl, or a pharmaceutically acceptable salt thereof.

18. The R １ and R ２ The compound of any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, wherein each is H.

19. The R １ and R ２ together with the carbon atoms to which they are attached, C ３－７ The compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, which forms a cycloalkyl group.

20. The R １ and R ２ 16. The compound of any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, wherein: together with the carbon atom to which they are attached, form a cyclobutyl group.

21. The R ３ and R ４ The compound according to any one of claims 1 to 8 and 16 to 20, or a pharmaceutically acceptable salt thereof, wherein each is H.

22. The R ２ and R ４ The compound according to any one of claims 1 to 8 and 16 to 20, or a pharmaceutically acceptable salt thereof, wherein each is H.

23. The R ５ and R ６ The compound according to any one of claims 1 to 8 and 16 to 20, or a pharmaceutically acceptable salt thereof, wherein each is H.

24. The R ９ , R １０ , R １１ , and R １２ are each independently selected from H and methyl, or a pharmaceutically acceptable salt thereof.

25. The R ９ , R １０ , R １１ , and R １２ 24. The compound of any one of claims 1 to 23, or a pharmaceutically acceptable salt thereof, wherein each is H.

26. The R ８ but, 【Chemistry 11】 The compound according to any one of claims 1 to 25,

27. Formula (II): 【Chemistry 12】 10. The compound of claim 1 having the formula: R ７ is a peptide, R ８ is a topoisomerase I inhibitor, Ring Z is a monocyclic C ５－７ a cycloalkyl ring or a monocyclic 5- to 7-membered heterocycloalkyl ring; Each RZ is independently １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ is selected from or two adjacent RZ together with the atoms to which they are attached form a fused monocyclic C ５－７ Cycloalkyl ring, fused monocyclic 5- to 7-membered heterocycloalkyl ring, fused C ６－１０ aryl ring, or a fused 6- to 10-membered heteroaryl ring, each of which is １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ and optionally substituted with 1, 2, or 3 substituents independently selected from R ａ１ , R ｂ１ , R ｃ１ , and R ｄ１ are independently H, C １－４ Alkyl, C ２－４ Alkenyl, C ２－４ alkynyl, halo, OH, CN, and NO, respectively. ２ and The compound or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, 2, or 3.

28. The R ７ 28. The compound of claim 27, or a pharmaceutically acceptable salt thereof, wherein: is a peptide comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

5.

29. The R ７ 28. The compound of claim 27, or a pharmaceutically acceptable salt thereof, wherein is Pv1, Pv2, Pv3, Pv4, or Pv5.

30. The R ７ is the R ７ and one of the sulfur atoms of the disulfide moiety in Formula II is derived from the cysteine residue, or a pharmaceutically acceptable salt thereof.

31. The R ８ The compound according to any one of claims 27 to 30, or a pharmaceutically acceptable salt thereof, wherein the compound is camptothecin, topotecan, irinotecan (CPT-11), ciratecan (DB-67, AR-67), cositecan (BNP-1350), lutotecan, gimatecan (ST1481), belotecan (CKD-602), rubitecan, topotecan, deruxtecan, or exatecan.

32. The R ８ The compound according to any one of claims 27 to 30, or a pharmaceutically acceptable salt thereof, wherein is exatecan.

33. The R ８ or a pharmaceutically acceptable salt thereof.

34. The ring Z is a monocyclic C ５－７ 34. The compound according to any one of claims 27 to 33, or a pharmaceutically acceptable salt thereof, which is a cycloalkyl ring.

35. The compound according to any one of claims 27 to 33, or a pharmaceutically acceptable salt thereof, wherein the ring Z is a cyclopentyl ring.

36. The compound according to any one of claims 27 to 33, or a pharmaceutically acceptable salt thereof, wherein the ring Z is a cyclohexyl ring.

37. The compound according to any one of claims 27 to 33, or a pharmaceutically acceptable salt thereof, wherein the ring Z is a cycloheptyl ring.

38. The compound according to any one of claims 27 to 33, or a pharmaceutically acceptable salt thereof, wherein ring Z is a monocyclic 5- to 7-membered heterocycloalkyl ring.

39. 34. The compound according to any one of claims 27 to 33, or a pharmaceutically acceptable salt thereof, wherein ring Z is a 5-membered heterocycloalkyl ring.

40. The compound according to any one of claims 27 to 30, or a pharmaceutically acceptable salt thereof, wherein ring Z is a 6-membered heterocycloalkyl ring.

41. The compound according to any one of claims 27 to 30, or a pharmaceutically acceptable salt thereof, wherein ring Z is a 7-membered heterocycloalkyl ring.

42. Two adjacent RZ, together with the atoms to which they are attached, form a fused monocyclic C ５－７ Cycloalkyl ring, fused monocyclic 5- to 7-membered heterocycloalkyl ring, fused C ６－１０ aryl ring, or a fused 6- to 10-membered heteroaryl ring, each of which is １－４ Alkyl, halo, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , and N.R. ｃ１ C(O)NR ｃ１ R ｄ１ 42. The compound according to any one of claims 27 to 41, or a pharmaceutically acceptable salt thereof, optionally substituted with 1, 2, or 3 substituents independently selected from:

43. 42. The compound according to any one of claims 27 to 41, wherein n is 0, or a pharmaceutically acceptable salt thereof.

44. The compound according to any one of claims 27 to 41, wherein n is 1, or a pharmaceutically acceptable salt thereof.

45. The compound according to any one of claims 27 to 42, or a pharmaceutically acceptable salt thereof, wherein n is 2.

46. The compound according to any one of claims 27 to 42, or a pharmaceutically acceptable salt thereof, wherein n is 3.

47. The compound is represented by formula (III), formula (IV) or formula (V): 【Chemistry 13】 The compound according to claims 27 to 33 and 42 to 46, or a pharmaceutically acceptable salt thereof, wherein

48. 【Chemical 14-1】 【Chemistry 14-2】 【Chemistry 14-3】 【Chemistry 14-4】 2. The compound of claim 1, selected from: or a pharmaceutically acceptable salt thereof.

49. 【Chemical 15-1】 【Chemistry 15-2】 【Chemistry 15-3】 【Chemistry 15-4】 【Chemistry 15-5】 【Chemistry 15-6】 2. The compound of claim 1, selected from: or a pharmaceutically acceptable salt thereof.

50. 50. A pharmaceutical composition comprising a compound according to any one of claims 1 to 49 or a pharmaceutically acceptable salt thereof.

51. 50. A method of treating cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a compound of any one of claims 1 to 49, or a pharmaceutically acceptable salt thereof.

52. 52. The method of claim 51, wherein the cancer is selected from bladder cancer, bone cancer, glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, epithelial cancer, esophageal cancer, Ewing's sarcoma, pancreatic cancer, gallbladder cancer, gastric cancer, gastrointestinal tumors, head and neck cancer, intestinal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liver cancer, lung cancer, melanoma, prostate cancer, rectal cancer, renal clear cell carcinoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer.

53. 52. The method of claim 51, wherein the cancer is selected from breast cancer, colorectal cancer, and gastric cancer.

54. 54. The method of claim 52 or 53, wherein the breast cancer is triple-negative breast cancer.

55. The following structure: 【Chemistry 16】 or a salt thereof.

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