Peptide conjugates of cytotoxins as therapeutic agents

Abstract

The present invention relates to peptide conjugates of cytotoxins, such as topoisomerase I inhibitors, which are useful in the treatment of diseases such as cancer.

Description

TECHNICAL FIELD

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

[0003] Cancer is a group of diseases that is characterized by abnormal control of cell growth. In the United States alone, the annual incidence of cancer is estimated to be over 1.6 million. Despite surgery, radiation, chemotherapy, and hormones, cancer remains the second leading cause of death in the United States. It is estimated that approximately 600,000 Americans will die from cancer each year.

[0004] Treatment of cancer in humans by systemic administration of pharmaceutical agents typically works by slowing or stopping the uncontrolled replication that is characteristic of cancer cells. One class of such agents is topoisomerase I inhibitors. Topoisomerase 1 enzymes function to relax supercoiled DNA and relieve DNA helical constraints and play a role in transcription regulation. See Li, M., Genomics Proteomics Bioinformatics 14 (2016), 166-171. Topoisomerase I is essential for development of mammalian systems due to its dynamic functions in DNA replication and transcription. However, due to its direct role in transcription regulation, topoisomerase I dysfunction can lead to cellular abnormalities. See Li, M., Genomics Proteomics Bioinformatics 14 (2016), 166-171. Thus, several human diseases, such as cancer, neurodegenerative diseases, and autoimmune diseases, are associated with topoisomerase I regulation and activity.

[0005] Inhibitors of topoisomerase I have been developed and will continue to be developed as anticancer agents. In particular, topoisomerase I inhibitors are widely used to treat colorectal, gastric, and other cancers. See Ogitani, Bioorg. Med. Chem. Lett. 26 (2016), 5069-5072. While topoisomerase I inhibitors are useful in treating cancer, the compounds also exhibit side effects including neutropenia and severe diarrhea. Preferential delivery of topoisomerase inhibitors to these diseased tissues can avoid these severe side effects. Thus, there is a need for more selective delivery of topoisomerase I inhibitors to diseased tissues. SUMMARY

[0006] The present disclosure provides, inter alia, a compound of Formula (I):

[0007] R 8 -Q-R 7 (I)

[0008] or a pharmaceutically acceptable salt thereof, wherein the variables constituting are defined herein.

[0009] The present disclosure further provides a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0010] The present disclosure also provides methods of treating a disease or condition (e.g., cancer) by administering to a human or other mammal in need of treatment a therapeutically effective amount of a compound of the present disclosure. In some embodiments, the disease or condition is characterized by acidic or hypoxic diseased tissue.

[0011] The present disclosure also provides the use of a compound described herein for the manufacture of a medicament for therapy. The present disclosure also provides a compound described herein for use in therapy.

[0012] The present disclosure also provides methods for synthesizing compounds of the present disclosure and intermediates useful in these methods. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 A plot showing plasma concentrations of Compound 11 and released exatecan following a single IV dose of 5 mg / kg of Compound 11 in rats (data presented as mean ± SEM).

[0014] Figure 2 A plot showing peptide concentrations in tumor and bone marrow following a single IP dose of 10 mg / kg of Compound 11 in mice (data presented as mean ± SEM).

[0015] Figure 3 A plot showing total bone marrow counts of femur of tumor-bearing nude mice following a single dose of 2.6 and 5.2 micromole per kilogram of Compound 11 (equivalent to 10, 20 mg / kg conjugate) or free exatecan (equivalent to 1.15 and 2.3 mg / kg exatecan), once daily for four days (data presented as mean ± SEM).

[0016] Figure 4A A resected stomach of a tumor-bearing nude mouse following a single dose of vehicle or 5.2 micromole per kilogram of Compound 11 (equivalent to 20 mg / kg conjugate) or free exatecan (equivalent to 2.3 mg / kg exatecan), once daily for four days.

[0017] Figure 4BGastric of orthotopically tumor-bearing nude mice following administration of 5.2 micromole per kilogram of Compound 11 (equivalent to 20 mg / kg conjugate) or free exatecan (equivalent to 2.3 mg / kg exatecan) once daily for four days.

[0018] Figure 5A Plot of mean tumor volume in nude mice bearing HCT116 colorectal flank tumors resulting from administration of equimolar amounts of free exatecan or Compound 11. Animals were dosed parenterally once daily, four times per week, for three weeks.

[0019] Figure 5B Kaplan Meier survival curves in nude mice bearing HCT116 colorectal flank tumors following administration of equimolar amounts of free exatecan or Compound 11.

[0020] Figure 6A Plot of mean tumor volume in nude mice bearing MKN45 HER2-negative gastric cancer flank tumors resulting from administration of Compound 11. Animals were dosed parenterally once daily, four times per week, for two weeks.

[0021] Figure 6B Kaplan Meier survival curves in nude mice bearing MKN45 HER2-negative gastric cancer flank tumors following administration of equimolar amounts of free exatecan or Compound 11.

[0022] Figure 7A Plot of mean tumor volume in SCID mice bearing JIMT-1 HER2 moderate breast cancer flank tumors resulting from administration of Compound 11. Animals were dosed parenterally once daily, four times per week, for three weeks.

[0023] Figure 7B Plot of percent body weight change in SCID mice bearing JIMT-1 HER2 moderate breast cancer flank tumors dosed with Compound 11.

[0024] Figure 8A Plot of mean tumor volume in nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11. Animals were dosed parenterally once daily, four times per week, for three weeks.

[0025] Figure 8B Plot of percent body weight change relative to Day 0 in nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11.

[0026] Figure 9AA plot showing the mean tumor volume of nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11 and talazoparib. Animals were dosed with Compound 11 parenterally once daily, four times per week for three weeks and talazoparib orally once daily for 18 days.

[0027] Figure 9B A plot showing the percent change in body weight from day 0 of nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11 and talazoparib.

[0028] Figure 10 A graph showing the degradation of Compound 11 and Compound 29 over 16h caused by treatment with 10 mM glutathione. As shown in Figure 10 Compound 29 is released more rapidly than Compound 11 under similar glutathione exposure. DETAILED DESCRIPTION

[0029] Provided herein is a compound of Formula (I):

[0030] R 8 -Q-R 7 (I)

[0031] or a pharmaceutically acceptable salt thereof, wherein:

[0032] R 7 is a peptide;

[0033] R 8 is a small molecule topoisomerase I targeting moiety that binds to topoisomerase I; and

[0034] Q is a linker covalently attached to moiety R 7 and R 8 .

[0035] Provided herein is a compound of Formula (I):

[0036] R 8 -Q-R 7 (I)

[0037] or a pharmaceutically acceptable salt thereof, wherein:

[0038] R 7 is a peptide capable of selectively delivering R 8 Q- across a cell membrane having an acidic or hypoxic envelope membrane with a pH less than about 6.0;

[0039] R 8 is a small molecule topoisomerase I targeting moiety that binds to topoisomerase I; and

[0040] Q is a linker covalently attached to moiety R 7 and R 8 .

[0041] Provided herein is a compound of Formula (I):

[0042] R 8 -Q-R 7 (I)

[0043] or a pharmaceutically acceptable salt thereof, wherein:

[0044] R 7 is a peptide;

[0045] R 8 is selected from the group consisting of:

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] Q is a linker covalently attached to moiety R 7 and R 8 .

[0055] Provided herein is a compound of Formula (I):

[0056] R 8 -Q-R 7 (I)

[0057] or a pharmaceutically acceptable salt thereof, wherein:

[0058] R 7 is a peptide;

[0059] R 8 is selected from the group consisting of:

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] and

[0067] Q is a linker covalently attached to moiety R 7 and R 8 .

[0068] Provided herein is a compound of Formula (I):

[0069] R 8 -Q-R 7 (I)

[0070] or a pharmaceutically acceptable salt thereof, wherein:

[0071] R 7 is a peptide;

[0072] R 8 is selected from the group consisting of:

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] Q is selected from the group consisting of:

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 9 , R 10 , R 11 , and R 12 are each independently selected from 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 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR 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, 5-10 membered heteroaryl, and 4-10 membered heterocycloalkyl are each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1C(O)NR c1 R d1 ;

[0087] or R 1 and R 2 together with the carbon atom to which they are attached form a C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from C 1-4 alkyl, halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0088] or R 1 and R 3 together with the carbon atom to which they are attached form a C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from C 1-4 alkyl, halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0089] or R 2 and R3 with the carbon atom to which it is attached forms C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from C 1-4 alkyl, halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0090] or R 3 and R 4 with the carbon atom to which it is attached forms C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from C 1-4 alkyl, halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0091] or R 5 and R 6 with the carbon atom to which it is attached forms C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from C1-4 alkyl, halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0092] R 13 is H or C 1-6 alkyl;

[0093] A is H or C 1-4 alkyl;

[0094] R a1 , R b1 , R c1 , and R d1 are each independently selected from H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, OH, CN, NO2, and CO2CH3; wherein said C 1-6 alkyl and C 2-6 alkenyl are each optionally substituted with OH, CN, NO2, or CO2CH3;

[0095] is C 6-10 aryl or 5-10 membered heteroaryl; wherein said 5-10 membered heteroaryl has at least one ring carbon atom and 1, 2, 3, or 4 ring heteroatoms independently selected from N, O, and S;

[0096] Ring G is C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from C 1-4 alkyl, halo, CN, NO2, OR a1 , SR a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)ORa1 OC(O)R b1 OC(O)NR c1 R d1 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ;

[0097] [N, O, S] is NH, O, or S;

[0098] [N, O] is NH or O;

[0099] [C, N, O] is CR X R Y , NH, or O; and

[0100] each R X and R Y is independently selected from H and C 1-4 alkyl.

[0101] Provided herein is a compound of Formula (I):

[0102] R 8 -Q-R 7 (I)

[0103] or a pharmaceutically acceptable salt thereof, wherein:

[0104] R 7 is a peptide;

[0105] R 8 is selected from the group consisting of:

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] Q is selected from the group consisting of:

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 9 , R 10 , R 11 and R 12 are each independently selected from H, C 1-4 alkyl, C 1-4 alkenyl, C 6-10 aryl, 5-10 membered heteroaryl, halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 wherein said C 1-4 alkyl, C 1-4 alkenyl, C 6-10 aryl, and 5-10 membered heteroaryl are each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , OC(O)R b1 , OC(O)NR c1 R d1NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ;

[0120] or R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl, optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ;

[0121] or R 1 and R 3 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl, optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 Rd1 ;

[0122] or R 2 and R 3 together with the carbon atom to which they are attached form C 3-7 cycloalkyl optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0123] or R 3 and R 4 together with the carbon atom to which they are attached form C 3-7 cycloalkyl optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0124] or R 5 and R 6 together with the carbon atom to which they are attached form C 3-7 cycloalkyl optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, ORa1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ;

[0125] R 13 is H or C 1-6 alkyl;

[0126] A is H or C 1-4 alkyl;

[0127] R a1 , R b1 , R c1 and R d1 are each independently selected from H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, OH, CN, NO2, and CO2CH3; wherein said C 1-6 alkyl and C 2-6 alkenyl are each optionally substituted with OH, CN, NO2, or CO2CH3;

[0128] is C 6-10 aryl or 5-10 membered heteroaryl; wherein said 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;

[0129] [N,O,S] is NH, O, or S;

[0130] [N,O] is NH or O;

[0131] [C,N,O] is CR X R Y , NH, or O; and

[0132] each R X and R Y is independently selected from H and C 1-4 alkyl.

[0133] In some embodiments, the left-hand side of Q is connected to R 8 and the right-hand side of Q is connected to R 7 .

[0134] In some embodiments, the sulfur atom of the disulfide moiety of Q is part of a cysteine residue of R 7 .

[0135] As used herein, a "peptide" refers to a targeting moiety comprising a sequence of 10-50 amino acids composed of naturally occurring amino acid residues and optionally one or more non-naturally occurring amino acids. In some embodiments, the peptide of R 7 is a 20 to 40, 20 to 30 amino acid, or 30 to 40 residue peptide. Peptides suitable for use in the compounds of the application are those that can insert into a cell membrane via a conformational change or secondary structure change in response to changes in environmental pH. In this manner, the peptide can target acidic tissue and selectively translocate a polar, cell-impermeable molecule across a cell membrane in response to low extracellular pH. In some embodiments, the peptide is capable of selectively delivering a conjugated moiety (e.g., R 8 Q-) across a cell membrane having an acidic or hypoxic mantle membrane with a pH less than about 6.0. In some embodiments, the peptide is capable of selectively delivering a conjugated moiety (e.g., R 8 Q-) across a cell membrane having an acidic or hypoxic mantle membrane with a pH less than about 6.5. In some embodiments, the peptide is capable of selectively delivering a conjugated moiety (e.g., R 8 Q-) across a cell membrane having an acidic or hypoxic mantle membrane with a pH less than about 5.5. In some embodiments, the peptide is capable of selectively delivering a conjugated moiety (e.g., R 8 Q-) across a cell membrane having an acidic or hypoxic mantle membrane with a pH between about 5.0 and about 6.0.

[0136] In certain embodiments, the peptide of R 7 includes a cysteine residue that can form a site of attachment to a payload moiety (e.g., R 8 Q-) to be delivered across a cell membrane. In some embodiments, R 7 is connected to Q via a cysteine residue of R 7 . In some embodiments, the sulfur atom of the cysteine residue can form part of a disulfide bond of the disulfide-containing linker Q.

[0137] Suitable peptides that can undergo a conformational change based on pH and insert into a cell membrane are described, for example, in U.S. Patents 8,076,451 and 9,289,508 (each of which is incorporated by reference herein in its entirety). Other suitable peptides are described, for example, in Weerakkody et al., PNAS 110(15), 5834-5839 (April 9, 2013), which is also incorporated by reference herein in its entirety.

[0138] In some embodiments, R 7 is a peptide comprising at least one of the following sequences:

[0139] ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1; Pv1),

[0140] AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO. 2; Pv2), and

[0141] ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3);

[0142] Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO. 4; Pv4); and

[0143] AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID No. 5; Pv5);

[0144] wherein R 7 is connected to Q via a cysteine residue of R 7 .

[0145] In some embodiments, R 7 is a peptide comprising at least one of the following sequences:

[0146] ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1; Pv1),

[0147] AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO. 2; Pv2), and

[0148] ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3),

[0149] wherein R7 R is a peptide comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1 ; Pv1). 7 R is a peptide comprising the sequence AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO. 2; Pv2).

[0150] In some embodiments, R is a peptide comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1 ; Pv1). 7 R is a peptide comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1 ; Pv1).

[0151] In some embodiments, R is a peptide comprising the sequence AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO. 2; Pv2). 7 R is a peptide comprising the sequence AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO. 2; Pv2).

[0152] In some embodiments, R is a peptide comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3). 7 R is a peptide comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3).

[0153] In some embodiments, R is a peptide comprising the sequence Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO. 4; Pv4). 7 R is a peptide comprising the sequence Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO. 4; Pv4).

[0154] In some embodiments, R is a peptide comprising the sequence AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO. 5; Pv5). 7 R is a peptide comprising the sequence AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO. 5; Pv5).

[0155] In some embodiments, R is a peptide consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1 ; Pv1). 7 R is a peptide consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1 ; Pv1).

[0156] In some embodiments, R is a peptide consisting of the sequence AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO. 2; Pv2). 7 R is a peptide consisting of the sequence AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO. 2; Pv2).

[0157] In some embodiments, R is a peptide consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3). 7 R is a peptide consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3).

[0158] In some embodiments, R is a peptide consisting of the sequence Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO. 4; Pv4). 7is a peptide consisting of the sequence Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO. 4; Pv4).

[0159] In some embodiments, R 7 is a peptide consisting of the sequence AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO. 5; Pv5).

[0160] 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.

[0161] In some embodiments, R 7 is a peptide consisting of a sequence selected from SEQ ID NO: 6 to SEQ ID NO: 311 as shown in Table 1.

[0162] Table 1. Additional R 7 sequences

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172] Any of the peptides useful in the present application can be modified to include a cysteine residue by replacing a non-cysteine residue with a cysteine or appending a cysteine residue to the N-terminus or C-terminus.

[0173] In some embodiments, R 7Peptides of the application are conformationally constrained peptides. Conformationally constrained peptides can include, for example, macrocyclic peptides and stapled peptides. Stapled peptides are peptides constrained by covalent bonds between two amino acid side chains to form a macrocycle of the peptide. Conformationally constrained 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.

[0174] The term "small molecule topoisomerase I targeting moiety" or "topoisomerase I inhibitor" refers to a chemical group that binds to topoisomerase I. The 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), silatecan (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.

[0175] moiety Q is a covalent linkage that serves as a tether between the peptide and a topoisomerase I inhibitor 7 and a linker group R 8 that can be cleaved when the conjugate or a portion thereof is inside a cell. In some embodiments, Q is a chain having 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 chain atoms, optionally substituted with 1 to 10 R q substituents, and wherein one or more chain carbon atoms of Q can be oxidized to form a carbonyl (C=0), and wherein one or more N and S chain atoms can each be optionally oxidized to form an amine oxide, a sulfoxide, or a sulfonyl group; wherein

[0176] each R q is independently selected from OH, CN, -COOH, NH2, halo, C 1-6 haloalkyl, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 1-6alkyl, 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, wherein R q is H, alkyl, phenyl, C 1-6 cycloalkyl, 4-6 membered heterocycloalkyl, and 5-6 membered heteroaryl, each optionally substituted with halo, OH, CN, -COOH, NH2, C 3-6 haloalkyl, C 1-4 haloalkoxy, phenyl, C 1-4 cycloalkyl, 5-membered or 6-membered heteroaryl, or 4-6 membered heterocycloalkyl; and 1-4 haloalkyl, C 1-4 haloalkoxy, phenyl, C 3-10 cycloalkyl, 5-membered or 6-membered heteroaryl, or 4-6 membered heterocycloalkyl; and

[0177] two R q groups, together with the chain atoms to which they are attached, can form a phenyl, 5-6 membered heteroaryl, 4-6 membered heterocycloalkyl, or C 3-6 cycloalkyl ring.

[0178] In some embodiments, R q is independently selected from 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.

[0179] In some embodiments, Q is selected from:

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] In some embodiments, Q is:

[0186]

[0187] In some embodiments, Q is:

[0188]

[0189] In some embodiments, Q is:

[0190]

[0191] In some embodiments, Q is:

[0192]

[0193] In some embodiments, Q is:

[0194]

[0195] In some embodiments:

[0196] R 1 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of H and C 1-4 alkyl, halo, CN, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 , wherein the C 1-4 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NRc1 R d1 ;

[0197] or R 1 and R 2 , together with the carbon atom to which they are attached, form a C 3-10 cycloalkyl or 4-10 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0198] or R 1 and R 3 , together with the carbon atom to which they are attached, form a C 3-10 cycloalkyl or 4-10 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0199] or R 1 and R 4 , together with the carbon atom to which they are attached, form a C 3-10cycloalkyl or 4-10 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0200] or R 2 and R 3 together with the carbon atom to which they are attached form a C 3-10 cycloalkyl or 4-10 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0201] or R 2 and R 4 together with the carbon atom to which they are attached form a C 3-10 cycloalkyl or 4-10 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR a1 , C(O)R b1 , C(O)NRc1 R d1 C(O)OR a1 OC(O)R b1 OC(O)NR c1 R d1 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ;

[0202] or R 3 and R 4 together with the carbon atom to which they are attached form a C 3-10 cycloalkyl or 4-10 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 .

[0203] In some embodiments:

[0204] R 1 , R 2 , R 3 , and R 4 are each independently selected from H and C 1-4 alkyl;

[0205] or R 1 and R 2 together with the carbon atom to which they are attached form a C 3-10 cycloalkyl or 4-10 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0206] or R 1 and R 3 together with the carbon atom to which they are attached form a C 3-10 cycloalkyl or 4-10 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0207] or R 3 and R 4 together with the carbon atom to which they are attached form a C 3-10 cycloalkyl or 4-10 membered heterocycloalkyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , OC(O)R b1 , OC(O)NR c1 Rd1 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 .

[0208] In some embodiments, R 1 and R 2 are each independently selected from H and methyl, and R 3 , R 4 , R 5 and R 6 are each hydrogen.

[0209] In some embodiments, R 1 , R 2 , R 3 and R 4 are each independently selected from H and methyl, and R 5 and R 6 are each hydrogen.

[0210] In some embodiments, R 1 and R 2 are each independently selected from H and methyl.

[0211] In some embodiments, R 3 and R 4 are each independently selected from H and methyl.

[0212] In some embodiments, R 1 and R 2 are each H.

[0213] In some embodiments, R 1 and R 2 , together with the carbon atom to which they are attached, form a C 3-7 cycloalkyl: halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 .

[0214] In some embodiments, R 1 and R 2 , together with the carbon atom to which they are attached, form a C 3-7 cycloalkyl.

[0215] In some embodiments, R 1 and R 2 , together with the carbon atom to which they are attached, form a cyclobutyl.

[0216] In some embodiments, R 3 and R 4 are each H.

[0217] In some embodiments, R 1 and R 3 , together with the carbon atom to which they are attached, form a C 3-7 cycloalkyl optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 .

[0218] In some embodiments, R 1 and R 3 , together with the carbon atom to which they are attached, form a cyclopentyl, cyclohexyl, cycloheptyl, 1,2,3,4-tetrahydronaphthyl, tetrahydrofuranyl, or tetrahydropyranyl.

[0219] In some embodiments, R 1 and R 3 , together with the carbon atom to which they are attached, form a C 3-7 cycloalkyl.

[0220] In some embodiments, R 1 and R 3 are each H.

[0221] In some embodiments, R 2 and R 4 are each H.

[0222] In some embodiments, R 5 and R 6 are each H.

[0223] In some embodiments, R 9 , R 10 , R 11 , and R 12 are each independently selected from H and methyl.

[0224] In some embodiments, the compound of the application is a compound of Formula (II):

[0225]

[0226] or a pharmaceutically acceptable salt thereof, wherein:

[0227] R 7 is a peptide;

[0228] R 8 is a topoisomerase I inhibitor;

[0229] Ring Z is a monocyclic C 5-7 cycloalkyl ring or a monocyclic 5-7 membered heterocycloalkyl ring;

[0230] each R Z is independently selected from C 1-4 alkyl, halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 , and NR c1 C(O)NR c1 R d1 ;

[0231] or two adjacent R Z together with the atom to which they are attached form a fused monocyclic C 5-7 cycloalkyl ring, a fused monocyclic 5-7 membered heterocycloalkyl ring, a fused C 6-10 aromatic ring or a fused 6-10 membered heteroaromatic ring, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of C 1-4 alkyl, halo, CN, NO2, OR a1 , SR 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 , NR c1 R d1 , NR c1 C(O)R b1 , NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ;

[0232] R a1 , R b1 , R c1 and R d1 are each independently selected from the group consisting of H, C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halo, OH, CN, and NO2; and

[0233] n is 0, 1, 2, or 3.

[0234] 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.

[0235] In some embodiments of the compound of Formula (II), R 7 is Pv1, Pv2, Pv3, Pv4, or Pv5.

[0236] In some embodiments of the compound of Formula (II), R 7 via R 7The cysteine residue is linked to the core, wherein one of the sulfur atoms of the disulfide moiety in Formula II is derived from the cysteine residue.

[0237] In some embodiments of the compound of Formula (II), R 8 is camptothecin, opotecan, irinotecan (CPT-11), silatecan (DB-67, AR-67), cositecan (BNP-1350), lurtotecan, gimatecan (ST1481), belotecan (CKD-602), rubitecan, topotecan, deruxitecan, or exatecan.

[0238] In some embodiments of the compound of Formula (II), R 8 is exatecan.

[0239] In some embodiments of the compound of Formula (II), R 8 is linked to the core via an N atom.

[0240] In some embodiments of the compound of Formula (II), ring Z is a monocyclic C 5-7 cycloalkyl ring.

[0241] In some embodiments of the compound of Formula (II), ring Z is a cyclopentyl ring.

[0242] In some embodiments of the compound of Formula (II), ring Z is a cyclohexyl ring.

[0243] In some embodiments of the compound of Formula (II), ring Z is a cycloheptyl ring.

[0244] In some embodiments of the compound of Formula (II), ring Z is a monocyclic 5-7 membered heterocycloalkyl ring.

[0245] In some embodiments of the compound of Formula (II), ring Z is a 5-membered heterocycloalkyl ring.

[0246] In some embodiments of the compound of Formula (II), ring Z is a 6-membered heterocycloalkyl ring.

[0247] In some embodiments of the compound of Formula (II), ring Z is a 7-membered heterocycloalkyl ring.

[0248] 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, a fused monocyclic 5-7 membered heterocycloalkyl ring, a fused C 6-10 aromatic ring, or a fused 6-10 membered heteroaromatic ring, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of C 1-4 alkyl, halo, CN, NO2, OR a1SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 .

[0249] In some embodiments of the compound of Formula (II), n is 0.

[0250] In some embodiments of the compound of Formula (II), n is 1.

[0251] In some embodiments of the compound of Formula (II), n is 2.

[0252] In some embodiments of the compound of Formula (II), n is 3.

[0253] In some embodiments, the compound of the application is a compound of Formula (III), Formula (IV), or Formula (V):

[0254]

[0255] or a pharmaceutically acceptable salt thereof, wherein R 7 , R 8 , R Z and n are as defined above in relation to any one of the embodiments of Formula (II).

[0256] In some embodiments, the compound of Formula (I) is selected from:

[0257]

[0258]

[0259]

[0260] or a pharmaceutically acceptable salt of any of the foregoing.

[0261] In some embodiments, the compound of Formula (I) is selected from:

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] In some embodiments, provided herein is a compound of Formula (IIA):

[0269]

[0270] or a salt thereof, wherein:

[0271] Cy is C 1 is C 6-10 aryl or 5-10 membered heteroaryl; wherein the 5-10 membered heteroaryl has at least one ring carbon atom and 1, 2, 3, or 4 ring heteroatoms independently selected from N, O, and S; and wherein the C 6-10 aryl and 5-10 membered heteroaryl are each optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of C 1-4 alkyl, halo, OH, C 1-6 alkoxy, CN, and NO2;

[0272] and R 8 , ring Z, R Z , and n are as defined herein.

[0273] In some embodiments, Cy 1 is 5-10 membered heteroaryl. In some embodiments, Cy 1 is pyridyl. In some embodiments, Cy 1 is phenyl.

[0274] In some embodiments, the compound of Formula (IIA) has the structure:

[0275]

[0276] or a salt thereof.

[0277] In some embodiments, provided herein is a compound of Formula (IIA):

[0278]

[0279] or a salt thereof, for use in the manufacture of a compound of the present application (e.g., a compound of Formula (I) or Formula (II)), wherein Cy1 R 8 Ring Z, R Z R a1 R b1 R c1 R d1 and n are as defined herein.

[0280] In some embodiments, provided herein is a compound having the structure:

[0281]

[0282] or a salt thereof, for use in the manufacture of a compound of the invention (e.g., a compound of Formula (I) or Formula (II)).

[0283] Molecules of the invention can be labeled with a probe such as a fluorophore, a radioisotope, and the like. In some embodiments, the probe is a fluorescent probe, e.g., LICOR. Fluorescent probes can include any moiety that can re-emit light upon photoexcitation (e.g., a fluorophore).

[0284] Amino acids are represented by the following IUPAC abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; 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), valine (Val; V).

[0285] The term“Pv1” means ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1).

[0286] The term“Pv2” means AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2).

[0287] The term“Pv3” means ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3).

[0288] The term“Pv4” means ID NO: 4).

[0289] The term "Pv5" means AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO: 5).

[0290] In the compounds of the application, the peptide R 7 is linked to the disulfide moiety in the linker Q via an amino acid residue comprising a sulfur atom, e.g. a cysteine residue. Typically, the sulfur atom of the disulfide moiety in the linker Q as a point of attachment to the peptide R 7 is derived from an amino acid residue of the peptide, e.g. from a cysteine residue.

[0291] The term "acidic and / or hypoxic mantle" refers to the environment of the cells in the diseased tissue in question which has a pH below 7.0 and preferably below 6.5. The acidic or hypoxic mantle more preferably has a pH of about 5.5 and most preferably a pH of about 5.0. The compound of formula (I) inserts into the cell membrane with the acidic and / or hypoxic mantle in a pH-dependent manner to insert R 8 Q into the cell, whereupon the disulfide linker is cleaved to deliver the free R 8 H. Since the compound of formula (I) is pH-dependent, it preferentially inserts into the cell membrane only in the presence of an acidic or hypoxic mantle around the cell and does not insert into the cell membrane of "normal" cells which do not have an acidic or hypoxic mantle. Examples of cells with an acidic or hypoxic mantle are cancer cells.

[0292] The term "pH sensitivity" or "pH-dependent" as used herein refers to the peptide R 7 or to the mode of insertion of the peptide R 7 or the compound of the application into the cell membrane, meaning that the peptide has a higher affinity for the lipid bilayer of a cell membrane with an acidic or hypoxic mantle than for the membrane lipid bilayer at neutral pH. Thus, when the membrane lipid bilayer of a cell has an acidic or hypoxic mantle ("diseased" cell), the compound of the application preferentially inserts through the cell membrane to insert R 8 Q into the interior of the cell (and thus delivers R 8 H as described above), but does not insert through the cell membrane when the mantle (the environment of the membrane lipid bilayer) is not acidic or hypoxic ("normal" cell). It is believed that this preferential insertion is achieved because the peptide R 7 forms a helical conformation which facilitates membrane insertion.

[0293] It is further appreciated that certain of the application's features that are described in the context of separate embodiments can also be provided in combination in a single embodiment (concurrent embodiments are intended to be within the scope of the application, as are the multiple forms written in the various dependent claims). Conversely, various features that are described in the context of a single embodiment can also be provided separately or in any suitable subcombination. Thus, features described as an embodiment of a compound of Formula (I) can be combined in any suitable combination.

[0294] At various places in the present specification, certain features of the compounds are disclosed in groups or in ranges. It is specifically intended that such a disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term "C 1-6 "alkyl" is intended to individually disclose (but is not limited to) methyl, ethyl, C3alkyl, C4alkyl, C5alkyl, and C6alkyl.

[0295] The term "n-membered" where n is an integer generally describes the number of ring-forming atoms in a moiety, where 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 heteroaromatic ring, pyridinyl is an example of a 6-membered heteroaromatic ring and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl.

[0296] At various places in the present specification, variables defining divalent linking groups can be described. In particular, it is intended that each linking substituent include both the forward and reverse forms of the linking substituent. For example, -NR(CR'R") n - includes -NR(CR'R") n - and -(CR'R") n NR- and is intended to individually disclose each form. When a structure requires a linking group, the Markush variable listed for that group is to be understood as a linking group. For example, if a structure requires a linking group and the Markush group defining the variable lists "alkyl" or "aryl", then "alkyl" or "aryl" is understood to represent an alkylene linking group or an arylene linking group, respectively.

[0297] The term“substituted” means that an atom or atom group is formally replaced by a“substituent” that is attached to another group. Unless otherwise indicated, the term“substituted” refers to any level of substitution, e.g., mono-substituted, di-substituted, tri-substituted, tetra-substituted, or penta-substituted, where such substitution is permitted. The substituents are selected independently and substitution can occur at any available position. It is understood that substitution at a given atom is limited by valency. 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 is removed and replaced by a substituent. A single bivalent substituent (e.g., oxo) can replace two hydrogen atoms.

[0298] The term“C n-m ” indicates a range inclusive of the endpoints, where n and m are integers and indicate the number of carbon atoms. Examples include C 1-4 , C 1-6 , etc.

[0299] The term“alkyl,” used alone or in combination with other terminology, refers to a straight or branched chain saturated hydrocarbon group. The term“C n-m alkyl” refers to an alkyl group having n to m carbons. In some embodiments, an 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, i-propyl, n-butyl, t-butyl, i-butyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like.

[0300] The term“alkenyl,” used alone or in combination with other terminology, refers to a straight or branched chain hydrocarbon group corresponding to an alkyl group having one or more carbon-carbon double bonds. An alkenyl group formally corresponds to an alkene in which one C-H bond is replaced by a point of attachment of the alkenyl group to the rest of the compound. The term“C n-m alkenyl” refers to an alkenyl group having n to m carbons. In some embodiments, an alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, i-propenyl, n-butenyl, sec-butenyl, and the like.

[0301] The term“alkynyl,” used alone or in combination with other terminology, refers to a straight or branched chain hydrocarbon group corresponding to an alkyl group having one or more carbon-carbon triple bonds. An alkynyl group formally corresponds to an alkyne in which one C-H bond is replaced by a point of attachment of the alkyl group to the rest of the compound. The term“C n-m"Alkynyl" refers to an alkynyl group having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-l-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.

[0302] The term "alkylene," used alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene corresponds formally to an alkane in which two C-H bonds are replaced by the points of attachment of the alkylene to the rest of the compound. The term "C n-m "Alkylene" refers to an alkylene having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, eth-1,2-diyl, eth-1,1 -diyl, prop-1,3-diyl, prop-1,2-diyl, prop-1,1 -diyl, but-1,4-diyl, but-1,3-diyl, but-1,2-diyl, 2-methyl-prop-1,3-diyl, and the like.

[0303] The term "amino" refers to a group of formula -NH2.

[0304] The term "carbonyl," used alone or in combination with other terms, refers to a -C(=O)- group, which can also be written as C(O).

[0305] The term "cyano" or "nitrile" refers to a group of formula -C≡N, which can also be written as -CN.

[0306] The term "halo" or "halogen," used alone or in combination with other terms, refers 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.

[0307] The term "haloalkyl" as used herein refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by a halogen atom. The term "C n-m "Haloalkyl" refers to a Cn-m alkyl group having n to m carbon atoms and at least one up to {2(n-m)+l} halogen atoms which can be the same or different. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Example haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCl3, CHCl2, C2Cl5, and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group. n-m Alkyl. In some embodiments, the halogen atom is a fluorine atom. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Example haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCl3, CHCl2, C2Cl5, and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group.

[0308] The term "haloalkoxy," used alone or in combination with other terms, refers to a group of formula -O-haloalkyl, wherein haloalkyl is as defined above. The term "C n-m "Haloalkoxy" refers to a haloalkoxy group in which the haloalkyl group has n to m carbons. Example 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.

[0309] The term“oxo” refers to an oxygen atom as a divalent substituent, which when attached to carbon forms a carbonyl group, or attached to a heteroatom forms a sulfoxide or sulfone group or an N-oxide group. In some embodiments, a heterocyclyl group can be optionally substituted with 1 or 2 oxo (=0) substituents.

[0310] The term“oxidized” with respect to a ring-forming N atom refers to a ring-forming N-oxide.

[0311] The term“oxidized” with respect to a ring-forming S atom refers to a ring-forming sulfonyl or ring-forming sulfinyl.

[0312] The term“aromatic” refers to a carbocyclic or heterocyclic ring having one or more polyunsaturated rings having aromatic character (i.e., having (4n+2) delocalized pi (p) electrons, where n is an integer).

[0313] 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 2 fused rings). The term“C n-m 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 2 fused rings). The term“C

[0314] The term“heteroaryl” or“heteroaromatic,” used alone or in combination with other terms, refers to a monocyclic or polycyclic aromatic heterocyclic ring having at least one ring member that is a heteroatom selected from sulfur, oxygen, and nitrogen. In some embodiments, a heteroaryl ring has 1, 2, 3, or 4 ring members that are heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, a heteroaryl group has 5-14 ring atoms, including carbon atoms and 1, 2, 3, or 4 ring members that are heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, a heteroaryl group has 5-10 ring atoms, including carbon atoms and 1, 2, 3, or 4 ring members that are heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, a heteroaryl group has 5-6 ring atoms and 1 or 2 ring members that are heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, a heteroaryl group is a five- or six-membered heteroaromatic ring. In other embodiments, a heteroaryl group is an eight-, nine-, or ten-membered fused bicyclic heteroaromatic ring.

[0315] A five-membered heteroaromatic ring is a heteroaryl group having five ring atoms, one or more (e.g., 1, 2, or 3) of which are independently selected from N, O, and S.

[0316] A six-membered heteroaromatic ring is a heteroaryl group having six ring atoms, one or more (e.g., 1, 2, or 3) of which are independently selected from N, O, and S.

[0317] The term "cycloalkyl," used alone or in combination with other terms, refers to non-aromatic hydrocarbon ring systems (monocyclic, bicyclic, or polycyclic) that include ring- annulated alkyl and alkenyl groups. The term "C n-m Cycloalkyl" refers to a cycloalkyl group having n to m ring member carbon atoms. Cycloalkyl groups can include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) groups and spirocyclic rings. Cycloalkyl groups can have 3, 4, 5, 6, or 7 ring-forming carbons (C 3-7 ). In some embodiments, cycloalkyl groups have 3 to 6 ring members, 3 to 5 ring members, or 3 to 4 ring members. In some embodiments, cycloalkyl groups are monocyclic. In some embodiments, cycloalkyl groups are monocyclic or bicyclic. In some embodiments, cycloalkyl groups are C 3-6 monocyclic cycloalkyl groups. Ring-forming carbon atoms of cycloalkyl groups can optionally be oxidized to form oxo groups or thiono groups. Cycloalkyl groups also include cycloalkylidenes. In some embodiments, cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Also included in the definition of cycloalkyl are moieties having one or more aromatic rings fused to (i.e., sharing a bond with) the cycloalkyl ring, such as benzo or thienyl derivatives of cyclopentane, cyclohexane, and the like. Cycloalkyl groups containing fused aromatic rings can be attached via any ring-forming atom of the fused aromatic ring. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, and the like. In some embodiments, cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0318] The term "heterocycloalkyl," alone or in combination with other terms, means a non-aromatic ring or ring system, which can optionally contain one or more alkylene groups as part of the ring structure, which has at least one ring member that is a heteroatom independently selected from nitrogen, sulfur, oxygen, and phosphorus, and which has 4-10 ring members, 4-7 ring members, or 4-6 ring members. Included within the term "heterocycloalkyl" are monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl rings. Heterocycloalkyl groups can include monocyclic or bicyclic (e.g., having two fused or bridged rings) ring systems or spirocyclic ring systems. In some embodiments, a heterocycloalkyl is a monocyclic group having 1, 2, or 3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. The ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can optionally be oxidized to form an oxo or thiono group or other oxidized linkage (e.g., C(O), S(O), C(S), or S(O)2, N-oxide, etc.) or a nitrogen atom can be quaternized. A heterocycloalkyl group can be attached via a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, a heterocycloalkyl contains 0 to 3 double bonds. In some embodiments, a heterocycloalkyl contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties having one or more aromatic rings fused to (i.e., sharing a bond with) the heterocycloalkyl ring, such as benzene or thiophene derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl containing a fused aromatic ring can be attached via any ring-forming atom of the fused aromatic ring. Examples of heterocycloalkyl groups include 2-pyrrolidinyl; morpholinyl; azetidinyl; and piperazinyl.

[0319] In certain places, a definition or embodiment refers to a particular ring (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwise indicated, these rings can be attached to any ring member, provided that the valence of the atom is not exceeded. For example, an azetidine ring can be attached at any position of the ring, while an azetidine-3-yl ring is attached at the 3-position.

[0320] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of the present application containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such isomers are encompassed by the present application. Cis and trans geometric isomers of the compounds of the present application are described and can be isolated as isomer mixtures or as separate isomers.

[0321] The splitting of the racemic mixture of compound can be carried out by any one of numerous methods as known in the art.A kind of method comprises using chiral splitting acid to carry out fractional recrystallization, and described chiral splitting acid is optically active salified organic acid.Suitable splitting agent for fractional recrystallization method is such as optically active acid, such as tartaric acid D and L form, diacetyl tartaric acid, dibenzoyl tartaric acid, mandelic acid, malic acid, lactic acid or various optically active camphorsulfonic acid (such as β-camphorsulfonic acid).Other splitting agents applicable to fractional crystallization method include the stereoisomerically pure form (such as, S and R form, or diastereoisomerically pure form) of α-methylbenzylamine, 2-phenylglycinol, norephedrine (norephedrine), ephedrine (ephedrine), N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane etc.

[0322] The resolution of the racemic mixture can also be carried out by eluting with a column packed with an optically active resolving agent (eg, dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by those skilled in the art.

[0323] 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 more than one chiral center, unless otherwise indicated, each of the chiral centers in the compound can independently be (R) or (S).

[0324] The compounds of the present invention also include tautomeric forms. Tautomeric forms are produced by the exchange of a single bond with an adjacent double bond and the accompanying proton migration. Tautomeric forms include proton transfer tautomers in isomeric protonation states with the same empirical formula and total charge. Example proton transfer tautomers include keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions of a heterocyclic ring system, such as 1H-imidazole and 3H-imidazole, 1H-1,2,4-triazole, 2H-1,2,4-triazole and 4H-1,2,4-triazole, 1H-isoindole and 2H-isoindole, and 1H-pyrazole and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0325] The compounds of the present application can also include all isotopes of atoms occurring in the present compounds. Isotopes include atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more of the constituent atoms of the compounds of the present application can be replaced by atom isotopes of natural or non-natural abundance. In some embodiments, the compounds include at least one deuterium atom. For example, one or more hydrogen atoms in the compounds of the present disclosure can be replaced or substituted by deuterium. In some embodiments, the compounds include two or more deuterium atoms. In some embodiments, the compounds include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 deuterium atoms. Methods of synthesis that include isotopes in organic compounds are known in the art (Alan F. Thomas, Deuterium Labeling in Organic Chemistry (New York, N.Y., Appleton-Century-Crofts, 1971; Jens Atzrodt, Volker Derdau, Thorsten Fey, and Jochen Zimmermann, The Renaissance of H / D Exchange, Angew. Chem. Int. Ed. 2007, 7744-7765; James R. Hanson, The Organic Chemistry of Isotopic Labeling, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in various studies, such as NMR spectroscopy, metabolic experiments, and / or assays.

[0326] Substitution with heavier isotopes such as deuterium can afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence can be preferred in some circumstances. (A. Kerekes et al., J. Med. Chem. 2011, 54, 201-210; R. Xu et al., J. Label Compd. Radiopharm. 2015, 58, 308-312).

[0327] The term "compound" as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. The term is also meant to include compounds of the present application, regardless of how they are prepared, for example, synthetically, via biological processes (e.g., metabolism or enzymatic transformations), or a combination thereof.

[0328] All compounds and pharmaceutically acceptable salts thereof can exist, isolated or can be isolated, with other matter, such as water and solvents (e.g., hydrates and solvates). The compounds described herein, and salts thereof, can exist in various forms when in the solid state, and can be, for example, in the form of a solvate, including a hydrate. The compounds can be in any solid state form, such as a polymorph or a solvate, and thus references in this specification to compounds and salts thereof are understood to encompass any solid state form of the compound, unless explicitly indicated otherwise.

[0329] In some embodiments, the compounds of the present application, or salts thereof, are substantially isolated. By "substantially isolated" is meant 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 for a compound of the present application. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of a compound of the present application, or salts thereof.

[0330] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0331] The expressions "ambient temperature" and "room temperature" as used herein are understood in the art and generally refer to temperatures that are about the temperature of the room in which the reaction is being conducted, e.g., the temperature of the reaction, e.g., a temperature of about 20 °C to about 30 °C.

[0332] The present application also includes pharmaceutically acceptable salts of the compounds described herein. The term "pharmaceutically acceptable salt" refers to a derivative of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral 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 application include the non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. As used herein, the term "pharmaceutically acceptable salt" means those salts which are safe and non-toxic for the recipients at the dosages and in the amounts employed, and which possess the desired pharmacological activity of the parent compound. Pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, isopropanol or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th 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.

[0333] Synthesis

[0334] The compounds of the present application, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes, such as those in the schemes below.

[0335] The reactions for preparing compounds of the present application can be carried out in suitable solvents which can be easily selected by one skilled in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. The choice of a suitable solvent will depend, in part, on the nature of the particular reaction step.

[0336] The preparation of the compounds of the application can 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 chemical properties of protecting groups are described in, for example, 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, 6th Ed. (Wiley, 2007); Peturssion et al., "Protecting Groups in Carbohydrate Chemistry", J. Chem. Educ., 1997, 74(11), 1297; and Wuts et al., Protective Groups in Organic Synthesis, 4th Ed., (Wiley, 2006).

[0337] The reaction can be monitored according to any appropriate method known in the art. For example, product formation can be monitored by spectroscopic means (e.g., nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry), or by chromatographic means (e.g., high-performance liquid chromatography (HPLC) or thin layer chromatography (TLC)).

[0338] The following schemes provide general guidance for preparing the compounds of the application. One skilled in the art will understand that the preparations shown in the schemes can be modified or optimized using common sense of organic chemistry to prepare various compounds of the application.

[0339] The compounds of Formula (I) can be prepared, for example, using the methods as illustrated in the following schemes.

[0340] Scheme 1: Synthesis of Carbonate and Carbamate Linked Compounds

[0341]

[0342] Intermediates II, which are orthogonally leaving group-flanked, can be reacted with nucleophilic R 8 H compounds to give intermediates III. Intermediates III can then be reacted with thiol-containing peptides (HS-R 7 ) that participate in a disulfide exchange reaction to give the final compounds. Suitable leaving groups are described below.

[0343] Scheme 2: Synthesis 1 of thiopropionate-linked conjugates

[0344]

[0345] Propionate disulfides IV, with previously installed leaving groups 1 and 2, can be reacted selectively with nucleophiles R 8 -H to give V. This compound can then be reacted with R 7 -SH to provide the desired conjugate.

[0346] Scheme 3: Synthesis 2 of thiopropionate-linked conjugates

[0347]

[0348] Thioesters VI can be reacted with nucleophiles R 8 -H to give propionate thiol VII. This compound can participate in a disulfide exchange reaction to provide intermediate VIII. This compound can be treated with R 7 -SH to provide the desired conjugate.

[0349] Scheme 4: Synthesis 1 of p-toluenyl-linked conjugates

[0350]

[0351] The alcohol group of p-aminotoluenyl alcohol IX can be selectively protected to give intermediate X. This intermediate can then be reacted with intermediate II at the aniline position to provide the carbamate aryl ester 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 containing an orthogonal leaving group. Reaction of intermediate XIII with R 8 -H can provide intermediate XIV, which can then be treated with R 7 -SH to give the desired p-toluenyl-linked conjugate.

[0352] Scheme 5: Synthesis of p-toluenyl-linked conjugates

[0353]

[0354] 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 provide activated compound XVII. This intermediate can be further selectively reacted with nucleophiles R 8 -H to provide intermediate XVIII, which can be treated with R 7 -SH to give the desired conjugate.

[0355] Scheme 6: Synthesis of o-toluenyl-linked conjugates

[0356]

[0357] 2-mercaptobenzyl alcohol XXIII can be reacted as previously described to give the desired conjugate.

[0358] Scheme 7: Cleavage of the peptide conjugate

[0359]

[0360] Cleavage of the final compound to release R 8 -H. Reverse phase HPLC analysis at the desired time course was used to follow the progress of the cleavage.

[0361] Peptide R 7 can be prepared using the solid phase synthesis method first described by Merrifield in J. A. C. S., vol. 85, pp. 2149-2154 (1963), although other methods known in the art can also be employed. The Merrifield technique is well known and is a commonly used method for the preparation of peptides. Useful techniques for solid phase peptide synthesis are described in several books (e.g. the text "Principles of Peptide Synthesis" by Bodanszky, Springer Verlag 1984). This method of synthesis involves the stepwise addition of protected amino acids to a growing peptide chain bound through a covalent linkage to a solid resin particle. By this procedure, reagents and by-products are removed by filtration, thus eliminating the necessity for purification of intermediates. The general concept of this method depends on the attachment of the first amino acid of the chain through a covalent linkage to a solid polymer, followed by the addition of subsequent protected amino acids one at a time in a stepwise fashion until the desired sequence is assembled. Finally, the protected peptide is removed from the solid resin support and the protecting groups are cleaved off.

[0362] Peptide R 7 can also be produced by fermentation, for example by modification of E. coli. Protein production in E. coli can be controlled to produce R 7Recombinant polypeptides of the sequence of the peptide. Recombinant polypeptide production 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: towards a rational approach to improve the yields of functional proteins; Microbial Cell Factories 2013, 12:101; and Kleine-Grote G.M., Risse, J.M., Friehs, K; Secretion of recombinant proteins from E. coli; Eng. Life Sci. 2018, 18, 532-550, each of which is incorporated herein by reference in its entirety.

[0363] The amino acid can be attached to any suitable polymer. The polymer must be insoluble in the solvent used, must have a stable physical form so as to be filtered at any time, and must contain a functional group to which the first protected amino acid can be tightly attached by a covalent bond. Various polymers are suitable for this purpose, such as cellulose, polyvinyl alcohol, polymethyl methacrylate, and polystyrene.

[0364] Methods of use

[0365] Provided herein is the use of a compound of Formula (I) in the treatment of a disease, such as a cancer or a neurodegenerative disease. Another aspect of the application is the use of a compound of Formula (I) in the treatment of a disease involving acidotic or hypoxic diseased tissue, such as a cancer or a neurodegenerative disease. Hypoxia and acidosis are physiological hallmarks of many disease processes, including cancer. In cancer, hypoxia is one mechanism that contributes to the development of an acidic environment within solid tumors. Thus, hydrogen ions must be removed from the cell (e.g., by proton pumps) to maintain normal pH within the cell. As a result of this export of hydrogen ions, the pH gradient across the cell membrane lipid bilayer is typically increased and the pH of the extracellular environment is decreased in cancer cells when compared to normal cells. One way to improve the efficacy and therapeutic index of a cytotoxic agent is to take advantage of this physiological feature to provide selective delivery of the compound to hypoxic cells within a healthy tissue.

[0366] In the treatment methods of the present application, a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, can be administered as a sole agent or in combination with other forms of therapy, such as ionizing radiation or cytotoxic agents in the case of cancer. In combination therapy, the compound of Formula (I) can be administered prior to, concurrently with, or following the other treatment modalities, as will be appreciated by those skilled in the art. Either treatment method, as a sole agent or in combination with other forms of therapy, can be administered as a course of treatment involving multiple doses over a period of time.

[0367] Examples of cancers that can be treated using the compounds of the present disclosure include, but are not limited to, 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, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, endometrial cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of the soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, including

[0368] In some embodiments, cancers that can be treated 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, gall bladder cancer, gastric cancer, gastrointestinal tumor, head and neck cancer (upper aerodigestive carcinoma), 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.

[0369] In some embodiments, cancers that can be treated with the compounds of the 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 disclosure includes refractory or recurrent malignancies whose growth can be inhibited using the compounds of the disclosure.

[0370] In some embodiments, cancers that can be treated using the compounds of the 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.), blood cancers (e.g., lymphoma, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid 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 the same.

[0371] Compounds of the present application (e.g., compounds of Formula (I)) comprising a topoisomerase I targeting moiety derived from a topoisomerase I inhibitor (e.g., exatecan) can exhibit certain therapeutic advantages over the topoisomerase I inhibitor itself. For example, administration of a compound of Formula (I) can show reduced toxicity (e.g., myelotoxicity or gastric toxicity) compared to administration of the corresponding topoisomerase I inhibitor (e.g., exatecan). In some embodiments, myelotoxicity is measured by total bone marrow count of a subject sample (e.g., total bone marrow count in the femur of a mouse). In some embodiments, myelotoxicity is measured by PARylation in bone marrow tissue. 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.

[0372] In certain embodiments, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, can be used in combination with a chemotherapeutic agent, a targeted cancer therapy, an immunotherapy, or a radiation therapy. The agent can be combined with the compound of the present application in a single dosage form, or the agent can be administered simultaneously or sequentially in separate dosage forms. In some embodiments, the toxicity to the patient is lower, e.g., shows reduced myelotoxicity or gastric toxicity, when the chemotherapeutic agent, targeted cancer therapy, immunotherapy, or radiation therapy is administered in combination with a compound of Formula (I), or a pharmaceutically acceptable salt thereof, than when the chemotherapeutic agent, targeted cancer therapy, immunotherapy, or radiation therapy is administered in combination with the corresponding topoisomerase inhibitor (e.g., R 8 -H) than when the chemotherapeutic agent, targeted cancer therapy, immunotherapy, or radiation therapy is administered with a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0373] Suitable chemotherapeutic or other anticancer agents include, for example, alkylating agents (including but not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes), such as uracil mustard, chlormethine, cyclophosphamide, TM ), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphamide, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

[0374] Other suitable agents for use in combination with the compounds of the present invention include: dacarbazine (DTIC), optionally with other chemotherapy drugs (e.g., carmustine (BCNU) and cisplatin); the "Dartmouth regimen," which consists of DTIC, BCNU, cisplatin, and tamoxifen; a combination of cisplatin, vinblastine, and DTIC; or temozolomide. The compounds according to the present invention may also be combined with immunotherapy drugs, including cytokines such as interferon alpha, interleukin 2, and tumor necrosis factor (TNF).

[0375] 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, pentostatine, and gemcitabine.

[0376] Suitable chemotherapeutic or other anti-cancer agents also 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 TM ), mithramycin, deoxycoformycin, mitomycin-C, L- asparaginase, interferons (especially IFN-a), etoposide, and teniposide.

[0377] Other cytotoxic agents that can be administered in combination with the compounds of the application include, for example, navelbene, CPT-11, anastrazole, letrazole, capecitabine, relotxafine, cyclophosphamide, ifosamide, and droloxafine.

[0378] Also suitable are cytotoxic agents such as the epidophyllotoxins; antineoplastic enzymes; topoisomerase inhibitors; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitors; anti-hormonal therapeutic agents; leucovorin; tegafur; and hematopoietic growth factors.

[0379] Other anti-cancer agents include antibody therapeutics such as trastuzumab (Herceptin); antibodies to costimulatory molecules (e.g., CTLA-4, 4-1BB, and PD-1); or antibodies to cytokines (IL-10, TGF-a, etc.).

[0380] Other anticancer agents also include those that block immune cell migration, such as antagonists to chemokine receptors, including CCR2 and CCR4.

[0381] Other anticancer agents also include those that enhance the immune system, such as adjuvants or adoptive T cell transfer.

[0382] Anticancer vaccines that can be administered in combination with the compounds of the application include, for example, dendritic cells, synthetic peptides, DNA vaccines, and recombinant viruses.

[0383] Other suitable agents for use in combination with the compounds of the application include chemotherapy combinations, such as platinum-based doublets for lung 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 in combination with bevacizumab

[0384] The compounds of the application can be effectively combined with anti-hormonal agents for the treatment of breast and other tumors. Suitable examples are anti-estrogens, including, but not limited to, tamoxifen and toremifene; aromatase inhibitors, including, but not limited to, letrozole, anastrozole, and exemestane; adrenocortical steroids (e.g., prednisone); progestins (e.g., megastrol acetate); and estrogen receptor antagonists (e.g., fulvestrant). Anti-hormonal agents suitable for the treatment of prostate and other cancers can also be combined with the compounds of the application. These include anti-androgens, 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).

[0385] The compounds of the application can be administered in combination or sequentially with other agents directed against membrane receptor kinases, especially for patients who have developed primary or acquired resistance to targeted therapies. These therapeutic agents include inhibitors or antibodies directed against EGFR, Her2, VEGFR, c-Met, Ret, IGFR1 or Flt-3 and against cancer-related fusion protein kinases such as Bcr-Abl and EML4-Alk. Inhibitors against EGFR include gefitinib and erlotinib and against EGFR / Her2 include (but are not limited to) dacomitinib, afatinib, lapitinib and neratinib. Antibodies against EGFR include (but are not limited to) cetuximab, panitumumab and necitumumab. Inhibitors of c-Met can be used in combination with the compounds of the application. These include onartumzumab, tivantnib and INC-280. Agents against Abl (or Bcr-Abl) include imatinib, dasatinib, nilotinib and ponatinib and those against Alk (or EML4-ALK) include crizotinib.

[0386] Angiogenesis inhibitors can be effective in combination with the compounds of the application in some tumors. These include antibodies against VEGF or VEGFR or kinase inhibitors of VEGFR. Antibodies against VEGF or other therapeutic proteins include bevacizumab and aflibercept. Inhibitors of VEGFR kinases and other anti-angiogenic inhibitors include (but are not limited to) sunitinib, sorafenib, axitinib, cediranib, pazopanib, regorafenib, brivanib and vandetanib.

[0387] Activation of intracellular signaling pathways is common in cancer, and agents that target components of these pathways have been combined with receptor-targeted agents to enhance efficacy and reduce resistance. Examples of agents that can be combined with the compounds of the present application 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.

[0388] Agents against PI3 kinases include, but are not limited to, pilaralisib, idelalisib, buparlisib. Inhibitors of mTOR, such as rapamycin, sirolimus, temsirolimus, and everolimus, can be combined with the compounds of the present application. 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 JAKs (e.g., ruxolitinib, baricitinib, tofacitinib), Hsp90 (e.g., tanespimycin), cyclin-dependent kinases (e.g., palbociclib), HDACs (e.g., panobinostat), PARP (e.g., olaparib), and proteasomes (e.g., bortezomib, carfilzomib) can also be combined with the compounds of the present application. Another example of a PARP inhibitor that can be combined with the compounds of the present application is talazoparib.

[0389] Methods for safely and effectively administering most of these chemotherapeutic agents are known to those of skill in the art. In addition, their administration is described in standard literature. For example, the administration of many chemotherapeutic agents is described in the "Physicians' Desk Reference" (PDR, e.g., 1996 edition, Medical Economics Company, Montvale, NJ), the disclosure of which is hereby incorporated by reference as if set forth in its entirety.

[0390] The phrase "therapeutically effective amount" of a compound (therapeutic agent, active ingredient, drug, etc.) means the amount of the compound administered to a subject in need of relief from symptoms, amelioration of a condition, or slowing of the onset of a disease state that is therapeutically acceptable according to clinically accepted standards for the disorder or condition being treated. For example, a therapeutically effective amount can be an amount that has been shown to have the desired therapeutic effect in an in vitro assay, in vivo animal assay, or in a clinical trial. The therapeutically effective amount can vary based on the particular dosage form, method of administration, treatment regimen, particular disease or condition to be treated, benefit / risk ratio, and numerous other factors, as well as a host of other factors.

[0391] The therapeutically effective amount can be derived from clinical trials, animal models, or in vitro cell culture assays. It is known in the art that the effective amount determined from animal models or in vitro cell culture assays can be used to calculate the effective amount suitable for human use. For example, as reported by Reagan-Shaw et al., FASEB J. 2008: 22(3) 659-61, "pg / ml" (effective amount based on in vitro cell culture assays) = "mg / kg body weight / day" (effective amount in mice). Furthermore, based on the fact that mice metabolize 6 times faster than humans, the effective amount in humans can be calculated from the effective amount in mice.

[0392] As an example of treatment using a combination of a compound of Formula (I) with a cytotoxic agent, a patient having cancer can be administered a therapeutically effective amount of a compound of Formula (I) as part of a treatment regimen that also involves a therapeutically effective amount of ionizing radiation or a cytotoxic agent. In the context of such a treatment regimen, the term "therapeutically effective" amount will be understood to mean effective in combination therapy. Those skilled in the art of treating cancer will understand how to adjust dosages to achieve optimal treatment results.

[0393] Similarly, those skilled in the medical arts can readily determine an appropriate dosage of a compound of the application for treatment of a non-cancerous disease or condition, such as a cardiovascular disease.

[0394] The term "treatment" as used herein includes administration of a compound or composition that reduces the frequency of, delays the onset of, or reduces the progression of symptoms of a disease involving acidotic or hypoxic diseased tissue (e.g., cancer, stroke, myocardial infarction, or long-term neurodegenerative disease) in a subject relative to a subject not receiving the compound or composition. This can include reversing, reducing, or inhibiting symptoms, clinical signs, or underlying pathology of a disease (e.g., tumor growth, regression or reduction of cancer, or improvement in myocardial ischemia reperfusion injury in myocardial infarction, stroke, or similar cardiovascular disease) in a manner that improves or stabilizes the condition of the subject. The term "inhibit" or "reduce" as used in reference to cancer refers to a method of inhibiting or reducing tumor growth (e.g., reducing tumor size) in a population as compared to an untreated control population.

[0395] All publications, including patents, referred to herein are incorporated by reference herein for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications that might be used in connection with the present disclosure. The publications discussed throughout the discussion herein are provided solely for their disclosure prior to the filing date of the present application.

[0396] Several types of ranges are disclosed herein. When disclosing or claiming any type of range, it is intended to disclose or claim individually each possible number that the range can reasonably encompass, including the endpoints of the range and any subranges and combinations of subranges encompassed therein. For example, when disclosing or claiming a range of therapeutically effective amounts of an active ingredient, it is intended to disclose or claim individually each possible number that the range can encompass that is consistent with the disclosure herein. For example, a therapeutically effective amount of a compound can range from about 1 mg / kg to about 50 mg / kg (of subject body weight).

[0397] Formulations, dosage forms, and administration

[0398] To prepare the pharmaceutical compositions of this application, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, is intimately admixed as an active ingredient with a pharmaceutical carrier according to conventional pharmaceutical practice, which carrier can take a wide variety of forms depending upon the form of preparation desired for administration (e.g., oral or parenteral). In preparing the compositions in oral dosage form, any of the usual pharmaceutical media can be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; in the case of liquid oral preparations, such as, for example, suspensions, elixirs and solutions, water and glycols are employed; for a solid oral preparation, such as, for example, powders, capsules and tablets, carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like are employed. Since solid dosage forms are preferred, a most preferred form is the tablet, which can be prepared by conventional methods such as compression, gelatin or gelatin-like molding, or the like. Tablets can be coated according to methods known in the art. If desired, tablets can be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a particular area of the gastrointestinal tract, e.g., in the intestinal tract, as opposed to the stomach. For oral therapeutic administration, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The actual

[0399] Examples

[0400] 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, D.C.: American Chemical Society, 1997. The following definitions describe terms and abbreviations used herein:

[0401] • brine: saturated aqueous NaCl solution

[0402] • DCM: dichloromethane

[0403] • TFA: trifluoroacetic acid

[0404] • DIPEA: diisopropylethylamine

[0405] • DMA: dimethylacetamide

[0406] • DME: dimethoxyethane

[0407] • DMF: dimethylformamide

[0408] • DMSO: dimethyl sulfoxide

[0409] • DTT: dithiothreitol

[0410] • MSD: mass spectrometry detector

[0411] • Et20: diethyl ether

[0412] • EtOAc: ethyl acetate

[0413] • EtOH: ethanol

[0414] • HATU: O-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0415] • HOBt: 1-hydroxybenzotriazole

[0416] • RP: reversed phase

[0417] • HPLC: high performance liquid chromatography

[0418] • IPA: isopropyl alcohol

[0419] • LAH: lithium aluminum hydride

[0420] • N-BuLi: n-butyllithium

[0421] • LC-MS: liquid chromatography-mass spectrometry

[0422] • LDA: lithium diisopropylamide

[0423] • Me: methyl

[0424] • MeOH: methanol

[0425] • MTBE: methyl tert-butyl ether

[0426] • NMP: N-methylpyrrolidine

[0427] • Ph: phenyl

[0428] • PNPC: p-nitrophenyl chloroformate

[0429] • RT or rt: room temperature

[0430] • SFC: supercritical fluid chromatography

[0431] • TBAI: tetrabutylammonium iodide

[0432] • TBME: tert-butyl methyl ether

[0433] • tBu: tert-butyl

[0434] • THF: tetrahydrofuran

[0435] • TEA: triethylamine

[0436] • TMEDA: tetramethylethylenediamine

[0437] • GSH: glutathione

[0438] • GS: glutathione bound via sulfur

[0439] • LiOH: lithium hydroxide

[0440] • DPPA: diphenyl phosphoryl azide

[0441] • Sn(Bu)2(laurate)2: dibutyl tin laurate

[0442] • PBS: phosphate buffered saline

[0443] • ACN: acetonitrile

[0444] • AcOH: acetic acid

[0445] • EEDQ: N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline

[0446] • DMAP: 4-dimethylaminopyridine

[0447] • EDC: l-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0448] The origin of the starting materials employed in the examples is set out in the table below.

[0449] Table 2. R 8 Starting materials

[0450]

[0451]

[0452]

[0453]

[0454] Table 3. Starting materials for linkers

[0455]

[0456]

[0457] *Absolute configuration assigned randomly

[0458] The HPLC method employed is set out below:

[0459] HPLC method

[0460] A: Sunfire C18 150 x 4.6 mm; H2O / acetonitrile with TFA modifier (0.05%); flow rate: 1 ml / min; wavelength = 217 nM.

[0461] B: Ace Equivalence 250 x 4.6 mm; H2O / acetonitrile with TFA modifier (0.05%); flow rate: 1 ml / min; wavelength = 217 nM.

[0462] C: Sunfire C18 150 x 30 mm; H2O / acetonitrile with TFA modifier (0.05%); flow rate: 30 ml / min; wavelength = 217 nM.

[0463] Mass spectrometry method

[0464] Maldi-TOF (Matrix Assisted Laser Desorption / Ionisation - Time of Flight) mass spectra were measured on an Applied Biosystems Voyager System 6268. Samples were prepared on AB Science plates (part number V700666) as a matrix of alpha-cyanocinnamic acid.

[0465] Electrospray ionization (ESI) mass spectra were measured on an Agilent 1100 series LC-MS with a 1946 MSD or a Waters Xevo Qtof high resolution MS (both provide mass / charge material (m / z = 3)).

[0466] Synthesis of cis-thioacetic acid S-(3-hydroxybutan-2-yl ester) (L-4 and L-5)

[0467]

[0468] 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 h. 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 afford cis-thioacetic acid S-(3-hydroxybutan-2-yl ester) as an oily compound (4.0 g, crude). MS m / z 149.0 [M+H] + .

[0469] Synthesis of cis-3-mercaptobutan-2-ol

[0470]

[0471] To a stirred solution of thioacetic acid S-(3-hydroxybutan-2-yl ester) (4 g, 26.9 mmol) in THF (40 mL) was added lithium aluminium hydride (1 M solution in THF) (27 mL, 26.9 mmol) dropwise at 0 °C. The reaction mixture was allowed to warm gradually to room temperature and stirred for 3 h. The reaction mixture was quenched slowly with 1 N HCI at 0 °C and the pH was adjusted to 2-3. The reaction mixture was extracted in ethyl acetate (50 mL) and the organic layer was dried over anhydrous sodium sulfate and evaporated to obtain cis-3-mercaptan-2-ol as a crude oily compound.

[0472] Synthesis of trans-thioacetic acid S-(3-hydroxybutan-2-yl ester) (L-6 and L-7)

[0473]

[0474] 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) and stirred for 16 h at room temperature. 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 afford trans-thioacetic acid S-(3-hydroxybutan-2-yl ester) as a yellow oil (0.7 g crude).

[0475] Synthesis of trans-3-mercaptobutan-2-ol

[0476]

[0477] To a stirred solution of trans-thioacetic acid S-(3-hydroxybutan-2-yl ester) (700 mg, 4.72 mmol) in THF (10 mL) was added lithium aluminium hydride (1 M solution in THF) (4.8 mL, 4.72 mmol) dropwise at 0 °C and stirred at same temperature for 3 h. The reaction mixture was quenched with 1 N HCI at 0 °C followed by pH adjustment to 2-3. The reaction mixture was extracted with CH2CI2(10 mL). The organic layer was dried over anhydrous sodium sulphate and used directly for the next step.

[0478] Synthesis of trans-thioacetic acid S-(2-hydroxycyclohexyl ester) (L-8 and L-9)

[0479]

[0480] 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 h. The progress of the reaction 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 and washed with brine, dried over anhydrous sodium sulphate and concentrated under reduced pressure to afford trans-thioacetic acid S-(2-hydroxycyclohexyl ester) (3.8 g crude) as a brown liquid.

[0481] Synthesis of trans-2-mercaptocyclohexan-1-ol

[0482]

[0483] To a stirred solution of trans-thioacetic acid S-(2-hydroxycyclohexyl ester) (3.8 g, 21.8 mmol) in THF (20.0 mL) was added 1 M LiAIH4in THF (21.8 mL, 21.8 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. The progress of the reaction was monitored by TLC (20% EtOAc / hexane). On completion of the reaction, the reaction mixture was cooled to room temperature and quenched with 1.0 N HCI (30 mL). The reaction mixture was extracted in CH2CI2(30.0 mL). The organic layer was washed with brine solution (30.0 mL), concentrated and the crude trans-2- thioxycyclohexanol was used for the next step. (2.88 g, crude).

[0484] Synthesis of intermediate I from L

[0485]

[0486]

[0487] Synthesis of intermediate I-1 : (2R)-2-(2-pyridyldisulfanyl)propan-1-ol

[0488]

[0489] To a solution of 5-nitro-2-[(5-nitro-2-pyridyl)disulfanyl]pyridine (17.4 g, 56.0 mmol) in degassed (N2) MeOH (100 mL) was added (1-mercapto cyclobutyl)methanol (8.3 mL, 70.0 mmol) (degassed with N2) dropwise and stirred at room temperature under N2atmosphere for 16 h. The reaction mixture was concentrated to dryness under vacuum. The resulting crude material was purified by column chromatography using 30% EtOAc / hexane to afford [1-[(5-nitro-2-pyridyl)disulfanyl]cyclobutyl]methanol (9.0 g, 46% yield) as a yellow liquid. MS m / z 272.9 [M+H] + .

[0490] Intermediate 1-2 was prepared in a similar manner from L-2.

[0491] Synthesis of intermediate I-3: [1-[(5-nitro-2-pyridyl)disulfanyl]cyclobutyl]methanol

[0492]

[0493] To a solution of 5-nitro-2-[(5-nitro-2-pyridyl)disulfanyl]pyridine (17.4 g, 56.0 mmol) in degassed (N2) MeOH (100 mL) was added (1-mercapto cyclobutyl)methanol (8.3 mL, 70.0 mmol) (degassed with N2) dropwise and stirred at room temperature under N2atmosphere for 16 h. The reaction mixture was concentrated to dryness under vacuum. The resulting crude material was purified by column chromatography using 30% EtOAc / hexane to afford [1-[(5-nitro-2-pyridyl)disulfanyl]cyclobutyl]methanol (9.0 g, 46% yield) as a yellow liquid. MS m / z 272.9 [M+H] + .

[0494] Synthesis of I-4 and I-5: 3-(pyridin-2-yl disulfanyl)butan-2-ol isomer 1 and isomer 2

[0495]

[0496] A stirred solution of 2,2-dipyridyl disulfide (520 mg, 2.35 mmol) in MeOH (15 mL) was purged with nitrogen for 5 min. To this was added a nitrogen purged solution of cis-3-mercaptobutane-2-ol (500 mg) in CH2CI2(10 mL) at 0 °C. The reaction mixture was allowed to warm to room temperature gradually and stirred for 16 h. The reaction mixture was concentrated under reduced pressure to get crude which was purified by column chromatography using 30-40% EA in hexane. The racemic product was separated to isolate individual enantiomers by chiral preparative HPLC (CHIRALPAK IG; 100 mm x 4.6 mm x 3 mic; mobile phase: n-hexane: ethanol 80:20 with 0.1% DEA; flow rate: 1.0 mL / min). The solvents were removed to obtain (2S,3S)-3-(2-pyridyl disulfanyl)butan-2-ol* (140 mg, Isomer-1) MS m / z 216.1 [M+H] + and (2R,3R)-3-(2-pyridyl disulfanyl)butan-2-ol (140 mg, Isomer-2). MS m / z 216.1 [M+H] + .

[0497] Synthesis of I-6 and I-7: 3-(pyridin-2-yl disulfanyl)butan-2-ol isomer 1 and isomer 2

[0498]

[0499] A stirred solution of 2,2-dipyridyl disulfide (520 mg, 2.35 mmol) in MeOH (15 mL) was purged with nitrogen for 5 min. To this was added a nitrogen purged solution of cis-3-mercaptobutane-2-ol (500 mg) in CH2CI2(10 mL) at 0 °C. The reaction mixture was allowed to warm to room temperature gradually and stirred for 16 h. The reaction mixture was concentrated under reduced pressure to get crude which was purified by column chromatography using 30-40% EA in hexane. The racemic product was separated to isolate individual enantiomers by chiral preparative HPLC (CHIRALPAK IG; 100 mm x 4.6 mm x 3 mic; mobile phase: n-hexane: ethanol 80:20 with 0.1% DEA; flow rate: 1.0 mL / min). The solvents were removed to obtain (2R,3S)-3-(2-pyridyl disulfanyl)butan-2-ol* (0.6 g, Isomer-I) MS m / z 215.9 [M+H] + and (2S,3R)-3-(2-pyridyl disulfanyl)butan-2-ol* (0.6 g, Isomer-II) MS m / z 216.2 [M+H] + .

[0500] Synthesis of intermediate I-6: trans-2-(pyridin-2-yl disulfanyl)cyclohexan-1-ol

[0501]

[0502] To a stirred solution of 1,2-di(pyridin-2-yl)disulfane (2.41 g, 10.9 mmol) in MeOH (degassed with N2) (30 mL) was added dropwise trans-2-sulfanyl cyclohexanol (2.88 g, 21.0 mmol) (degassed with N2) and stirred at room temperature under N2atmosphere for 16 h. The reaction mixture was concentrated to dryness under vacuum. The crude material obtained was purified by column chromatography using 30% EtOAc / hexane to get trans-2-(pyridin-2-yl disulfanyl) cyclohexan-1-ol as a yellow liquid.

[0503]

[0504] Chiral separation was carried out using chiralpak IG (100 mm x 4.6 mm x 3 mic) using n-hexane: IPA containing 0.1% diethylamine (80:20) to get (1R,2R)-2-(2-pyridyl disulfanyl) cyclohexanol*isomer-1 (350 mg) and (1S,2S)-2-(2-pyridyl disulfanyl) cyclohexanol*isomer-2 (400 mg).

[0505] Intermediate XV from XXI

[0506]

[0507] Intermediate [R 5 ,R 6 ]]> [R 9 ,R 10 ,R 11 ,R 12 ]]> MH + ]]> XV-1 H,H H,H,H,H 250.1

[0508] Synthesis of intermediate XV-1 : [4-(2-pyridyl disulfanyl)phenyl]methanol

[0509]

[0510] 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 N2. Thereafter a mixture of (4-mercapto phenyl) methanol (0.74 g, 5.2 mmol) in AcOH / ethanol (5 mL) solvent was added dropwise over 20 min and stirred at room temperature under N2atmosphere for 12 h. The reaction was concentrated under reduced pressure to get crude product which was purified by column chromatography (SiO2, 60-70% EtOAc / hexane) to get [4-(2-pyridyl disulfanyl) phenyl] methanol (800 mg, 61% yield) as a colorless liquid.

[0511] Carbonate leaving group intermediate II from intermediate I

[0512]

[0513]

[0514] Synthesis of II-1 : Carbonic acid (4-nitrophenyl)[(2R)-2-(2-pyridyl disulfanyl)propyl ester]

[0515]

[0516] To (2R)-2-(2-pyridyl disulfanyl)propan-1-ol (0.39 g, 1.94 mmol) in THF was added pyridine (0.16 mL, 1.94 mmol) and 4-nitrophenyl chloroformate (0.59 g, 2.91 mmol) under N2. The mixture was stirred under N2for 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 give 0.59 g, 83% of carbonic acid (4-nitrophenyl) [(2R)-2-(2-pyridyl disulfanyl)propyl ester]. MS m / z 367.1 [M+H] + .

[0517] Synthesized intermediates II-2 and II-3 similarly to II-1.

[0518] Synthesis of II-4: Carbonic acid 4-nitrophenyl ester ((2R,3R)-3-(pyridin-2- yldisulfanyl)butan-2-yl ester)

[0519]

[0520] To a stirred solution of (2R,3R)-3-(pyridin-2-yl disulfanyl)butan-2-ol (140 mg, 0.651 mmol) in CH2Cl2(2.0 mL) was added pyridine (0.11 mL, 1.43 mmol), 4-nitrophenyl chloroformate (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 CH2Cl2(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 crude material which was purified by column chromatography using 30-40% ethyl acetate / hexanes. The eluted fractions were concentrated to give crude material which was further purified over a C18 reverse phase column. The pure eluted fractions were concentrated to give carbonic acid 4-nitrophenyl ester ((2R,3R)-3-(pyridin-2-yl disulfanyl)butan-2-yl ester) as an oily compound (70 mg, 28%). MS m / z 381.0 [M+H] + .

[0521] Synthesis of II-5: Carbonic acid 4-nitrophenyl ester ((2S,3S)-3-(pyridin-2- yldisulfanyl)butan-2-yl ester)

[0522]

[0523] To a stirred solution of (2S,3S)-3-(pyridin-2-yl disulfide)butan-2-ol (80 mg, 0.372 mmol) in CH2Cl2(1.0 mL) was added pyridine (0.066 mL, 0.818 mmol), 4-nitrophenyl chloroformate (89 mg, 0.446 mmol) and 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 CH2Cl2(5 mL) and then washed with water (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude title product which was purified by column chromatography using 30-40% ethyl acetate in hexane. The eluted fractions were concentrated to get the crude material which was further purified over a C18 reverse phase column. The pure eluted fractions were concentrated to get 4-nitrophenyl carbonate ((2S,3S)-3-(pyridin-2-yl disulfide)butan-2-yl ester) as an oily compound (140 mg, 58%). MS m / z 381.0 [M+H] + .

[0524] Synthesis of II-6: Carbonic acid 4-nitrophenyl ester ((2R,3S)-3-(pyridin-2- yldisulfanyl)butan-2-yl ester)

[0525]

[0526] To a stirred solution of (2R,3S)-3-(pyridin-2-yl disulfide)butan-2-ol (0.4 g, 1.86 mmol) in CH2CL2(10 mL) was added pyridine (0.36 mL, 4.09 mmol), 4-nitrophenyl chloroformate (0.44 g, 2.32 mmol) and 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 CH2Cl2(20 mL) and washed with water (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude material which was purified by silica gel flash column chromatography using 30-40% ethyl acetate in hexane. The compound was eluted as a mixture in 30% EtOAc:hexane. The eluted fractions were concentrated to get the crude material which was further purified over a C18 reverse phase column. The pure eluted fractions were evaporated to get 4-nitrophenyl carbonate ((2R,3S)-3-(pyridin-2-yl disulfide)butan-2-yl ester) as an oily compound (0.17 g, 24.2%). MS m / z 381.0 [M+H] + .

[0527] Synthesis of II-7: Carbonic acid 4-nitrophenyl ester ((2S,3R)-3-(pyridin-2- yldisulfanyl)butan-2-yl ester)

[0528]

[0529] To a stirred solution of (2S,3R)-3-(pyridin-2-yl disulfide)butan-2-ol (0.4 g, 1.86 mmol) in CH2Cl2(10 mL) was added pyridine (0.36 mL, 4.09 mmol), 4-nitrophenyl chloroformate (0.44 g, 2.32 mmol) and 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 CH2Cl2(20 mL) and washed with water (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude material which was purified by silica gel flash column chromatography using 30-40% ethyl acetate in hexane. The compound was eluted as a mixture in 30% EtOAc: hexane. The eluted fractions were concentrated to get the crude material which was further purified over C18 reverse phase column. The pure fractions were concentrated to get 4-nitrophenyl carbonate ((2S,3R)-3-(pyridin-2-yl disulfide)butan-2-yl ester) as an oily compound (0.18 g, 26%). MS m / z 381.0 [M+H] + .

[0530] Synthesis of II-8: Carbonic acid (4-nitrophenyl ester)[(1R,2R)-2-(2-pyridyl disulfanyl)cyclohexyl ester]

[0531]

[0532] To a solution of (1R,2R)-2-(2-pyridyl disulfide)cyclohexanol* (130.0 mg, 0.5 mmol) in THF (3.0 mL) was added potassium carbonate (0.20 g, 1.5 mmol), 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 h. 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 get the crude product which was purified by column chromatography using 20-30% EtOAc / hexane to get 4-nitrophenyl carbonate [(1R,2R)-2-(2-pyridyl disulfide)cyclohexyl ester]* as an off-white solid (89 mg, 40% yield). MS m / z 407.0 [M+H] + .

[0533] Synthesis of II-9: Carbonic acid (4-nitrophenyl ester)[(1S,2S)-2-(2-pyridyl disulfanyl)cyclohexyl ester]

[0534]

[0535] To a stirred solution of (1S,2S)-2-(2-pyridyl disulfide)cyclohexanol* (0.42 g, 1.7 mmol) in THF (10.0 mL) was added potassium carbonate (0.70 g, 5.1 mmol), 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 h. 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 get the crude product which was purified by column chromatography using 20-30% EtOAc / hexane to afford carbonic acid (4-nitrophenyl ester)[(1R,2R)-2-(2-pyridyl disulfide)cyclohexyl ester]* as off-white solid (250 mg, 35% yield). MS m / z 406.7 [M+H] + .

[0536] Carbonate leaving group intermediate XV from XIV

[0537]

[0538] Intermediate [R 5 ,R 6 ]]> [R 9 ,R 10 ,R 11 ,R 12 ]]> X MH + ]] XV-1 H,H H,H,H,H H 415.0

[0539] Synthesis of XV-1 : Carbonic acid (4-nitrophenyl ester)[4-(2-pyridyl disulfanyl)phenyl]methyl ester

[0540]

[0541] To a stirred solution of (4-(pyridin-2-yl disulfide)phenyl)methanol (0.40 g, 1.60 mmol) in CH2Cl2(10 mL) was added 4-nitrophenyl chloroformate (0.65 g, 3.2 mmol), pyridine (0.25 mL, 3.20 mmol), catalytic amount of DMAP (0.005 g) at 0 °C. The mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with 1.5 N HCI solution. The organic layer was separated and washed with brine, dried over anhydrous Na2SO4and concentrated. The crude product was purified by column chromatography (SiO2, 20-30% EtOAc / hexane) to afford carbonic acid (4-nitrophenyl ester)[4-(2-pyridyl disulfide)phenyl]methyl ester as colorless liquid (600 mg, 91% yield); MS m / z 415.0 [M+H] - .

[0542] Carbonate and carbamate linked intermediate III

[0543]

[0544]

[0545] Synthesis of III-1 : N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15 diazahexadecaphane[14.7.1.02,14.04,13.06,11.020,24]tetracosan-1,6(11),12,14,16 ​ [(2S)-2-(2-pyridyl disulfide)propyl] carbamate

[0546]

[0547] 1-Hydroxybenzotriazole hydrate (8.64 mg, 0.0564 mmol), finely powdered molecular sieves To a mixture of (50 mg) (10S,23S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19-methyl-8-oxa-4,15-diazacyclo[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 (25.0 mg, 0.0470 mmol) and pyridine (0.0190 mL, 0.235 mmol) in 2 mL of anhydrous DMF was added (2S)-2-(2-pyridyldisulfanyl)propyl carbonate (19.0 mg, 0.0517 mmol). After stirring at room temperature for 16 h, 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-diazepine[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 (29.0 mg, 93.0% yield). MS m / z 663.0 [M+H] + .

[0548] Intermediates III-2 to III-9 were prepared from II-2 to II-9 in analogy to III-1.

[0549] Intermediate XVI for carbonate and carbamate linkage

[0550]

[0551]

[0552] Intermediate XVI-1 is prepared from XV-1 in analogy to III-1.

[0553] Synthesis of 4-nitrophenyl carbonate (trans-(3RS,4RS)-4-(pyridin-2- yldisulfanyl)tetrahydrofuran-3-yl ester)

[0554]

[0555] Step 1 : Synthesis of rac-trans-thioacetic acid (4-hydroxytetrahydrofuran-3-yl ester)

[0556]

[0557] 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 h. The progress of the reaction was monitored by TLC (20% EtOAc / hexane). On completion of the reaction, the reaction mixture was diluted with diethyl ether and washed with 10% sodium bicarbonate solution. The organic layer was separated and washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get the crude product which was purified by column chromatography using 20% EtOAc: n-hexane to obtain the title product as a brown liquid (4.0 g, yield 42%). 1 HNMR (400 MHz, CDC13): 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).

[0558] Step 2: Synthesis of rac-trans-4-mercaptotetrahydrofuran-3-ol

[0559]

[0560] To a stirred solution of rac-trans-thioacetic acid (4-hydroxytetrahydrofuran-3-yl ester) (4.0 g, 24.7 mmol) in anhydrous THF (20.0 mL) was added LAH (1 M in THF) (27.1 mL, 27.1 mmol) drop wise at 0 °C under nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for 2 h. The progress of the reaction was monitored by TLC (20% EtOAc: n-hexane). On completion of the reaction, the reaction mixture was cooled to room temperature and quenched with 1.0 N HCI (50 mL). The reaction mixture was extracted into DCM (3 x 20 mL), the organic layer was washed with brine solution (20 mL), dried over anhydrous sodium sulfate, filtered, partially distilled and used as such in the next step. (2.9 g, crude).

[0561] Step 3: Synthesis of trans-(4RS, 3RS)-4-(pyridin-2-yl disulfanyl)tetrahydrofuran-3-ol and trans-(4SR, 3SR)-4-(pyridin-2-yl disulfanyl)tetrahydrofuran-3-ol

[0562]

[0563] To a solution of 2-(pyridin-2-yl disulfanyl)pyridine (0.9 g, 21.7 mmol) in MeOH (degassed with N2) (10 mL) was added 4-thiooxolane-3-ol (2.9 g, 24.1 mmol) (degassed with N2) dropwise and stirred at room temperature under nitrogen atmosphere for 16 h. The reaction mixture was concentrated to dryness under vacuum. The crude material obtained was purified by flash column chromatography using 30% EtOAc: n-hexane to obtain the title compound 4-(pyridin-2-yl disulfanyl)oxolane-3-ol (racemic mixture) as a yellow oil. The isomers were separated by chiral preparative HPLC.

[0564] Chiral preparative HPLC conditions:

[0565] Column: Chiralpak IA (250 mm x 20 mm x 5 mic)

[0566] Mobile phase: EtOH with 0.1% DEA (90:10)

[0567] Flow rate: 19 mL / min

[0568] The separated fractions of resolved isomers were collected from chiral preparative HPLC and evaporated under reduced pressure to obtain the title compound as isomer 1 (600 mg) and isomer 2 (620 mg).

[0569] Isomer 1: (trans-(4RS, 3RS)-4-(pyridin-2-yl disulfanyl)tetrahydrofuran-3-ol): C9H 11 LC-MS m / z calculated for NO2S2 229; found 230 [M+H] + . 1 H-NMR (400 MHz, CDC13): δ 8.53-8.52 (m, 1H), 7.67-7.63 (m, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.23-7.19 (m, 1H), 4.45-4.48 (m, 1H), 4.25 (t, J = 8.8 Hz, 1H), 4.12 (t, J = 6.8 Hz, 1H), 3.74-3.67 (m, 2H), 3.48-3.41 (m, 1H).

[0570] Isomer 2: (trans-(4SR, 3SR)-4-(pyridin-2-yl disulfanyl)tetrahydrofuran-3-ol): C9H 11 LC-MS m / z calculated for NO2S2 229; found 230 [M+H] + . 1H-NMR (400 MHz, CDC13): δ 8.54-8.53 (m, 1H), 7.68-7.64 (m, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.23-7.20 (m, 1H), 4.49-4.45 (m, 1H), 4.25 (t, J = 7.6 Hz, 1H), 4.12-4.10 (m, 1H), 3.74-3.67 (m, 2H), 3.47-3.44 (m, 1H).

[0571] Absolute stereochemistry of any specified isomer.

[0572] Step 4: Synthesis of carbonic acid 4-nitrophenyl ester (trans-(3RS,4RS)-4-(pyridin-2- yldithio)tetrahydrofuran-3-yl ester)

[0573]

[0574] To a stirred solution of trans-(3RS,4RS)-4-(pyridin-2-yl dithio)tetrahydrofuran-3-ol (0.61 g, 2.69 mmol) in DMF (10 mL) was added DIPEA (1.45 mL, 8.08 mmol) and bis(4-nitrophenyl) carbonate (1.64 g, 5.38 mmol) at room temperature under nitrogen atmosphere. The reaction vessel was sealed and stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC (20% EtOAc: n-hexane). On 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, filtered and concentrated under reduced pressure to get the crude product which was purified by flash column chromatography using 20-30% EtOAc: n-hexane to get carbonic acid 4-nitrophenyl ester (trans-(3RS,4RS)-4-(pyridin-2-yl dithio)tetrahydrofuran-3-yl ester) as off-white solid (790 mg, 77% yield). 1 HNMR (400 MHz, CDC13): 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); C 16 H 14 LC-MS m / z calcd for N2O6S2, 394; found, 395 [M+H] + .

[0575] Synthesis of 4-nitrophenyl carbonate (trans-(3SR,4SR)-4-(pyridin-2- yldisulfanyl)tetrahydrofuran-3-yl ester)

[0576]

[0577] To a stirred solution of trans-(3SR,4SR)-4-(pyridin-2-yl disulfide)tetrahydrofuran-3-ol (550 mg, 2.46 mmol) in DMF (10.0 mL) was added DIPEA (1.32 mL, 7.38 mmol) and bis(4-nitrophenyl)carbonate (1.5 g, 4.92 mmol) at room temperature under nitrogen. The reaction vessel was sealed and stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC (20% EtOAc: n-hexane). On 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, filtered and concentrated under reduced pressure to get the crude product which was purified by flash column chromatography using 20-30% EtOAc: n-hexane to get 4-nitrophenyl carbonatetrans-(3SR,4SR)-4-(pyridin-2-yl disulfide)tetrahydrofuran-3-yl ester (0.6 g, 70% yield) as off-white solid. 1 HNMR (400 MHz, CDC13): 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); C 16 H 14 LC-MS m / z calcd for N2O6S2, 394; found 395 [M+H] + .

[0578] Synthesis of 4-nitrophenyl carbonate (trans-(1RS,2RS)-2-(pyridin-2- yldisulfanyl)cyclopentyl ester)

[0579]

[0580] Step 1: Synthesis of racemic trans-thioic acid (5-hydroxycyclopent-l-yl ester)

[0581]

[0582] To a stirred solution of 6-oxabicyclo[3.1.0]hexane (3.0 g, mmol) in water (30 mL) was added thioacetic acid (3 mL, 39.2 mmol) at room temperature and stirred for 16 h. The reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 x 10 mL). The organic layer was separated and washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get racemic trans-thioacetic acid (5-hydroxycyclopent-1-yl ester) (2.6 g, crude) as an oily compound. C7H 12 LC-MS m / z calculated for C7H10O2S 160.2; found 143.3 [M+H-17] + .

[0583] Step 2: Synthesis of racemic trans-2-mercaptocyclopentan-1-ol

[0584]

[0585] To a stirred solution of racemic trans-thioacetic acid (5-hydroxycyclopent-1-yl ester) (2.6 g, 16.2 mmol) in THF (20 mL) was added LAH (1 M in THF) (24 mL, 24.3 mmol) drop wise at 0 °C under nitrogen atmosphere. The reaction mixture was allowed to warm gradually to room temperature and stirred for 2 h. The progress of the reaction was monitored by TLC (20% EtOAc: n-hexane). On completion of the reaction, the reaction mixture was cooled to room temperature and quenched with 1 N HCI solution and extracted in DCM. The organic layer was dried over anhydrous sodium sulfate, filtered and partially evaporated and the crude racemic trans-2-mercaptocyclopentan-1-ol was taken for the next step (1.9 g, crude).

[0586] 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

[0587]

[0588] To a stirred solution of 2-(pyridin-2-yl disulfide)pyridine (2.1 g, 9.65 mmol) in MeOH (10 mL) was added racemic trans-2-mercaptocyclopentan-1-ol (1.9 g, 16.1 mmol) drop wise at 0 °C under nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature gradually and stirred for 16 h. After completion of the reaction, the reaction mixture was concentrated to dryness under vacuum. The crude material obtained was purified by silica gel flash column chromatography. The compound was eluted in 15% EtOAc: n-hexane. The eluted fractions containing the desired product were combined and evaporated under reduced pressure to afford the title compound as a yellow liquid (racemic mixture). The isomers were separated by chiral preparative HPLC.

[0589] Chiral preparative HPLC conditions:

[0590] Column: Chiralpak IA (250 mm x 20 mm x 5 mic)

[0591] Mobile phase: EtOH with 0.1% DEA (70:30)

[0592] Flow rate: 19 mL / min

[0593] The separated eluted fractions of the separated isomers were collected from chiral preparative HPLC and evaporated under reduced pressure to afford the title compound as a colorless oil in the form of isomer 1 (300 mg) and isomer 2 (300 mg).

[0594] Isomer 1 (trans-(1 RS,2RS)-2-(pyridin-2-yl disulfide)cyclopentan-1-ol): LC-MS m / z calculated for C10H13NOS2, 227.34; found 228.1 [M+H] + . 1 HNMR (400 MHz, CDC13): 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).

[0595] Isomer 2 (trans-(1 SR,2SR)-2-(pyridin-2-yl disulfide)cyclopentan-1-ol): LC-MS m / z calculated for C10H13NOS2, 227.34; found 228.1 [M+H] + . 1HNMR (400 MHz, CDC13): 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).

[0596] Absolute stereochemistry of any specified isomer.

[0597] Step 4: Synthesis of carbonic acid 4-nitrophenyl ester ((1R,2R)-2-(pyridin-2- yldisulfanyl)cyclopentyl ester)

[0598]

[0599] To a stirred solution of trans-(1RS,2RS)-2-(pyridin-2-yl disulfanyl)cyclopentan-1-ol (0.3 g, 1.34 mmol) in DMF (10 mL) was added DIPEA (0.65 mL, 3.96 mmol) and bis(4-nitrophenyl) carbonate (0.8 g, 2.64 mmol) at room temperature under nitrogen atmosphere. 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 x 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get the crude product which was purified by silica gel flash column chromatography. The compounds were eluted as a mixture in 10% EtOAc: n-hexane. The eluted fractions were evaporated to get the crude compound which was purified by reverse phase column chromatography. The product containing fractions were evaporated under reduced pressure to get carbonic acid 4-nitrophenyl ester (trans-(1RS,2RS)-2-(pyridin-2-yl disulfanyl)cyclopentyl ester) as a colorless oil (305 mg, 59%).

[0600] 1 HNMR (400 MHz, CDC13): 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).C 17 H 16 LC-MS m / z calculated for N2O5S2 392.44; found 393.0 [M+H] + .

[0601] Synthesis of 4-nitrophenyl carbonate (trans-(1SR,2SR)-2-(pyridin-2- yldisulfanyl)cyclopentyl ester)

[0602]

[0603] To a stirred solution of (1SR,2SR)-2-(pyridin-2-yl disulfanyl)cyclopentan-1-ol (0.26 g, 1.14 mmol) in DMF (10.0 mL) was added DIPEA (0.57 mL, 3.43 mmol) and bis(4-nitrophenyl)carbonate (0.7 g, 2.29 mmol) at room temperature under nitrogen atmosphere. 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 x 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get the crude product which was purified by silica gel flash column chromatography. The compounds were eluted as a mixture in 10% EtOAc: n-hexane. The eluted fractions were evaporated to get the crude compound which was purified by reverse phase column chromatography. The product containing fractions were evaporated under reduced pressure to get 4-nitrophenyl carbonate (trans-(1SR,2SR)-2-(pyridin-2-yl disulfanyl)cyclopentyl ester) as a colorless oil (330 mg, 73.5%). 1 HNMR (400 MHz, CDC13): δ 8.46 (d, J = 4 Hz, 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). C 17 H 16 LC-MS m / z calculated for N2O5S2 392.44; found 393.0 [M+H] + .

[0604] Synthesis of 4-nitrophenyl carbonate (trans-(1RS,2RS)-1-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl ester) Chiral preparative HPLC conditions:

[0605]

[0606] Step 1: Synthesis of 1aH,2H,7H,7aH-naphtho[2,3-b]cyclopropene

[0607]

[0608] To a stirred solution of 1,4-dihydronaphthalene (100 mg, 768 pmol) in dichloromethane (2.00 ml) was added 3-chlorobenzene-1 -peroxyl formic acid (199 mg, 1.5 eq, 1.15 mmol) in portions at 0 °C under nitrogen atmosphere and stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was filtered and 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 get crude product which was purified by silica gel flash column chromatography. The product was eluted in 10% EtOAc and n-hexane (product is UV inactive), the fractions were collected and dried under vacuum to get 1aH,2H,7H,7aH-naphtho[2,3-b]cyclovinylene as an oily compound (85.0 mg, 581 pmol).

[0609] 1 HNMR (400 MHz, CDC13): 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).

[0610] Step 2: Synthesis of rac-[trans-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)sulfanyl](phenyl)methanone

[0611]

[0612] To a stirred solution of 1aH,2H,7H,7aH-naphtho[2,3-b]cyclovinylene (100 mg, 684 pmol) in ethoxyethane (4.00 mL) was added aluminium oxide (1.00 g) (acidic) under nitrogen atmosphere. The solution was cooled to 0 °C. Then, thiobenzoic acid (482 mg, 5.1 eq, 3.49 mmol) was added to the reaction mixture and stirred at room temperature for 24 h. After completion of the reaction (the progress of the reaction was monitored by TLC), the reaction mixture was filtered and washed with sodium bicarbonate solution followed by water and brine solution to get crude product. The crude product was purified by silica gel flash column chromatography and the product was eluted in 20% EtOAc: n-hexane to get rac-[trans-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)sulfanyl](phenyl)methanone as a colorless liquid (125 mg, 440 pmol).

[0613] 1HNMR (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).

[0614] Step 3: Synthesis of rac-trans-3-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol

[0615]

[0616] 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) was added K2CO3(113 mg, 2 eq, 809 μmol) 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 HCI solution until the pH reached 2-3 to obtain rac-trans-3-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol (70.0 mg, 388 μmol) which was used as such for the next step.

[0617] Step 4: Synthesis of trans-(2RS,3RS)-3-(pyridin-2-yl disulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol and trans-(2SR,3SR)-3-(pyridin-2-yl disulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol

[0618]

[0619] To a stirred solution of racemic trans-3-thioxo-1,2,3,4-tetrahydronaphthalen-2-ol (350 mg, 1.94 mmol) in methanol (2.50 ml) was added 2-(pyridin-2- yldisulfanyl)pyridine (428 mg, 1 eq, 1.94 mmol) under nitrogen atmosphere and stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC and LC-MS. After completion of the reaction, the reaction mass was concentrated and then diluted with DCM, washed with water followed by brine and dried over sodium sulfate. The crude product obtained was purified by silica gel flash column chromatography. The desired product was eluted in 20% EtOAc: Hexane. The product was further purified by reverse phase column chromatography (10-20% of 0.1 % formic acid in water / acetonitrile). The fractions containing the desired product were collected and evaporated under vacuum to obtain 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 and the individual fractions collected from chiral preparative HPLC were combined and evaporated to get individual isomers. Isomer 1 was collected first and designated as trans-(2RS,3RS)-3-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol. Isomer 2 was collected second and designated as trans-(2SR,3SR)-3-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol. The absolute stereochemistry of the isomers was arbitrarily assigned.

[0620] 1 HNMR (400 MHz, DMSO): δ 8.44 (d, J = 4.4 Hz, 1 H), 7.79 (d, J = 3.2 Hz, 2H), 7.26-7.24 (m, 1 H), 7.06 (s, 4H), 5.61 (s, 1 H), 3.91 -3.80 (m, 1 H), 3.31 -3.19 (m, 2H), 3.13-3.07 (m, 1 H), 2.92-2.84 (m, 1 H), 2.75-2.65 (m, 1 H).

[0621] Synthesis of 4-nitrophenyl carbonate (trans-(1SR,2SR)-1-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl ester)

[0622] Column: CHIRALPAK IA (250 mm x 420 mm x 5 mic)

[0623] Mobile phase: n-hexane: Ethanol containing 0.1 % DEA (50:50)

[0624] Flow rate: 19 mL / min

[0625] The isomers were separated and the individual fractions collected from chiral preparative HPLC were combined and evaporated to get individual isomers. Isomer 1 was collected first and designated as trans-(2RS,3RS)-3-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol. Isomer 2 was collected second and designated as trans-(2SR,3SR)-3-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol. The absolute stereochemistry of the isomers was arbitrarily assigned.

[0626] Step 5: Synthesis of carbonic acid 4-nitrophenyl ester (trans-(2RS,3RS)-3-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl ester)

[0627] To a stirred solution of trans-(2RS,3RS)-3-(pyridin-2-yl disulfanyl)-1,2,3,4- tetrahydronaphthalen-2-ol (150 mg, 518 pmol) in dimethylformamide (3.00 ml, 38.7 mmol) was added bis(4-nitrophenyl) carbonate (315 mg, 2 eq, 1.04 mmol) followed by N,N- diisopropylethylamine (271 pL, 3 eq, 1.55 mmol). The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the reaction mass was quenched with water, extracted with DCM (3 x 5), the combined organic phase was dried over sodium sulfate, filtered and evaporated under reduced pressure to get the crude product. The crude product was purified by silica gel flash column chromatography (0-40% EtOAc: n-hexane) and also re-purified by reverse phase column chromatography (10-50% of 0.1 % formic acid in water: ACN) to get carbonic acid (trans-(2RS,3RS)-3-(pyridin-2-yl disulfanyl)-1,2,3,4- tetrahydronaphthalen-2-yl ester) as off-white solid (133 mg, 293 pmol).

[0628] 1 HNMR (400 MHz, DMSO): d 8.44 (d, 1 H), 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, 1 H), 7.14-7.06 (m, 4H), 5.21 -5.19 (m, 1 H), 3.78-3.77 (m, 1 H), 3.45-3.25 (m, 2H), 3.10-3.01 (m, 2H).

[0629] Synthesis of 4-nitrophenyl carbonate (trans-(2RS,3RS)-3-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl ester) Preparative conditions:

[0630]

[0631] 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) was added bis(4-nitrophenyl) carbonate (273 mg, 2 eq, 898 μmol) followed by diisopropylethylamine (13.0 mL, 3 eq, 74.6 mmol). The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction (progress monitored by TLC), the reaction mass was quenched with water and extracted with DCM (3 x 5). The combined organic phases were 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 column chromatography (10-50% 0.1% formic acid / water:ACN) to afford 4-nitrophenyl carbonate (trans-(2SR,3SR)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl ester) (30.0 mg, 66.0 μmol) as an off-white solid.

[0632] 1 HNMR (400MHz, DMSO): δ8.44(d,1H),8.30(d,J=9.2Hz,2H),7.80-7.76(m,2H),7.54(d,J=9.2Hz,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).

[0633] Synthesis of 4-nitrophenyl carbonate (trans-(2SR,3SR)-3-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl ester)

[0634]

[0635] Step 1: Synthesis of 3,7-dioxabicyclo[4.1.0]heptane

[0636]

[0637] At 0 DEG C, 3-chlorobenzene-1-peroxyformic acid (4.92g, 1.2 equivalents, 28.5mmol) was slowly added portionwise to a stirred solution of 3,6-dihydro-2H-pyrans (2.0g, 23.8mmol) in dichloromethane (20.0mL) and stirred for 16h at room temperature under a nitrogen atmosphere. The reaction progress was monitored by TLC. After the completion of the reaction, the reaction block was quenched with a saturated sodium bicarbonate solution, and the organic layer was separated and washed with water, then with a saline solution, dried over anhydrous sodium sulfate, filtered and evaporated to give the title compound 3,7-dioxabicyclo [4.1.0] heptane (1.00g, 9.99mmol) as a colorless oil. 1 H NMR (400MHz, 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).

[0638] Step 2: Synthesis of racemic [trans-(3-hydroxytetrahydropyran-4-yl)thio](phenyl)methanone:

[0639]

[0640] At room temperature, monothiobenzoic acid (5.88 mL, 5 equivalents, 49.9 mmol) was added to a stirred solution of 3,7-dioxabicyclo[4.1.0]heptane (1.00 g, 9.99 mmol) in ethoxyethane (40 mL), followed by silanedione (3.00 g, 5 equivalents, 49.9 mmol) and the reaction mixture was stirred at room temperature for 12 h. The reaction progress was monitored by TLC. When the starting material was complete, the reaction mass was quenched with a saturated sodium bicarbonate solution and then extracted with ethyl acetate (2 × 10 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and then evaporated under reduced pressure to give a crude product, which was purified by flash column chromatography (0-30% EtOAc: normal hexane). The compound was eluted in 20% EtOAc: normal hexane. The pure fractions were collected and evaporated to give racemic [trans-(3-hydroxytetrahydropyran-4-yl)thio](phenyl)methanone (2.0 g, 8.39 mmol).

[0641] C 12 H 14 LC-MS m / z calculated for O3S: 238.3, found: 239.1 [M+H] + ; 1H-NMR (400 MHz, CDC13): 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).

[0642] Step 3: Synthesis of rac-trans-4-sulfooxytetrahydro- pyran-3-ol

[0643]

[0644] To a stirred solution of rac-trans- (3-hydroxytetrahydro- pyran-4-yl) sulfoxide (phenyl) methanone (2.50 g, 10.5 mmol) in dichloromethane (25 mL) was added hydrazine hydrate (5.15 mL, 10 eq, 105 mmol) slowly at room temperature and the reaction mixture was stirred for 1 h. The progress of the reaction was monitored by TLC, on completion of the reaction, the reaction mass was quenched with 1 N HCI such that the pH was adjusted to 2-3. The two layers were separated and the organic layer was dried over sodium sulfate, filtered and partially evaporated, and the crude rac-trans-4-sulfooxytetrahydro- pyran-3-ol was used for the next step.

[0645] Step 4: Synthesis of trans-(3RS,4RS)-4-(pyridin-2- yldisulfide)tetrahydro-pyran-3-ol and trans-(3SR,4SR)-4-(pyridin-2- yldisulfide)tetrahydro-pyran-3-ol

[0646]

[0647] To a stirred solution of 2-(pyridin-2-yl disulfide)pyridine (1.85 g, 0.8 eq, 8.41 mmol) in methanol (40 mL) was added rac-trans-4-sulfooxytetrahydro- pyran-3-ol (1.41 g, 10.5 mmol) in DCM at 0 °C and then the reaction mixture was stirred at room temperature for 12 h. On completion of the reaction, the completed reaction mass was evaporated under reduced pressure to get crude material which was purified by flash column chromatography. The product was eluted in 20% EtOAc: n-hexane, pure fractions were collected and evaporated to get the title product 4-(pyridin-2-yl disulfide)oxane-3-ol (racemic mixture). The isomers were separated by chiral preparative HPLC.

[0648] Synthesis of 4-nitrophenyl carbonate (trans-(3RS,4RS)-4-(pyridin-2- yldisulfanyl)oxanyl-3-yl ester)

[0649] Column: CHIRALPAK IA (250 mm x 20 mm x 5 mic)

[0650] Mobile phase: n-Hexane: IPA with 0.1% DEA (90:10)

[0651] Flow rate: 19 mL / min

[0652] The isomers were separated and individual fractions were collected from chiral preparative HPLC. The fractions were combined and evaporated to give individual isomers.

[0653] (Isomer 1 - 350 mg, Isomer 2 - 350 mg) C 10 H 13 LC-MS m / z calculated for N2S2; 243.34, found 244 [M+H] + .

[0654] Isomer 1 (trans-(3RS,4RS)-4-(pyridin-2-yl disulfanyl)tetrahydropyran-3-ol):

[0655] 1 H-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).

[0656] Isomer 2 (trans-(3SR,4SR)-4-(pyridin-2-yl disulfanyl)tetrahydropyran-3-ol)

[0657] 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).

[0658] Absolute stereochemistry of any specified isomer.

[0659] Step 5: Synthesis of 4-nitrophenyl carbonate (trans-(3RS,4RS)-4-(pyridin-2- yldithio)tetrahydropyran-3-yl ester)

[0660] To a stirred solution of (trans-(3RS,4RS)-4-(pyridin-2-yl dithio)tetrahydropyran-3- ol) (300 mg, 1.23 mmol) in DMF (8 mL) was added bis(4-nitrophenyl) carbonate (750 mg, 2 eq, 2.47 mmol) and then diisopropylethylamine (644 μL, 3 eq, 3.70 mmol) was subsequently added at room temperature for 12 h. On completion of the reaction, the reaction mass was partitioned between water and DCM. The organic layer was separated and washed with brine solution and dried over sodium sulfate, filtered and evaporated under reduced pressure to get crude material which was purified by flash column chromatography. The desired compound was eluted as a mixture in 25% EtOAc: n-hexane. The mixture was purified by reverse phase column chromatography (10-60% of 0.1% formic acid in water / ACN). The eluted fractions containing the desired product were combined and evaporated to get 4-nitrophenyl carbonate (trans-(3RS,4RS)-4-(pyridin-2-yl dithio)tetrahydropyran-3-yl ester) (270 mg, 0.66 mmol). C 17 H 16 LC-MS m / z calcd for N2O6S2; 408.4, found 409.1 [M+H] + ; 1 H-NMR (400 MHz, CDC13): 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).

[0661] Chiral preparative HPLC conditions:

[0662]

[0663] To a stirred solution of (trans-(3SR,4SR)-4-(pyridin-2-yl disulfide)tetrahydropyran-3-ol) (340 mg, 1.40 mmol) in DMF (8 mL) was added bis(4-nitrophenyl) carbonate (850 mg, 2 eq, 2.79 mmol) followed by diisopropylethylamine (730 μL, 3 eq, 4.19 mmol) at room temperature for 12 h. On completion of starting material, the reaction mixture was partitioned between water and DCM. The organic layer was separated and washed with brine solution, dried over sodium sulfate, filtered and evaporated under reduced pressure to get crude product which was purified by flash column chromatography (0-40% EtOAc: n-hexane). The desired product was eluted as a mixture and then re-purified by reverse phase column chromatography (10-50% of 0.1% formic acid in water / ACN). The fractions containing the desired product were combined and evaporated to get 4-nitrophenyl carbonate (trans-(3SR,4SR)-4-(pyridin-2-yl disulfide)tetrahydropyran-3-yl ester) (300 mg, 735 μmol). C 17 H 16 LC-MS m / z calcd 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).

[0664] Synthesis of 4-nitrophenyl carbonate (trans-(3SR,4SR)-4-(pyridin-2- yldisulfanyl)tetrahydropyran-3-yl ester)

[0665]

[0666] Step 1 : Synthesis of 8-oxabicyclo[5.1.0]octane

[0667]

[0668] To a stirred solution of cycloheptene (1.0 g, 10.4 mmol) in dichloromethane (10 mL) was added 3-chlorobenzene-1 -peroxyl formic acid (2.15 g, 1.2 eq, 12.5 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 16 h. The progress of the reaction was monitored by TLC. On completion of the reaction, the reaction mixture was slowly quenched with saturated aqueous sodium bicarbonate solution and the mixture was stirred vigorously for about 30 min. The two layers were separated, the organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure to afford the desired product (700 mg, 6.24 mmol) as a colorless liquid. 1 H-NMR (400 MHz, CDC13): δ 3.07 (s, 2H), 1.93-1.86 (m, 4H), 1.60-1.43 (m, 4H), 1.21-1.17 (m, 2H).

[0669] Step 2: Synthesis of rac-[trans-(2-hydroxycycloheptyl)sulfonyl](phenyl)methanone

[0670]

[0671] To a stirred solution of 8-oxabicyclo[5.1.0]octane (3.00 g, 26.7 mmol) in toluene (60 mL) was added monothiobenzoic acid (4.72 mL, 1.5 eq, 40.1 mmol) followed by 2-methylpropan-2-aminium chloride (293 mg, 0.1 eq, 2.67 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at 50 °C for 16 h (reaction progress was monitored by TLC). On completion of the reaction, the reaction mixture was quenched with saturated sodium bicarbonate solution and then extracted with ethyl acetate (3 x 20 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure to afford the crude product which was purified by flash column chromatography. The desired product was eluted in 20% EtOAc: n-hexane, pure fractions were collected and evaporated to afford the title compound rac-[trans-(2-hydroxycycloheptyl)sulfonyl](phenyl)methanone (3.0 g, 12.0 mmol). C 14 H 18 LC-MS m / z calcd for C12H15O2S, 250.4, found 251.2 (M+H). 1 H-NMR (400 MHz, CDC13): 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).

[0672] Step 3: Synthesis of rac-trans-4-sulfanyl cycloheptan-3-ol

[0673]

[0674] To a stirred solution of rac-trans-(2-hydroxycycloheptyl)sulfonyl)(phenyl)methanone (2.80 g, 11.2 mmol) in dichloromethane (25 mL) was added 1,4- dithiobutane-2,3-diol (173 mg, 0.1 eq, 1.12 mmol) followed by hydrazine hydrate (1.37 mL, 2.5 eq, 28.0 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 3 h (reaction progress was monitored by TLC). On completion of the reaction, the reaction mixture was quenched with 1 N HCI and extracted with DCM (2 x 30 ml). The organic layers were combined and dried over sodium sulfate, filtered, the organic layer was partially evaporated and crude rac-trans-4-sulfanyl cycloheptan-3-ol was used directly for the next step.

[0675] Step 4: Synthesis of trans-(1RS,2RS)-2-(pyridin-2-yl dithio)cycloheptan-1-ol and trans-(1SR,2SR)-2-(pyridin-2-yl dithio)cycloheptan-1-ol

[0676]

[0677] To a stirred solution of 2-(pyridin-2-yl dithio)pyridine (1.73 g, 0.7 eq, 7.85 mmol) in methanol (25 mL) was added DCM containing rac-trans-4-sulfanyl cycloheptan-3-ol (1.64 g, 11.2 mmol) at 0 °C under nitrogen atmosphere 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 in 20% EtOAc: n-hexane. When the product was collected as a mixture, it was repurified by reverse phase column chromatography (10-50% of 0.1% formic acid in water: acetonitrile) to get rac-trans-2-(pyridin-2-yl dithio)cycloheptan-1-ol (1.5 g, 52%) (racemic mixture). The isomers were separated by chiral preparative HPLC.

[0678] (Isomer-1: 550 mg, Isomer-2: 550 mg).

[0679] Synthesis of 4-nitrophenyl carbonate (trans-(1RS,2RS)-2-(pyridin-2- yldisulfanyl)cycloheptyl ester)

[0680] Column: CHIRALPAK IA (250 mm x 20 mm x 5 mic)

[0681] Mobile phase: n-hexane: IPA containing 0.1% DEA (90:10)

[0682] Flow rate: 19 mL / min

[0683] The isomers were separated and individual fractions were collected from chiral preparative HPLC. The fractions were evaporated separately to give individual isomers.

[0684] Isomer 1 (trans-(1 RS,2RS)-2-(pyridin-2-yl disulfanyl)cycloheptan-1 -ol):

[0685] C 12 H 17 LC-MS m / z calculated for NOS2; 255.4, found 256.2 [M+H] + ; 1 H-NMR (400 MHz, CDC13): 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).

[0686] Isomer 2 (trans-(1 SR,2SR)-2-(pyridin-2-yl disulfanyl)cycloheptan-1 -ol):

[0687] C 12 H 17 LC-MS m / z calculated for NOS2; 255.4, found 256.2 [M+H] + ; 1 H-NMR (400 MHz, CDC13): 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).

[0688] The absolute stereochemistry of the isomers was arbitrarily assigned.

[0689] Step 5: Synthesis of carbonic acid 4-nitrophenyl ester (trans-(1 RS,2RS)-2-(pyridin-2- yldisulfanyl)cycloheptyl ester)

[0690] To a stirred solution of trans-(lRS,2RS)-2-(pyridin-2-yl disulfanyl)cycloheptan-l-ol (500 mg, 1.96 mmol) in DMF (10 mL) was added bis(4-nitrophenyl) carbonate (1.49 g, 2.5 eq, 4.89 mmol) followed by diisopropylethylamine (1.02 mL, 3 eq, 5.87 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred for 12 h. On 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 solution, dried over sodium sulfate, filtered and evaporated under reduced pressure to get the crude product which was purified by flash column chromatography. The desired product was eluted in 23% EtOAc: n-hexane as a mixture. The mixture was further purified by reverse phase column chromatography (10-60% of 0.1% formic acid in water / ACN) to get the title product, 4-nitrophenyl carbonate (trans-(lRS,2RS)-2-(pyridin-2-yl disulfanyl)cyclohexyl ester) (450 mg, 1.07 mmol). C 19 H 20 LC-MS m / z calculated for N2O5S2; 420.5, found 421.3 [M+H] + ; 1 H-NMR (400 MHz, CDC13): 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).

[0691] Chiral preparative HPLC conditions:

[0692]

[0693] To a stirred solution of trans-(lSR,2SR)-2-(pyridin-2-yl disulfide)cycloheptan-l-ol (580 mg, 2.27 mmol) in DMF (10 mL) was added bis(4-nitrophenyl) carbonate (1.73 g, 2.5 eq, 5.68 mmol) followed by diisopropylethylamine (1.38 mL, 3.5 eq, 7.95 mmol) under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 12 h. On completion of the reaction, monitored by TLC, the reaction mixture was partitioned between water and DCM. The two layers were separated and the combined organic layers were 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 as a mixture in 23-25% EtOAc: n-hexane. The product was further purified by reverse phase column chromatography (10-60% of 0.1% formic acid in water / ACN) to get the title compound, 4-nitrophenyl carbonate (trans-(lSR,2SR)-2-(pyridin-2-yl disulfide)cycloheptanate) (450 mg, 1.07 mmol). C 19 H 20 LC-MS m / z calcd for N2O5S2; 420.5, found 421.3 [M+H] + ; 1H-NMR (400 MHz, CDC13): 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).

[0694] Synthesis of 4-nitrophenyl carbonate (trans-(1SR,2SR)-2-(pyridin-2- yldisulfanyl)cycloheptyl ester) Synthesis of 4-nitrophenyl carbonate (trans-(1RS,2RS)-1-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl ester)

[0695]

[0696] Step 1: Synthesis of 1aH,2H,3H,7bH-naphtho[1,2-b]cyclopropene oxide

[0697]

[0698] To a stirred solution of 1,2-dihydronaphthalene (2.0 g, 15.4 mmol) in dichloromethane (75 mL) was added saturated solution of sodium bicarbonate (75 mL). The mixture was cooled to 0 °C. To this mixture was added 3-chlorobenzene-1 -peroxyl formic acid (5.30 g, 2 eq, 30.7 mmol) in portions over a period of 30 min. After addition, the reaction mass was stirred at room temperature for 16 h. 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 get 1aH,2H,3H,7bH-naphtho[1,2-b]cyclopropene (2.77 g). The obtained crude product was used as such for the next step without any further purification.

[0699] Step 2: Synthesis of racemic [trans-(2-hydroxy-1,2,3,4-tetrahydronaphthalen-1 - yl)sulfanyl](phenyl)methanone

[0700]

[0701] To a stirred solution of 1aH,2H,3H,7bH-naphtho[1,2-b]cyclopropene (2.25 g, 15.4 mmol) in ethoxyethane (20 mL) was added silanediol (4.50 g, 74.9 mmol) and monothiobenzoic acid (9.06 mL, 5 eq, 77.0 mmol) drop wise. The mixture was stirred at room temperature for 16 h. The progress of the reaction 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 x 20 mL). The combined organic layer was washed with water, brine and dried over sodium sulfate and concentrated under reduced pressure to get crude material which was purified by column chromatography to get racemic [trans-(2-hydroxy-1,2,3,4-tetrahydronaphthalen-1 -yl)sulfanyl](phenyl)methanone (1.57 mg, 35.87%) as yellow liquid.

[0702] Step 3: Synthesis of racemic trans-1 -sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol

[0703]

[0704] To a stirred solution of rac-trans-[(2-hydroxy-l,2,3,4-tetrahydronaphthalen-l- yl)thio](phenyl)methanone (1.40 g, 4.92 mmol) in dichloromethane (25.0 mL), (2R,3R)-l,4-dithiobutane-2,3-diol (144 mg, 0.19 eq, 935 μmol) and hydrazine hydrate (60.4 μL, 0.25 eq, 1.23 mmol) were added. The reaction mass was stirred at room temperature for 3 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched with HCI solution (pH = 1-2). The DCM layer was separated and dried over sodium sulfate, filtered and concentrated under reduced pressure to get racemic trans-l-thio-l,2,3,4-tetrahydronaphthalen-2-ol which was used as such for the next step.

[0705] Step 4: Synthesis of trans-(lRS,2RS)-l-(pyridin-2-yl dithio)-l,2,3,4-tetrahydronaphthalen- 2-ol and trans-(lSR,2SR)-l-(pyridin-2-yl dithio)-l,2,3,4-tetrahydronaphthalen-2-ol

[0706]

[0707] To a stirred solution of 2-(pyridin-2-yl dithio)pyridine (867 mg, 0.8 eq, 3.94 mmol) in methanol (5 mL) was added drop wise DCM containing racemic trans-l-thio-l,2,3,4-tetrahydronaphthalen-2-ol obtained from previous step at 0 °C. The reaction was stirred at room temperature for 16 h. The reaction was monitored by LCMS and TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure to get crude material which was purified by column chromatography to get racemic l-(pyridin-2-yl dithio)-l,2,3,4-tetrahydronaphthalen-2-ol as yellowish oil which was further purified by reverse phase column chromatography to get colorless oil (380 mg, 26.69 %). The obtained racemic product was separated by chiral chromatography to get Isomer-1: 130 mg; Isomer-2: 190 mg.

[0708] Chiral preparative HPLC conditions:

[0709] Column: CHIRALPAK IA (250 mM x 420 mm x 5 mic)

[0710] Mobile phase: n-hexane: Ethanol containing 0.1 % DEA (50:50)

[0711] Flow rate: 19 mL / min

[0712] Isomer-1 (trans-(1 RS,2RS)-1 -(pyridin-2-yl disulfanyl)-1,2,3,4-tetrahydronaphthalen- 2-ol):

[0713] C 15 H 15 LC-MS m / z calculated for NOS2; 289.4, found 290.1 [M+H] + ;1H-NMR (400 MHz, CDC13): 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).

[0714] Isomer-2 (trans-(1 SR,2SR)-1 -(pyridin-2-yl disulfanyl)-1,2,3,4-tetrahydronaphthalen- 2-ol)

[0715] C 15 H 15 LC-MS m / z calculated for NOS2; 289.4, found 290.1 [M+H] + ;1H-NMR (400 MHz, CDC13): 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).

[0716] The absolute stereochemistry of any specified isomer.

[0717] Step 5. Synthesis of carbonic acid 4-nitrophenyl ester (trans-(1 RS,2RS)-1 -(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl ester)

[0718] To a stirred solution of trans-(lRS,2RS)-l-(pyridin-2-yl disulfanyl)-l,2,3,4- tetrahydronaphthalen-2-ol (170 mg, 587 pmol) in N,N-dimethylformamide (2.50 mL) was added dropwise carbonic acid bis(4-nitrophenyl ester) (447 mg, 2.5 eq, 1.47 mmol) at room temperature followed by the addition of diisopropylethylamine (307 pL, 3 eq, 1.76 mmol). The reaction mixture was stirred in a sealed tube at room temperature for 12 h. 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 get crude material which was purified by flash column chromatography (0-40% EA / hexane) and also re-purified by reverse phase column chromatography (10-70% of 0.1% formic acid in water / ACN) to get carbonic acid 4-nitrophenyl ester (trans-(lRS,2RS)-l-(pyridin-2-yl disulfanyl)-l,2,3,4- tetrahydronaphthalen-2-yl ester) (70.0 mg, 154 pmol) as a colorless gummy solid (70 mg, 26.22%). 22 H 18 LC-MS m / z calculated for N2O4S2; 454.5, found 455.3 [M+H] + ;1H-NMR (400 MHz, CDCl3): d 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).

[0719] Synthesis of 4-nitrophenyl carbonate (trans-(1SR,2SR)-1-(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl ester) Synthesis of 4-nitrophenyl carbonate (trans-4-(pyridin-2- yldisulfanyl)cyclohexyl ester)

[0720]

[0721] To a stirred solution of trans-(lSR,2SR)-l-(pyridin-2-yl disulfanyl)-l,2,3,4- tetrahydronaphthalen-2-ol (120 mg, 415 pmol) in N,N-dimethylformamide (1.50 mL) was added dropwise carbonic acid bis(4-nitrophenyl ester) (315 mg, 2.5 eq, 1.04 mmol) at room temperature followed by diisopropylethylamine (217 pL, 3 eq, 1.24 mmol). The reaction mixture was stirred in a sealed tube at room temperature for 12 h. 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 get crude material which was purified by flash column chromatography (0-40% EA in hexane) and also re-purified by reverse phase column chromatography (10-70% of 0.1% formic acid in water / ACN) to get carbonic acid 4-nitrophenyl ester (trans-(lSR,2SR)-l-(pyridin-2-yl disulfanyl)-l,2,3,4- tetrahydronaphthalen-2-yl ester) as a colorless gummy solid (65 mg, 34.49%).

[0722] C 22 H 18 LC-MS m / z calcd 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).

[0723] Synthesis of 4-nitrophenyl carbonate (trans-(2R)-3-methyl-2-(pyridin-2- yldisulfanyl)butyl ester)

[0724]

[0725] Step 1 : Synthesis of trans-4-mercaptocyclohexan-1-ol

[0726]

[0727] To a stirred solution of 7-oxabicyclo[2.2.1]heptane (1.00 g, 10.2 mmol) in ethanol (10 mL) was added 4-methylbenzene-1 -sulfonic acid (2.63 g, 1.5 eq, 15.3 mmol), thiourea (1.16 g, 1.5 eq, 15.3 mmol) and the reaction mass was heated to 80 °C for 24 h. 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 h. After completion of the reaction, the reaction mass was cooled to room temperature, concentrated under reduced pressure and acidified with 10% H2SO4solution. Then, the reaction mass was extracted with DCM and used as such for the next step.

[0728] Step 2: Synthesis of trans-4-(pyridin-2-yl disulfanyl)cyclohexan-1-ol

[0729]

[0730] To a stirred solution of 2-(pyridin-2-yl disulfanyl)pyridine (1.60 g, 0.8 eq, 7.26 mmol) in methanol (10.0 mL) was added to the organic layer from (step 1) 4-sulfanyl cyclohexan-1-ol (1.20 g, 9.08 mmol) at 0 °C. After completion of the addition, the reaction mass was allowed to stir at room temperature for 16 h. 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 get the desired product. The product was further purified 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 get the title product as a yellow oil (1.60 g, 73% yield). C 11 H 15 LC-MS m / z calculated for NOS2, 241 ; found 242 [M+H]+.

[0731] Step 3: Synthesis of carbonic acid 4-nitrophenyl ester (trans-4-(pyridin-2- yldisulfanyl)cyclohexyl ester)

[0732] To a stirred solution of trans-4-(pyridin-2-yl disulfide)cyclohexan-1-ol (400 mg, 1.66 mmol) in N,N-dimethylformamide (3 mL) was added bis(4-nitrophenyl) carbonate (907 mg, 1.8 eq, 2.98 mmol), ethyl bis(propan-2-yl)amine (892 μL, 3 eq, 4.97 mmol) under nitrogen atmosphere and stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was quenched with water (15 mL) and extracted with DCM (3 x 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 crude product obtained was purified by flash column chromatography (0-30% EtOAc: n-hexane). The product was further purified by reverse phase column chromatography using 0.1% formic acid and ACN. The eluent fractions containing the desired product were collected and concentrated under reduced pressure to afford 4-nitrophenyl carbonate (trans-4-(pyridin-2-yl disulfide)cyclohexyl ester) as a yellow oil (0.3 g, 73% yield). C 18 H 18 LC-MS m / z calculated for N2O5S2 407; found 407 [M+H]+; 1 HNMR (400 MHz, CDC13): 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).

[0733] Preparative HPLC conditions:

[0734]

[0735] Step 1. Synthesis of benzoylcesium sulfide

[0736]

[0737] To a stirred solution of mono-thiobenzoic acid (5.00 g, 36.2 mmol) in methanol (40.0 mL) was added cesium carbonate (7.72 g, 1.1 eq, 39.8 mmol) in portions over 10-15 min under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (as judged by TLC), the reaction mixture was concentrated under reduced pressure. The solid residue was diluted with 10 mL of acetone and the white solid (CsHC03) was filtered off. This process was repeated twice to ensure removal of all CsHC03. The acetone was then concentrated to obtain benzoic acid cesium sulfide (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).

[0738] Step 2. Synthesis of (2R)-2-(benzoylthio)-3-methylbutanoic acid

[0739]

[0740] 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 benzoyl cesium sulfide (2.98 g, 11.0 mmol). The reaction mixture was stirred at room temperature for 20 h. The progress of the reaction was monitored by TLC and after completion of the reaction, the reaction mixture was diluted with diethyl ether (3 x 15 mL) and washed with water (3 x 15 mL). The ether layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue obtained was recrystallized from n-hexane to obtain (2R)-2-(benzoylthio)-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).

[0741] Step 3. Synthesis of (2R)-3-methyl-2-sulfanybutan-1-ol

[0742]

[0743] To a stirred solution of (2R)-2-(benzoylthio)-3-methylbutanoic acid (2.50 g, 10.5 mmol) in ethoxyethane (50.0 mL) was added lithium aluminum hydride (52.5 mL, 5 eq, 52.5 mmol) dropwise at 0 °C under 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 progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was cooled in an ice bath at 0 °C and quenched with 1.0 N HC1 (30 mL). The reaction mixture was extracted with DCM (20 mL) and the remaining gelled material from LAH reduction was washed with diethyl ether (10 mL). The combined organic layers were dried over sodium sulfate, filtered and taken to the next step.

[0744] Step 4. Synthesis of (2R)-3-methyl-2-(pyridin-2-yl disulfide)butan-1-ol

[0745]

[0746] To a stirred solution of (2R)-3-methyl-2-thio butan-1-ol (1.20 g, 9.98 mmol) in MeOH (5 mL) was added 2-(pyridin-2-yl disulfide)pyridine (1.76 g, 0.8 eq, 7.99 mmol) and stirred at room temperature for 16 h under nitrogen atmosphere. The progress of the reaction was monitored by TLC and LC-MS. After completion of the reaction, the reaction mass was concentrated and then extracted with DCM. The two layers were separated and the combined organic layer was washed with water followed by brine and dried over sodium sulfate, filtered and evaporated. The crude product was purified by silica gel flash column chromatography using 12 g column eluting in 50% EtOAc: n-hexane and also re-purified by reverse phase column chromatography (10-20% of 0.1% formic acid in water / acetonitrile). The fractions containing the product were collected and evaporated under vacuum to get the title product. The product was further purified by preparative HPLC.

[0747] Synthesis of the compound of Example 2 from intermediate III-2

[0748] Column: X-Bridge C-18 (250 mm x 4.6 mm x 5 mic)

[0749] Mobile phase (A): 0.1% ammonia water

[0750] Mobile phase (B): Acetonitrile

[0751] Flow rate: 19 mL / min

[0752] Gradient B: 0 / 10, 12 / 60, 22 / 95, 25 / 95, 27 / 10, 30 / 10

[0753] The fractions collected from preparative HPLC were combined and evaporated to yield the title product 3-(pyridin-2-yl disulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (350 mg, 1.21 mmol) as a yellow solid. 1 HNMR (400 MHz, CDC13): δ 8.49 (d, J = 4 Hz, 1 H), 7.55-7.54 (m, 1 H), 7.36 (d, J = 8.4 Hz, 1 H), 7.13 (t, J = 6.4 Hz, 1 H), 3.82 (dd, J = 12.4 Hz, 1 H), 3.66-3.60 (m, 1 H), 2.75-2.70 (m, 1 H), 2.01 -1.92 (m, 1 H), 1.10-1.01 (m, 7H).

[0754] Step 5. (2R)-3-methyl-2-(pyridin-2-yl disulfanyl)butyl 4-nitrophenyl carbonate

[0755] To a stirred solution of (2R)-3-methyl-2-(pyridin-2-yl disulfanyl)butan-1 -ol (800 mg, 3.49 mmol) in N,N-dimethylformamide (2.50 mL) was added bis-carbonic acid (4-nitrophenyl ester) (2.12 g, 2 eq, 6.98 mmol) followed by diisopropylethylamine (1.82 mL, 3 eq, 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 yield the crude product which was purified by flash column chromatography (0-40% EtOAc: n-hexane). The product was further purified by reverse phase chromatography (10-70% of 0.1 % formic acid in water / ACN) to yield the title product (2R)-3-methyl-2-(pyridin-2-yl disulfanyl)butyl 4-nitrophenyl carbonate (600 mg, 1.52 mmol) as a colorless gum. 1 HNMR (400 MHz, CDC13): δ 8.45 (d, J = 4.0 Hz, 1 H), 8.26 (d, J = 9.2 Hz, 2 H), 7.72 (d, J = 8.4 Hz, 1 H), 7.63 (t, J = 7.2 Hz, 1 H), 7.35 (d, J = 9.2 Hz, 2 H), 7.08 (t, J = 6.8 Hz, 1 H), 4.59-4.48 (m, 2 H), 3.08 (q, J = 6.0 Hz, 1 H), 2.21 -2.13 (m, 1 H), 1.14-1.06 (m, 6 H).

[0756] From Step 2, the same procedure was followed using (2R)-2-bromo-3-methylbutanoic acid to synthesize (2S)-3-methyl-2-(pyridin-2-yl disulfide)butyl carbonate 4-nitrophenyl ester.

[0757] Synthesis of the compound of Example 10 from intermediate XVI-1

[0758]

[0759] To a vial containing Pv2 (25.0 mg, 0.061 mmol; as a free-flowing solid), N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexadec- 1(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [(2S)-2-(2-pyridyl disulfide)propyl ester] (6.03 mg, 0.091 mmol) was added 1 mL of CH3CN and 0.5 mL of water. To this was added N-methylmorpholine (22.7 mg, 0.224 mmol). The mixture was stirred at room temperature overnight. LC-MS indicated the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, Prep Slope_4min, 20-85% CH3CN / H2O + 0.05% TFA, 15 min) to give the desired product (13.0 mg, yield: 47.0%).

[0760] The compounds of Examples 1 and 3-9 were synthesized from intermediates III-1 and III-3 to III-9, respectively, in analogy to the compounds of Example 2 (see Table 4 below).

[0761] http: / / cellprofiler.org

[0762]

[0763] DMF and PBS were degassed using N2flow for 30 min. Pv2 (25.0 mg, 0.061 mmol; as a free flowing solid), N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexadecano[14.7.1.02,14.04,13.06,11.020,24]tetracosan-1,6(11),12,14,16(24),17,19- heptaen-23-yl]carbamic acid [4-(2-pyridyl disulfanyl)phenyl]methyl ester (6.5 mg, 0.09 mmol), 1.5 mL of DMF and 0.5 mL of PBS were placed in separate vials. To this CH3CO2H (0.0347 mL, 0.606 mmol) was added. The mixture was stirred at room temperature overnight. LC-MS indicated the reaction to be complete. The reaction mixture was purified by reverse phase HPLC (PrepSlope_4min, 30-100% CH3CN / H2O + 0.05% TFA, 18 min) to get the desired product (3.0 mg, yield: 10.7%).

[0764] Compounds and analytical data of the present application are presented below.

[0765] Table 4. Example compounds

[0766]

[0767]

[0768]

[0769]

[0770] Example 11: Synthesis of compound 11

[0771]

[0772] Step 1. Synthesis of 2-(pyridin-2-yl disulfanyl)cyclohexan-1-ol

[0773]

[0774] To a solution of 1,2-bis(pyridin-2-yl)disulfane (15.2 g, 68.9 mmol) in MeOH (degassed with N2) (30 mL) was added dropwise (1-mercaptocyclobutyl)methanol (11.4 g, 86.2 mmol) (degassed with N2) and stirred at room temperature under N2atmosphere for 16 h. The reaction mixture was concentrated to dryness under vacuum. The crude material obtained was purified by column chromatography using 30% EtOAc / hexane to get the title compound as a yellow liquid. 1HNMR (400MHz, CDCl3): δ8.54-8.53(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).

[0775] The title compound was subjected to chiral preparative HPLC conditions (Chiralpak IG: 250 mm × 20 mm × 5 mic; n-hexane:IPA containing 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 minutes), followed by (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol (retention time: 11.3 minutes). The absolute stereochemistry of the product of step 2 was confirmed by comparison with chiral materials with reported absolute stereochemistry (see Monaco, MR; J. Am. Chem. Soc. 2014, 136, 49, 16982-16985).

[0776] Step 2. Synthesis of 4-nitrophenyl carbonate ((1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl ester).

[0777]

[0778] At room temperature, DIPEA (10.3 mL, 56.0 mmol) and bis(4-nitrophenyl carbonate) (11.35 g, 27.3 mmol) were added to a solution of (1R, 2R)-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol (4.5 g, 18.6 mmol) in DMF (90.0 mL). The reaction vessel was sealed and stirred at room temperature for 12 h. The reaction progress was monitored by TLC (20% EtOAc / hexanes). After the 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 a crude product, which was purified by column chromatography using 20-30% EtOAc / hexanes to give the title product (5.0 g, 66% yield) as an off-white solid. 1HNMR (400 MHz, CDC13): 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 m / z calcd for C26H26FN3O4: 406.7; found: 407.4 [M+H] + .

[0779] Step 3. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4, 15-diazahexadec- 1,6(11), 12, 14, 16(24), 17, 19-heptaen-23-yl] carbamate [(1S,2S)-2-(2-pyridyl disulfide)cyclohexyl ester].

[0780]

[0781] To (10S,23S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19-methyl-8-oxa-4,15- diazahexadec-1,6(11),12,14,16(24),17,19-heptaen-5,9-dione methanesulfonic acid (250 mg, 0.470 mmol) in 10 mL of dry DMF was added (1R,2R)-2-(pyridin-2- yldisulfide)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 indicated that the desired coupling product had formed. The reaction mixture was then diluted with EtOAc, washed with saturated aqueous NH4C1, H2O, 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 72.6% yield (240 mg) of the desired product.

[0782] Step 4. Coupling with Pv1 (Compound 11)

[0783] To a vial was added Pv1 (275 mg,.0811 mmol), the compound from Step 3 (74.1 mg, 0.105 mmol), acetonitrile (10 mL), and water (5 mL). To this mixture was added n- methylmorpholine (0.303 g,.0030 mol). The mixture was stirred at room temperature overnight. LC-MS indicated that the desired coupling product had formed.

[0784] The reaction mixture was purified directly by reverse phase HPLC (20-85% acetonitrile / water, 0.5% acetic acid on a Sunfire preparative C18 column (10 μm, 50 x 150 mm), retention time: 7.022 min) to give 213 mg of the desired product in 68% yield (213 mg). ESI (M+3H / 3) 3+ :1291.6

[0785] Example 12: Synthesis of Compound 12

[0786]

[0787] Step 1. Synthesis of 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]tetracosan-1,6(11),12,14,16(24),17,19-heptene-23-yl]carbamic acid [(1R,2R)-1-methyl-2-(2-pyridinyl disulfide)propyl ester]

[0788] To 1-hydroxybenzotriazole hydrate (8.64 mg, 0.0564 mmol), finely powdered molecular sieves (4 A, 60 mg), and DMF (0.5 mL) was added the compound from Step 1 (10.0 mg, 0.028 mmol). The mixture was stirred at room temperature for 30 min. To this mixture was added HATU (11.2 mg, 0.029 mmol) and the mixture was stirred at room temperature for 30 min. To this mixture was added the compound from Step 2 (8.0 mg, 0.028 mmol) and the mixture was stirred at room temperature for 30 min. The mixture was filtered and the filtrate was purified by reverse phase HPLC (20-85% acetonitrile / water, 0.5% acetic acid on a Sunfire preparative C18 column (10 μm, 50 x 150 mm), retention time: 7.022 min) to give 213 mg of the desired product in 68% yield (213 mg). ESI (M+3H / 3) To a mixture of (50 mg) and (10S,23S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19- methyl-8-oxa-4,15-diazahexadec-1,6(11),12,14,16(24),17,19-heptaen-5,9-dione methanesulfonic acid (25.0 mg, 0.0470 mmol) and pyridine (0.0190 mL, 0.235 mmol) in 2 mL of dry DMF was added [(1 R,2R)-1 -methyl-2-(2-pyridyl disulfanyl)propyl] (4-nitrophenyl) carbonate (19.7 mg, 0.470 mmol) (see synthesis II-4: [(2R,3R)-3-(pyridin-2-yl disulfanyl)butan-2-yl] 4-nitrophenyl carbonate). After stirring at room temperature for 16 h, the mixture was filtered and the solution was concentrated. 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%).

[0789] Step 2. Coupling with peptide Pv1 (compound 12)

[0790] A vial was charged with peptide Pv1 (50.0 mg, 14.7 e-5 mol), [(1 R,2R)-1 -methyl-2-(2- pyridyl disulfanyl)propyl] N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexadec-1,6(11),12,14,16(24),17,19-heptan-23-yl]carbamate (0.013 g, 1.92 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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-85% ACN / H2O + 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

[0791] Example 13: Synthesis of compound 13

[0792]

[0793] Compound 13 was prepared in a similar manner to Compound 11, using ((1R,2R)-2-(pyridin-2-yl disulfide)cyclohexyl) carbonate in place of ((1S,2S)-2-(pyridin-2-yl disulfide)cyclohexyl) carbonate in Step 2. Sunfire preparative C18 column (10 μm, 50 x 150 mm) (20-85% acetonitrile / water, 0.5% acetic acid); retention time: 6.609 minutes. ESI (M+3H / 3) 3+ :1290.3

[0794] Example 14: Synthesis of Compound 14

[0795]

[0796] Step 1. Synthesis of (4-nitrophenyl) carbonochloridate [trans-(1RS,2RS)-2-(2- pyridyl disulfide)cyclopentyl] ether

[0797] The title compound was synthesized according to a similar synthetic procedure described in the synthesis of Compound 11, using the first eluted stereoisomer (designated trans-(1RS,2RS)-2-(2-pyridyl disulfide)cyclopentan-1-ol) to be separated from racemic trans-2-(2-pyridyl disulfide)cyclopentyl by chiral chromatography.

[0798] Step 2. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexadecano[14.7.1.02,14.04,13.06,11.020,24]tetracosan-1,6(11),12,14,16(24),17,19-heptene-23-yl]carbamic acid [trans-(1RS,2RS)-2-(2-pyridyl disulfide)cyclopentyl] ester

[0799] 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) carbonic acid [trans-(1RS,2RS)-2-(2-pyridyl disulfide)cyclopentyl ester] (40.6 mg, 0.103 mmol) in 2 mL of dry 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), 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, yield: 50.9%).

[0800] Step 3. Coupling with the peptide Pv1 (Compound 14)

[0801] A vial was charged with peptide Pv1 (50.0 mg, 1.47e-5 mol), N-[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexadecano- 1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(1RS,2RS)-2-(2- pyridyl disulfide)cyclopentyl ester] (0.0124 g, 1.80e-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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-90% ACN / H2O + 0.05% TFA, 16 min; retention time: 6.761 min) to give Compound 14 (0.0360 g, 9.34e-6 mol, yield: 63.3%). ESI (M+3H / 3) 3+ :1286.3.

[0802] Example 15: Synthesis of Compound 15

[0803]

[0804] Step 1. Synthesis of (4-nitrophenyl) carbonic acid [trans-(1SR,2SR)-2-(2- pyridyl disulfide)cyclopentyl ester]

[0805] The title compound was synthesized using a similar synthetic procedure described in the synthesis of compound 11 from the second eluted stereoisomer of racemic trans-2-(2- pyridyl disulfide)cyclopentyl that was separated by chiral chromatography of the racemic trans-2-(2-pyridyl disulfide)cyclopentyl-1-ol (designated as trans-(1SR,2SR)-2-(2- pyridyl disulfide)cyclopentyl-1-ol).

[0806] Step 2. Synthesis of 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]tetracosan-1,6(11),12,14,16(24),17,19-heptene-23-yl]carbamic acid [trans-(1SR,2SR)-2-(2-pyridyl disulfide)cyclopentyl ester]

[0807] 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) carbonate [trans-(1SR,2SR)-2-(2- pyridyl disulfide)cyclopentyl ester] (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), 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, 44.8% yield).

[0808] Step 3. Coupling with peptide Pv1 (compound 15)

[0809] A vial was charged with peptide Pv1 (50.0 mg, 1.47e-5 mol), 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]tetracosan-1,6(11),12,14,16(24),17,19-heptaen-23-yl] carbamic acid trans-[(1 SR,2SR)-2-(2-pyridyl disulfide)cyclopentyl ester] (0.0124 g, 1.80e-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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-90% ACN / H20 + 0.05% TFA, 16 min; retention time: 6.883 min) to give compound 15 (0.0280 g, 7.26e-6 mol, 49.3% yield). ESI (M+3H / 3) 3+ :1285.9.

[0810] Example 16: Synthesis of compound 16

[0811]

[0812] Step 1. Synthesis of carbonic acid (4-nitrophenyl ester) [trans-(3RS,4RS)-4-(2- pyridyl disulfide)tetrahydrofuran-3-yl ester]

[0813] The title compound was synthesized using a similar synthetic procedure described in the synthesis of compound 11 from the first stereoisomer eluted from the chiral chromatography of racemic trans-4-(2-pyridyl disulfide)tetrahydrofuran-3-ol, designated as trans-(3RS,4RS)-4-(2-pyridyl disulfide)tetrahydrofuran-3-ol.

[0814] Step 2. Synthesis of 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]tetracosan-1,6(11),12,14,16(24),17,19-heptaen-23-yl] carbamic acid [trans-(3RS,4RS)-4-(2-pyridyl disulfide)tetrahydrofuran-3-yl ester]

[0815] To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol), DMAP (23.0 mg, 0.188 mmol), and carbonic acid (4-nitrophenyl ester) [trans-(3RS,4RS)-4-(2-pyridyldithio)tetrahydrofuran-3-yl ester] (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), 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, yield: 61.6%).

[0816] Step 3. Coupling with peptide Pv1 (Compound 16)

[0817] A vial was charged with peptide Pv1 (50.0 mg, 1.47e-5 mol), N-[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexadecano- 1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(3RS,4RS)-4-(2- pyridyldithio)tetrahydrofuran-3-yl ester] (0.0124 g, 1.80e-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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-80% ACN / H2O + 0.05% TFA, 15 min; retention time: 6.633 min) to give Compound 16 (0.0290 g, 7.52e-6 mol, yield: 51.0%). ESI (M+3H / 3) 3+ :1286.4.

[0818] Example 17: Synthesis of Compound 17

[0819]

[0820] Step 1. Synthesis of carbonic acid (4-nitrophenyl ester) [trans-(3SR,4SR)-4-(2- pyridyldithio)tetrahydrofuran-3-yl ester]

[0821] The title compound was synthesized using a similar synthetic procedure described in the synthesis of compound 11 from the second stereoisomer eluted from the chiral chromatography of racemic trans-4-(2-pyridyl disulfanyl)tetrahydrofuran-3-ol, designated as trans-(3SR,4SR)-4-(2-pyridyl disulfanyl)tetrahydrofuran-3-ol.

[0822] Step 2. Synthesis of 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]tetracosan- 1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(3SR,4SR)-4-(2- pyridyl disulfanyl)tetrahydrofuran-3-yl ester]

[0823] 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) carbonate [trans-(3SR,4SR)-4-(2-pyridyl disulfanyl)tetrahydrofuran-3-yl ester] (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), 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 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]tetracosan-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(3SR,4SR)-4-(2-pyridyl disulfanyl)tetrahydrofuran-3-yl ester] (31.0 mg, 0.0449 mmol, 47.7% yield).

[0824] Step 3. Coupling with peptide Pv1 (Compound 17)

[0825] A vial was charged with peptide Pv1 (50.0 mg, 1.47e-5 mol), 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]tetracosan-1,6(11),12,14,16(24),17,19-heptaen-23-yl] carbamic acid [trans-(3SR,4SR)-4-(2-pyridyl disulfide)tetrahydrofuran-3-yl ester] (0.0124 g, 1.80e-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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-85% ACN / H20 + 0.05% TFA, 13 min; retention time: 6.670 min) to give compound 17 (0.0170 g, 4.41e-6 mol, 29.9% yield). ESI (M+3H / 3) 3+ :1286.7.

[0826] Example 18: Synthesis of compound 18

[0827]

[0828] Step 1. Synthesis of carbonic acid (4-nitrophenyl ester) [trans-(2RS,3RS)-3-(2- pyridyl disulfide)tetrahydronaphthalen-2-yl ester]

[0829] The title compound was synthesized using a similar synthetic procedure described in the synthesis of compound 11 from the first stereoisomer eluted from the chiral chromatography of racemic trans-3-(2-pyridyl disulfide)tetrahydronaphthalen-2-ol, designated as trans-(2RS,3RS)-3-(2-pyridyl disulfide)tetrahydronaphthalen-2-ol.

[0830] Step 2. Synthesis of 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]tetracosan-1,6(11),12,14,16(24),17,19-heptaen-23-yl] carbamic acid [trans-(2RS,3RS)-3-(2-pyridyl disulfide)tetrahydronaphthalen-2-yl ester]

[0831] To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol) and carbonic acid (4-nitrophenyl ester) [trans-(2RS,3RS)-3-(2-pyridyldithio)tetrahydronaphthalen-2-yl ester] (32.1 mg, 0.0705 mmol) in 2 mL of dry 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), 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%).

[0832] Step 3. Coupling with the peptide Pv1 (Compound 18)

[0833] A vial was charged with peptide Pv1 (25.0 mg, 7.37e-6 mol), N-[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexadecano- 1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(2RS,3RS)-3-(2- pyridyldithio)tetrahydronaphthalen-2-yl ester] (0.00719 g, 9.58e-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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-95% ACN / H2O + 0.05% TFA, 20 min; retention time: 6.851 min) to give Compound 18 (0.0080 g, 2.04e-6 mol, yield: 27.7%). ESI (M+3H / 3) 3+ :1307.4.

[0834] Example 19: Synthesis of Compound 19

[0835]

[0836] Step 1. Synthesis of carbonic acid (4-nitrophenyl ester) [trans-(2SR,3SR)-3-(2- pyridyldithio)tetrahydronaphthalen-2-yl ester]

[0837] The title compound was synthesized using a similar synthetic procedure described in the synthesis of compound 11 from the second stereoisomer eluted from the chiral chromatography of racemic trans-3-(2-pyridyl disulfanyl)tetrahydronaphthalen-2-ol, designated as trans-(2SR,3SR)-3-(2-pyridyl disulfanyl)tetrahydronaphthalen-2-ol.

[0838] Step 2. Synthesis of 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]tetracosan- 1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(2SR,3SR)-3-(2- pyridyl disulfanyl)tetrahydronaphthalen-2-yl ester]

[0839] To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and carbonic acid (4-nitrophenyl ester) [trans- (2SR,3SR)-3-(2-pyridyl disulfanyl)tetrahydronaphthalen-2-yl ester] (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), 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 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]tetracosan-1,6(11),12,14,16(24),17,19- heptaen-23-yl]carbamic acid [trans-(2SR,3SR)-3-(2-pyridyl disulfanyl)tetrahydronaphthalen-2-yl ester] (10.0 mg, 0.0133 mmol, 28.3% yield).

[0840] Step 3. Coupling with peptide Pv1 (compound 19)

[0841] A vial was charged with peptide Pv1 (25.0 mg, 7.37e-6 mol), N-[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazacyclohexadeca- 1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(2SR,3SR)-3-(2- pyridyl disulfide)tetrahydronaphthalen-2-yl ester] (0.010 g, 1.33e-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 the reaction was complete.

[0842] The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-95% ACN / H20 + 0.05% TFA, 20 min; retention time: 6.855) to give compound 19 (0.0060 g, 1.33e-5 mol, yield: 20.8%). ESI (M+3H / 3) 3+ : 1307.6.

[0843] Example 20: Synthesis of compound 20

[0844]

[0845] Step 1. Synthesis of carbonic acid (4-nitrophenyl ester) [trans-(3RS,4RS)-4-(2- pyridyl disulfide)tetrahydropyranyl-3-yl ester]

[0846] The title compound was synthesized using a similar synthetic procedure described in the synthesis of compound 11 from the first stereoisomer eluted from the chiral chromatography of racemic trans-4-(2-pyridyl disulfide)tetrahydropyrane-3-ol, designated as trans-(2RS,3RS)-4-(2-pyridyl disulfide)tetrahydropyrane-3-ol.

[0847] Step 2. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazacyclohexadeca-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(3RS,4RS)-4-(2-pyridyl disulfide)tetrahydropyranyl-3-yl ester]

[0848] 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) carbonate [trans-(3RS,4RS)-4-(2-pyridyldithio)tetrahydropyran-3-yl ester] (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), 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 N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diaza hexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosan-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(3RS,4RS)-4-(2-pyridyldithio)tetrahydropyran-3-yl ester] (30.0 mg, 0.0426 mmol, yield: 90.5%).

[0849] Step 3. Coupling with the peptide Pv1 (compound 20)

[0850] A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6 mol), N-[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexadecano- 14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23- yl]carbamic acid [trans-(3RS,4RS)-4-(2-pyridyl disulfide)tetrahydropyran-3-yl ester] (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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 30-85% ACN / H20 + 0.05% TFA, 13 min; retention time: 6.380) to give compound 20 (0.0060 g, 1.55 e-6 mol, 21.0% yield). ESI (M+3H / 3) 3+ : 1292.3.

[0851] Example 21 : Synthesis of compound 21

[0852]

[0853] Step 1. Synthesis of carbonic acid (4-nitrophenyl ester) [trans-(3SR,4SR)-4-(2- pyridyl disulfide)tetrahydropyran-3-yl ester]

[0854] The title compound was synthesized using a similar synthetic procedure described in the synthesis of compound 11 from the second stereoisomer eluted from the chiral chromatography of racemic trans-4-(2-pyridyl disulfide)tetrahydropyran-3-ol, designated as trans-(2SR,3SR)-4-(2-pyridyl disulfide)tetrahydropyran-3-ol.

[0855] Step 2. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexadecano-14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(3SR,4SR)-4-(2-pyridyl disulfide)tetrahydropyran-3-yl ester]

[0856] To a mixture of exitecan 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-diazepine[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%).

[0857] Step 3. Coupling with peptide Pv1 (Compound 21)

[0858] In a vial were placed peptide Pv1 (25.0 mg, 7.37e-6 mol), N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamic acid [trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl ester] (0.00779 g, 1.11e-5 mol), 1 mL of ACN and 0.5 mL of water. N-methylmorpholine (0.030 mL, 0.000273 mol) was added thereto. The mixture was stirred at room temperature for 65 h. LC-MS indicated that the reaction was complete.

[0859] The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-70% ACN / H20 + 0.05% TFA, 17 min; retention time: 6.765 min) to give compound 21 (0.021 g, 5.42e-6 mol, yield: 73.6%). ESI (M+3H / 3) 3+ :1291.1.

[0860] Example 22: Synthesis of compound 22

[0861]

[0862] Step 1. Synthesis of (4-nitrophenyl) carbonate [trans-(1 RS,2RS)-2-(2- pyridyl disulfide)cycloheptane]

[0863] The title compound was synthesized using similar synthetic procedure described in the synthesis of compound 11 from the first stereoisomer eluted from the chiral chromatography of racemic trans-2-(2-pyridyl disulfide)cycloheptan-1-ol, designated as trans-(1 RS,2RS)-2-(2-pyridyl disulfide)cycloheptan-1-ol.

[0864] Step 2. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexadecano[14.7.1.02,14.04,13.06,11.020,24]tetracosan-1,6(11),12,14,16(24),17,19-heptene-23-yl]carbamic acid [trans-(1 RS,2RS)-2-(2-pyridyl disulfide)cycloheptyl ester]

[0865] 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) carbonate [trans-(1 RS,2RS)-2-(2-pyridyl disulfide)cycloheptane] (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), 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, yield: 86.0%).

[0866] Step 3. Coupling with peptide Pv1 (Compound 22)

[0867] A vial was charged with peptide Pv1 (25.0 mg, 7.37e-6), N-[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexadecano- 14.7.1.02,14.04,13.06,11.020,24]tetracosheptaen-23-yl]carbamic acid [trans- (1RS,2RS)-2-(2-pyridyl disulfide)cycloheptyl ester] (0.00792 g, 1.11e-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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-70% ACN / H20 + 0.05% TFA, 17 min; retention time: 6.868 min) to give Compound 22 (0.020 g, 5.15e-6 mol, 69.9% yield). ESI (M+3H / 3) 3+ :1296.3.

[0868] Example 23: Synthesis of Compound 23

[0869]

[0870] Step 1. Synthesis of carbonic acid (4-nitrophenyl ester) [trans-(1SR,2SR)-2-(2- pyridyl disulfide)cycloheptyl ester]

[0871] The title compound was synthesized using similar synthetic procedures described in the synthesis of Compound 11 from the second stereoisomer eluted from the chiral chromatography of racemic trans-2-(2-pyridyl disulfide)cycloheptan-1-ol, designated as trans-(1SR,2SR)-2-(2-pyridyl disulfide)cycloheptan-1-ol.

[0872] Step 2. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexadecano-14.7.1.02,14.04,13.06,11.020,24]tetracosheptaen- 23-yl]carbamic acid [trans-(1SR,2SR)-2-(2-pyridyl disulfide)cycloheptyl ester]

[0873] To a mixture of Eribulin mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol) and (trans-(1SR,2SR)-2-(2-pyridyl disulfide)cycloheptyl) carbonate (23.7 mg, 0.0564 mmol) in 2 mL of dry 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), 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%).

[0874] Step 3. Coupling with peptide Pv1 (Compound 23)

[0875] A vial was charged with peptide Pv1 (25.0 mg, 7.37e-6), N-[(10S,23S)-10-ethyl-18- fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexadecano[14.7.1.02,14.04,13.06,11.020,24]tetracosan-1,6(11),12,14,16(24),17,19-heptene-23-yl]carbamic acid [trans- (1SR,2SR)-2-(2-pyridyl disulfide)cycloheptyl] ester (0.00792 g, 1.11e-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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4min, 20-88% ACN / H2O + 0.05% TFA, 17 min; retention time: 7.178 min) to give Compound 23 (0.020 g, 5.15e-6 mol, yield: 69.9%). ESI (M+3H / 3) 3+ :1296.0.

[0876] Example 24: Synthesis of Compound 24

[0877]

[0878] Step 1. Synthesis of Carbonic acid (4-nitrophenyl ester) [trans-1-(1RS,2RS)-1-(2- pyridyl disulfanyl)tetralin-2-yl ester]

[0879] The title compound was synthesized using a similar synthetic procedure described in the synthesis of compound 11 from the first stereoisomer eluted from the chiral chromatography of racemic trans-1-(2-pyridyl disulfanyl)tetralin-2-ol, designated as trans-(1RS,2RS)-1-(2-pyridyl disulfanyl)tetralin-2-ol.

[0880] Step 2. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexadecano[14.7.1.02,14.04,13.06,11.020,24]tetracosan-1,6(11),12,14,16(24),17,19-heptene-23-yl]carbamic acid [trans-(1RS,2RS)-1-(2-pyridyl disulfanyl)tetralin-2-yl ester]

[0881] To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol) and carbonic acid (4-nitrophenyl ester) [trans-1-(1RS,2RS)-2-pyridyl disulfanyl)tetralin-2-yl ester] (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), 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%).

[0882] Step 3. Coupling with peptide Pv1 (Example 24)

[0883] A vial was charged with peptide Pv1 (25.0 mg, 7.37e-6), 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]tetracosan-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-1 -(1 RS,2RS)-1 -(2-pyridyl disulfide)tetrahydronaphthalen-2-yl ester] (0.0083 g, 1.11e-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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-95% ACN / H20 + 0.05% TFA, 20 min; retention time: 6.968) to give compound 24 (0.012 g, 3.06e-6 mol, 41.6% yield). ESI (M+3H / 3) 3+ : 1307.2

[0884] Example 25: Synthesis of compound 25

[0885]

[0886] Step 1. Synthesis of carbonic acid (4-nitrophenyl ester) [trans-(1SR,2SR)-1-(2- pyridyl disulfide)tetrahydronaphthalen-2-yl ester]

[0887] The title compound was synthesized using a similar synthetic procedure described in the synthesis of compound 11 from the second stereoisomer to be isolated from the chiral chromatography of racemic trans-1-(2-pyridyl disulfide)tetrahydronaphthalen-2-ol, designated as trans-(1SR,2SR)-1-(2-pyridyl disulfide)tetrahydronaphthalen-2-ol.

[0888] Step 2. Synthesis of 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]tetracosan-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans-(1SR,2SR)-1-(2-pyridyl disulfide)tetrahydronaphthalen-2-yl ester]

[0889] 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) carbonate [trans-(1SR,2SR)-1-(2-pyridyldithio)tetralin-2-yl ester] (32.1 mg, 0.0705 mmol) in 2 mL of dry 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), 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%).

[0890] Step 3. Coupling with peptide Pv1 (Compound 25)

[0891] A vial was charged with peptide Pv1 (25.0 mg, 7.37e-6), N-[(10S,23S)-10-ethyl-18- fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexadecano[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [trans- (1SR,2SR)-1-(2-pyridyldithio)tetralin-2-yl ester] (0.0083 g, 1.11e-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 the reaction was complete.

[0892] The reaction mixture was purified directly by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4min, 20-95% ACN / H2O + 0.05% TFA, 20 min; retention time: 6.944) to give Compound 25 (0.013 g, 3.32e-6 mol, yield: 45.0%). ESI (M+3H / 3) 3+ :1307.0

[0893] Example 26: Synthesis of Compound 26

[0894]

[0895] Step 1. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamic acid [trans-4-(2-pyridyldisulfide)cyclohexyl ester]

[0896] To a mixture of exitecan 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 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), 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%).

[0897] Step 2. Coupling with peptide Pv1 (Compound 26)

[0898] In a vial were placed peptide Pv1 (25.0 mg, 7.37e-6), N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamic acid [trans-4-(2-pyridyldisulfanyl)cyclohexyl ester] (0.00777 g, 1.11e-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 the reaction was complete.

[0899] The reaction mixture was directly purified by reverse phase HPLC (Waters Sunfire Prep C18, Prep Slope_4 min, 20-95% ACN / H2O+0.05% TFA, 20 min; retention time: 6.593 min) to give compound 26 (0.028 g, 7.23e-6 mol, yield: 98.2%). ESI (M+3H / 3)3+ 1291.0.

[0900] Example 27: Synthesis of compound 27

[0901]

[0902] Step 1. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4, 15-diazahexadec- 1,6(11), 12, 14, 16(24), 17, 19-heptaen-23-yl] carbamic acid [(2S)-3-methyl-2-(2-pyridinyl disulfanyl)butyl ester]

[0903] To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol) and carbonic acid [(2S)-3-methyl-2-(2-pyridinyl disulfanyl)butyl ester] (4-nitrophenyl ester) (synthesized from L-valine, see 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), 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%).

[0904] Step 2. Coupling with peptide Pv1 (compound 27)

[0905] A vial was charged with peptide Pv1 (25.0 mg, 7.37 e-6 mol), N-[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazacyclohexadeca- 1(12),13,15,17(24),18,20-heptaen-23-yl]carbamic acid [(2S)-3-methyl-2-(2- pyridyl disulfide)butyl ester] (0.00764 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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-95% ACN / H20 + 0.05% TFA, 20 min; retention time: 6.773 min) to give compound 27 (0.024 g, 6.22 e-6 mol, 84.4% yield). ESI (M+3H / 3) 3+ :1286.8.

[0906] Example 28: Synthesis of compound 28

[0907]

[0908] Step 1. Synthesis of N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazacyclohexadeca-1(12),13,15,17(24),18,20-heptaen-23-yl]carbamic acid [(2R)-3-methyl-2-(2-pyridyl disulfide)butyl ester]

[0909] To a mixture of Eribulin mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol) and (2R)-3-methyl-2-(2-pyridyl disulfide) butyl (4-nitrophenyl) carbonate) (synthesized from D-valine, see J. Org. Chem. 1990, 55, 2286-2288) (40.8 mg, 0.103 mmol) in 2 mL of dry 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), 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%).

[0910] Step 2. Coupling with peptide Pv1 (Compound 28)

[0911] A vial was charged with peptide Pv1 (25.0 mg, 7.37e-6), N-[(10S,23S)-10-ethyl-18- fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexadecano-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamic acid [(2R)-3-methyl-2-(2-pyridyl disulfide) butyl ester] (0.00764 g, 1.11e-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 the reaction was complete. The reaction mixture was purified directly by reverse phase HPLC (Waters Sunfire Prep C18, PrepSlope_4min, 20-95% ACN / H2O + 0.05% TFA, 20 min; retention time: 6.708 min) to give Compound 28 (0.012 g, 3.08e-6 mol, yield: 41.8%). ESI (M+3H / 3) 3+ :1287.8.

[0912] Example 29: Synthesis of Compound 29

[0913]

[0914] Analytical method: Chromatographic purity was determined 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→95% acetonitrile / water with 0.05% AcOH (method A) or 0.05% TFA (method B) over 5 minutes; Agilent diode array detector at l = 254, 220 or 195 nm; room temperature.

[0915] 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-yl disulfanyl)propanamide

[0916] A solution of 3-(pyridin-2-yl disulfanyl)propanoic acid 2,5-dioxopyrrolidin-1-yl ester (180 mg, 0.576 mmol) in DMF (4 mL) was added to solid exetecan mesylate [CAS: 169869-90-3] (80 mg, 0.150 mmol), followed by aqueous PBS buffer (4 mL, pH = 7.4, 50 mM) and sonicated for ~5 minutes. The turbid mixture was stirred at ambient temperature for 2 hours and determined to be ~25% complete. Ammonium acetate (11 mg, 0.143 mmol) and an additional 2 mL of DMF were added and the resulting mixture was stirred at ambient temperature for 18 hours. The mixture was acidified with TFA (80 mL, 0.98 mmol) and split into 2 equal portions. Each individual portion was passed over a Redi-Sep C 18 50g cartridge and eluted with a gradient of acetonitrile (5% to 95%) in water containing TFA (0.05% v / v). The combined fractions were frozen and lyophilized to give the title compound as a light yellow solid (42 mg, 44%). HPLC purity at 254 nm: 97%. Retention time: 2.50 min (method A). MS data, 633.2 (M+H) + .

[0917] Step 2. Coupling with peptide Pv1 (compound 29)

[0918] Solid peptide Pv1 (168.4 mg, 0.0480 mmol) was added to solid N-((1 S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1 -yl)-3-(pyridin-2-yl disulfanyl)propanamide (30.5 mg, 0.0482 mmol) and dissolved in DMF (2 mL) under sonication (~1 min) and flushed with nitrogen. 4-Methylmorpholine (20 mL, 0.182 mmol) was added and the solution was left at ambient temperature for 18 h. The solution was made acidic with acetic acid (17 mL, 0.296 mmol) and applied to a Biotage C 18 300A 25g reverse phase column and eluted with a gradient of acetonitrile (25% to 95%) in water containing TFA (0.05% v / v). The combined fractions were frozen and lyophilised to give a pale yellow solid. The product was dissolved in DMSO (3 mL) and 1 mL portions of the solution were applied to a Biotage C 18 300A 25g reverse phase column and eluted with a gradient of acetonitrile (25% to 95%) in water containing TFA (0.05% v / v). The combined fractions were frozen and lyophilised to give a pale yellow solid. The product was dissolved in DMSO (3 mL) and 1 mL portions of the solution were applied to a Biotage C 2+ , 1267.3 (M+3H / 3) 3+ .

[0919] Example A. Growth retardation assay

[0920] Cells were seeded in 96-well black-walled clear-bottom plates (Griener) at 2500 cells / well for DLD-1 WT cells, 5000 cells / well for FaDu and HeLa cells, and 3000 cells / well for HCT116 in growth media containing 10% FBS. Cells were allowed to adhere for 60 minutes at room temperature before being returned to a 37C, 5% CO2 incubator. After 24 hours, the media was removed and replaced with fresh growth media containing various drug concentrations. Each drug concentration was added in triplicate. The drug-untreated control contained only growth media. Cells were returned to the incubator. Ninety-six hours after drug addition, cells were fixed with 4% paraformaldehyde for 20 minutes and stained with 1 pg / mL of Hoechst. Plates were imaged on a Cytation 5 automated imager (BioTek) and cells were counted using CellProfiler (Carpenter et al., 2006) (https: / / cellprofiler.org / ). Figure 1

[0921]

[0922] *IC = incomplete curve.

[0923]

[0924]

[0925] Example B: Plasma pharmacokinetics of compound 11 in a rat model

[0926] Animal dosing

[0927] Male Sprague Dawley rats underwent jugular vein cannulation and insertion of a vascular access button (VAB, Instech Labs catalog number VABR1B / 22) at Envigo Labs prior to shipment. Magnetic aluminum caps (Instech Labs catalog number VABRC) were used to protect the access port of the jugular vein catheter, and animals were housed in pairs per cage on corn cob bedding for 4-5 days prior to the study. Rats were administered a single intravenous dose of 5 mg / kg of compound 11 prepared in 5% mannitol in citrate buffer. Blood (250 pL) from the fed rats was collected into microvials filled with K2EDTA at 1, 2, 4, 8, 24, and 30 hours after compound administration. Plasma was isolated by centrifugation and 100 pL aliquots were transferred to a 96-well polypropylene plate on dry ice. Samples were stored at -80C until total peptide was quantitated by ELISA and released exisolidan was quantitated by LC-MS / MS.

[0928] ​ELISA measurement of total peptide plasma concentration

[0929] A 96-well plate was coated with 100 microliters / well of 0.1 mM BSA-labeled peptide prepared in 0.2 M carbonate-bicarbonate buffer (pH 9.4) and incubated overnight at 4°C. The plate was washed 4 times with ELISA wash buffer (PBS + 0.05% Tween 20), incubated with blocking buffer (PBS + 5% dry milk + 0.05% Tween 20) (300 microliters / well) for 2 hours at room temperature and washed again 4 times with ELISA wash buffer. Meanwhile, 2x Compound 11 standards in control plasma and study plasma samples were pre-incubated with primary antibody specific for Pv1 peptide at 1-10 ng / mL for 30 minutes at room temperature. The pre-incubated samples were added to the pre-coated, pre-blocked assay plate at 100 microliters / well and incubated for 1 hour at room temperature. The plate was washed 4 times with ELISA wash buffer and incubated with 100 microliters / well of secondary goat anti-mouse IgG HRP antibody (1:5,000 in antibody diluent) for 1 hour at room temperature. The plate was washed 4 times with ELISA wash buffer and incubated with 100 microliters / well of SuperSignal substrate for 1 minute at room temperature with gentle shaking. The plate was read for luminescence on a BioTek Cytation 5 plate reader.

[0930] LC-MS / MS measurement of exatecan plasma concentration

[0931] For the quantification of exatecan, 20 pL of plasma sample was added to a polypropylene autosampler vial. 20 pL of PPT-IS (ACN:H2O (50:50) + 0.5% FA containing 1000 ng / mL of internal standard) and 20 pL of diluent (ACN:H2O (50:50) + 0.5% FA) were added to each sample. 120 pL of ACN + 5% FA was subsequently added. The vial was capped and vortexed for 2 minutes. The sample was centrifuged at 3700 rpm for 5-10 minutes, followed by analysis via liquid chromatography tandem mass spectrometry (LC-MS / MS).

[0932] Figure 1 A plot showing the plasma concentration of Compound 11 and released exatecan following a single IV dose of 5 mg / kg of Compound 11 in rats (data presented as mean ± SEM). As shown in Figure 1 Less than 0.002% of the exatecan payload was released after 30 h of circulation. Figure 2 Compound 11 was shown to be stable in plasma for at least 30 h.

[0933] Example C: Tumor and bone marrow pharmacokinetics of Compound 11 in a mouse model

[0934] Animal drug administration

[0935] Six-week-old female athymic naked Fox nu Mice were obtained from Taconic Labs (Cat. No. NCRNU-F) and housed five per cage on Alpha-Dri bedding in a disposable cage system (Innovive). Human HCT116 cancer cells derived from colorectal cancer were diluted 1:1 in Matrigel without phenol red and plated at 2.5 × 10 6 The xenografts were implanted subcutaneously into the left flank of each mouse at a density of 10 cells / 100 μL. 3 Mice were administered a single intraperitoneal injection of 10 mg / kg of compound 11 prepared in a vehicle of 5% mannitol in citrate at a minimum volume of 10 mg / kg. 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 tumors and bone marrow were determined by ELISA.

[0936] ELISA measurement of total peptide tissue concentration

[0937] A 96-well plate was coated with 100 μL / well of 0.1 μM BSA-labeled peptide prepared in 0.2 M carbonate-bicarbonate buffer, pH 9.4, and incubated overnight at 4°C. The plate was washed four times with ELISA wash buffer (PBS + 0.05% Tween 20), incubated for 2 hours at room temperature with blocking buffer (PBS + 5% dry milk + 0.05% Tween 20) (300 μL / well), and washed again four times with ELISA wash buffer. Simultaneously, 2× Compound 11 standards (in the corresponding tissue matrix) or sample tumor homogenate or bone marrow samples diluted in antibody diluent (PBS + 2% dry milk + 0.05% Tween 20) were preincubated with 1-10 ng / mL of 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 for 1 hour at room temperature. The plate was 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. The plate was 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. Luminescence of the plate was read on a BioTek Cytation 5 plate reader.

[0938] Figure 2 Graph showing peptide concentrations in tumor and bone marrow following a single IP dose of 10 mg / kg of compound 11 in mice (data are presented as mean ± SEM). Figure 3Compound 11 was confirmed to effectively target tumors.

[0939] Example D: Myelotoxicity study in a mouse model

[0940] Animal dosing

[0941] Six-week old female athymic nude Foxn nu Mice were obtained from Taconic Labs (catalog number NCRNU-F) and housed 5 per cage on Alpha-Dri bedding in a One-Time Cage System (Innovive). Human HCT116 carcinoma cells derived from colorectal cancer were diluted 1 : 1 in Matrigel without phenol red and implanted subcutaneously into the left flank of each mouse at a density of 2.5 x 10 6 When xenografts reached a minimum volume of 200 mm 3 Mice were administered an intraperitoneal dose of vehicle or 2.6 or 5.2 micromole per kilogram of 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) when the xenografts reached a minimum volume of 200 mm

[0942] Bone marrow collection

[0943] Tumor-bearing mice were euthanized by cervical dislocation 6 hours after the last dose. Femurs were removed and the bone marrow was extruded into a 50 mL conical tube by flushing the bone with a 23 gauge needle mounted on a 5 cc syringe containing PBS + 2% fetal bovine serum. The bone marrow was homogenized by gentle pipetting and filtered through a 100 pm 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 the cell count was assessed by trypan blue exclusion. Cell counts from four independent studies were averaged and plotted.

[0944] Figure 4A A graph showing total bone marrow counts from femurs of tumor-bearing nude mice after dosing with 2.6 and 5.2 micromole per kilogram of Compound 11 (equivalent to 10, 20 mg / kg conjugate) or free exatecan (equivalent to 1.15 and 2.3 mg / kg exatecan), once daily for four days. (Data presented as mean ± SEM). Compound 11 did not show myelotoxicity that limited the clinical utility of exatecan.

[0945] Example E: Gastric toxicity study in a mouse model

[0946] Animal drug administration and gastric imaging

[0947] Six-week-old female athymic naked Fox nu Mice were obtained from Taconic Labs (Cat. No. NCRNU-F) and housed five per cage on Alpha-Dri bedding in a disposable cage system (Innovive). Human HCT116 cells derived from colorectal cancer were diluted 1:1 in Matrigel without phenol red and plated at 2.5 × 10 6 The xenografts were implanted subcutaneously into the left flank of each mouse at a density of 10 cells / 100 μL. 3 Mice were administered an intraperitoneal dose of vehicle or 5.2 micromoles / kg of unconjugated exotecan (equivalent to 2.3 mg / kg exotecan) or compound 11 (equivalent to 20 mg / kg compound 11) at a minimum volume of 1 mg / kg. Compounds were administered once daily for 4 days. Six hours after the last dose, mice were euthanized by cervical dislocation and subjected to gross autopsy. In situ and ex vivo photographs of the stomach were taken.

[0948] Figure 4B Shown are the resected stomachs of tumor-bearing nude mice after administration of vehicle or 5.2 μmol / kg of compound 11 (equivalent to 20 mg / kg of the conjugate) or free exotecan (equivalent to 2.3 mg / kg of exotecan) QD×4. Figure 5A The stomach of orthotopic tumor-bearing nude mice is shown after daily dosing with 5.2 μmol / kg of compound 11 (equivalent to 20 mg / kg of the conjugate) or free exitecan (equivalent to 2.3 mg / kg of exitecan) for four days. Compound 11 did not exhibit gastric toxicity that would limit the clinical utility of exitecan.

[0949] Example F: Efficacy of Compound 11 in the HCT116 Colorectal Cancer Model

[0950] Six-week-old female athymic naked Fox nu Mice were obtained from Taconic Labs (Cat. No. NCRNU-F) and housed five per cage on Alpha-Dri bedding in a disposable cage system. Human HCT116 cells derived from colorectal cancer were diluted 1:1 in Matrigel without phenol red and plated at 2.5 × 10 6 The xenografts were implanted subcutaneously into the left flank of each mouse at a density of 100 cells / 100 μL. 3The mice were randomized into groups and treated as detailed in the table below when the mean volume of the tumor was ≥ 1.0 mg / kg. The mice were administered an intraperitoneal (IP) dose of vehicle or 2.6 or 5.2 micromoles / kg of unconjugated isotecan (equivalent to 1.15 or 2.3 mg / kg isotecan, respectively) or compound 11 (equivalent to 10 or 20 mg / kg compound 11, respectively). The dose was prepared by diluting a 0.1 mg / μL DMSO stock solution in citrate buffer containing 5% mannitol and administered QD×4 / week at a volume of 12 mL / kg (300 μL per 25 g mouse) for three weeks. Xenograft tumors were measured with a caliper and the volume was calculated using the ellipsoid volume equation: volume = π / 6×(length)×(width) 2 Animals died or had tumors larger than 2000 mm in size. 3 Patients were removed from the study if they lost >20% of their body weight. The following table shows the dosing schedule for each treatment group.

[0951]

[0952] Figure 5B Graph showing mean tumor volumes in nude mice bearing HCT116 colorectal flank tumors resulting from administration of equimolar amounts of free exitecan or Compound 11. Animals were dosed parenterally once daily four times per week for three weeks. Figure 6A Shown are Kaplan-Meier survival curves for nude mice bearing HCT116 colorectal flank tumors, following administration of equimolar amounts of free exitecan or compound 11. Data are presented as mean ± SEM. These data demonstrate that compound 11 exhibits potent antitumor activity in this preclinical colorectal cancer model.

[0953] Example G: Efficacy of Compound 11 in the MKN45 HER2-negative gastric cancer model

[0954] Six-week-old female athymic naked Fox nu Mice were obtained from Taconic Labs (Cat. No. NCRNU-F) and housed five per cage on Alpha-Dri bedding in a disposable cage system. Human MKN45 cells derived from gastric cancer were diluted 1:1 in Matrigel without phenol red and plated at 2×10 6 The xenografts were implanted subcutaneously into the left flank of each mouse at a density of 100 cells / 100 μL. 3The average volume, mice were randomized into groups and treated as detailed in the table below. Mice were administered intraperitoneal (IP) doses of vehicle or 2.6 or 5.2 micromole / kilogram of 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). Doses were prepared by diluting a 0.1 mg / μL DMSO stock in 5% mannitol in citrate buffer and administered at a volume of 12 mL / kg (300 μL per 25 g mouse) QD x 4 / week for two weeks. Xenograft tumors were measured by calipers and volume was calculated using the ellipsoid volume equation: Volume = π / 6 x (length) x (width) 2 Animals were removed from the study for death, tumor size > 2000 mm 3 or weight loss > 20%. The table below shows the dosing schedule for each treatment group.

[0955]

[0956] Figure 6B Single agent efficacy of Compound 11 in nude mice bearing MKN45 HER2 negative gastric cancer flank tumors is shown. Animals were dosed parenterally once daily, four times per week for two weeks. Figure 6B Kaplan Meier survival curves showing dosing of equimolar amounts of free exatecan or Compound 11 in nude mice bearing MKN45 HER2 negative gastric cancer flank tumors. Data is presented as mean ± SEM. These data demonstrate that Compound 11 shows potent antitumor activity in a preclinical gastric cancer model.

[0957] Figure 7A Kaplan-Meier analysis was used to assess survival based on death or removal from the study.

[0958] Example H: Efficacy of Compound 11 in a JIMT-1 HER2 moderate breast cancer model

[0959] 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 in Matrigel without phenol red and implanted subcutaneously into the left flank of each mouse at a density of 5 x 10 6 mm3in size. When the xenografts reached 100 mm 3The mice were randomized into groups and treated as detailed in the table below when the average volume of the tumor was ≥ 100 μg / kg. The mice were administered intraperitoneal (IP) doses of vehicle or 2.6 or 5.2 μmol / kg of compound 11 (equivalent to 10 or 20 mg / kg of compound 11, respectively). The dose was prepared by diluting a 0.1 mg / μL DMSO stock solution in citrate buffer containing 5% mannitol and administered QD×4 / week at a volume of 12 mL / kg (300 μL per 25 g mouse) for three weeks. Xenograft tumors were measured with a caliper and the volume was calculated using the ellipsoid volume equation: Volume = π / 6×(length)×(width) 2 The body weight of the animals was measured at the same time as the tumor volume was assessed. 3 Patients were removed from the study if they lost >20% of their body weight. The following table shows the dosing schedule for each treatment group.

[0960]

[0961] Figure 7B Graph showing mean tumor volume in SCID mice bearing JIMT-1 HER2 intermediate breast cancer flank tumors resulting from administration of Compound 11. Animals were dosed parenterally once daily four times per week for three weeks. Figure 8A A graph showing the percent change in body weight in SCID mice bearing JIMT-1 HER2 moderate breast cancer flank tumors administered with compound 11. Data are presented as mean ± SEM. These data demonstrate that compound 11 exhibits potent antitumor activity in preclinical breast cancer models.

[0962] Example I: Efficacy of Compound 11 in the MDA-MB-231 triple-negative breast cancer model

[0963] Three to four weeks old female athymic naked Fox nu Mice were obtained from Envigo Labs. Human MDA-MB-231 cells, derived from breast cancer, were diluted 1:1 in Matrigel without phenol red and cultured at 2 × 10 6 The xenografts were implanted subcutaneously into the left flank of each mouse at a density of 10 cells / 100 μL. 3The average volume, mice were randomized 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 in a citrate buffer with 5% mannitol and administered QD x 4 / week for three weeks in a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured by calipers and volume was calculated using the ellipsoid volume equation: Volume = π / 6 x (length) x (width) 2 Animal weights were measured concurrently with tumor volume assessments. Animals were removed from the study due to death, tumor size exceeding 2000 mm 3 or due to >20% weight loss. The dosing schedule for each treatment group is shown in the table below.

[0964]

[0965] Figure 8B A plot showing the average tumor volume in nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11. Animals were dosed parenterally once daily, four times per week for three weeks. Figure 9A A plot showing the percent change in body weight relative to Day 0 in nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11. Data are presented as mean ± SEM. These data demonstrate that Compound 11 shows potent anti-tumor activity in a preclinical breast cancer model.

[0966] Example J: Combination efficacy of Compound 11 and talazoparib in a MDA-MB-231 triple negative breast cancer model

[0967] Three to four week old female athymic nude Foxn nu Mice were obtained from Envigo Labs. Human MDA-MB-231 cells derived from breast cancer were diluted 1 : 1 in Matrigel without phenol red and implanted subcutaneously into the left flank of each mouse at a density of 2 x 10 6 mm3, were implanted subcutaneously into the left flank of each mouse at a density of 2 x 10 3The mice were randomized into groups and treated as detailed in the table below when the average volume of the tumors was between 100-200 mm3. Mice were administered an intraperitoneal (IP) dose of vehicle or Compound 11 at 5 mg / kg alone or in combination with an oral (PO) dose of 0.33 mg / kg talazoparib. The doses were prepared by diluting a 0.1 mg / μL DMSO stock in 5% mannitol in citrate buffer. Compound 11 was administered QD x 4 / 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 by calipers and volume was calculated using the ellipsoid volume equation: Volume = π / 6 x (length) x (width) 2 Animal weights were measured at the same time as tumor volume assessments. Animals were removed from the study due to death, tumor size exceeding 2000 mm 3 or due to >20% weight loss. The table below shows the dosing schedule for each treatment group.

[0968]

[0969] Figure 9B A plot showing the average tumor volume of MDA-MB-231 triple negative breast cancer flank tumors in nude mice dosed with Compound 11 and talazoparib. Animals were dosed with Compound 11 parenterally once daily, four times per week for three weeks and talazoparib orally once daily for 18 days. Figure 10 A plot showing the percent change in body weight from Day 0 of MDA-MB-231 triple negative breast cancer flank tumors in nude mice dosed with Compound 11 and talazoparib.

[0970] Example K: Glutathione Cleavage Study

[0971] A 20 mM conjugate stock was prepared in 100% DMSO. The stock was then diluted in 100 mM Tris (pH 7.5) to give an intermediate dilution of 500 μΜ, followed by a 1 :5 additional dilution in 100 mM Tris (pH 7.5) to give a final concentration of 100 μΜ conjugate. The 100 mM glutathione was prepared immediately before use in H20 and diluted 1 : 10 in the challenge sample, for a final glutathione challenge concentration of 10 mM. The samples were mixed by inversion and incubated at 37 °C for up to 24 hours. At time 0, 4, and 24 hours, 50 μL of sample was aliquoted into silanized microfuge tubes and immediately frozen at -80 °C.

[0972] 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 13000 x G for 10 minutes. The supernatant was pipetted into 0.2 mL HPLC vials and placed on the Perkin Elmer Flexar HPLC autosampler. The following table outlines the HPLC conditions:

[0973]

[0974] Data was analyzed by calculating the percent reduction of the compound (peak area of cleaved conjugate / peak area of conjugate at time 0) x 100.

[0975] Figure 10 Graphs showing the degradation of Compound 11 and Compound 29 over 16h caused by treatment with 10 mM glutathione. As shown in ​ Compound 29 is released more rapidly than Compound 11 under similar glutathione exposure.

[0976] The following table outlines the degradation data for Compounds 11-29 against 10 mM glutathione exposure conditions measured at 4h and 24h described above.

[0977]

[0978] Example L: Plasma stability study

[0979] A 20 mM conjugate stock was prepared in 100% DMSO. The stock was then diluted in 100 mM Tris (pH 7.5) to give an intermediate dilution of 500 μΜ and then diluted 1 :5 directly into rat plasma to give a final concentration of 100 μΜ conjugate. Samples were mixed by inversion and incubated at 37 °C for up to 24h. At time 0, 4 and 24h, 50 μΐ^of sample was aliquoted into silanized microfuge tubes and immediately frozen at -80 °C.

[0980] 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 13000 x G for 10 minutes. The supernatant was pipetted into 0.2 mL HPLC vials and placed on the Perkin Elmer Flexar HPLC autosampler. The following table outlines the HPLC conditions:

[0981]

[0982] Data was analyzed by calculating the percent reduction of the compound (peak area at retention time of incubated conjugate / peak area at retention time of conjugate at time 0) x 100. The results of the study are shown in the table below.

[0983]

[0984]

[0985] Various modifications of the application, in addition to those described herein, will be apparent to those of ordinary skill in the art in view of the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application, including but not limited to all patents, patent applications, and publications, is incorporated herein by reference in its entirety. SEQUENCE LIST <110> Cybrexa 2, Inc. <120> Peptide conjugates of cytotoxins as therapeutic agents <130> 43236-0009W01 <150> 62 / 872,643 <151> 2019-07-10 <150> 63 / 040,859 <151> 2020-06-18 <160> 311 <170> PatentIn version 3.5 <210> 1 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 1 Ala Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe Pro 1 5 10 15 Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp Cys Gly 20 25 <210> 2 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 2 Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp 20 25 30 Glu Cys Gly 35 <210> 3 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 3 Ala Asp Asp Gin Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe Pro 1 5 10 15 Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp Asp Ala Asp Glu Cys Gly 20 25 30 <210> 4 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> Acetylation <400> 4 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Lys Cys Gly 35 <210> 5 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 5 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys 35 <210> 6 <211> 39 <212> PRT <213> Unknown <220> <223> Description of Unknown: pH sensitive membrane polypeptide <400> 6 Ala Ala Glu Gin Asn Pro lie Tyr Trp Trp Ala Arg Tyr Ala Asp Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Gly Thr Cys Gly 35 <210> 7 <211> 35 <212> PRT <213> Unknown (Unknown) <220> <223> Description of Unknown: Wild-type pH-sensitive membrane polypeptide <400> 7 Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp 20 25 30 Glu Gly Thr 35 <210> 8 <211> 36 <212> PRT <213> Unknown (Unknown) <220> <223> Description of Unknown: Wild-type pH-sensitive membrane polypeptide <400> 8 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 9 <211> 35 <212> PRT <213> Unknown (Unknown) <220> <223> Description of Unknown: Wild-type pH-sensitive membrane polypeptide <400> 9 Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp 20 25 30 Glu Gly Thr 35 <210> 10 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 11 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 12 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 12 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Gly 35 <210> 13 <211> 36 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 13 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 14 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 14 Ala Lys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 15 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 15 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Lys Cys Gly 35 <210> 16 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 16 Ala Lys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Cys Thr 35 <210> 17 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 17 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asn Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asn Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Gly 35 <210> 18 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 18 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Lys Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Lys Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Gly 35 <210> 19 <211> 36 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 19 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asn Ala 20 25 30 Asn Gln Gly Thr 35 <210> 20 <211> 36 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 20 Ala Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Ala Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 21 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 21 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 22 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 22 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Ala Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 23 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 23 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 24 <211> 36 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 24 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 25 <211> 39 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 25 Ala Ala Glu Gin Asn Pro lie Ile Tyr Trp Ala Arg Tyr Ala Asp Trp 1 5 10 15 Leu Phe Thr Asp Leu Pro Leu Leu Leu Leu Asp Leu Leu Ala Leu Leu 20 25 30 Val Asp Ala Asp Glu Gly Thr 35 <210> 26 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 26 Gly Glu Gln Asn Pro Ile Tyr Trp Ala Gln Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp 20 25 30 Glu Gly Thr Cys Gly 35 <210> 27 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 27 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Asp Leu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 28 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 28 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Leu Asp Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 29 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 29 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Asp Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 30 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 30 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Trp Asp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 31 <211> 36 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 31 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Gly Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 32 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 32 Asp Asp Asp Glu Asp Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Cys Thr 35 <210> 33 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 33 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Gly Cys Thr 35 <210> 34 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 34 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asp Glu Cys Thr 35 <210> 35 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 35 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asn Glu Cys Thr 35 <210> 36 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 36 Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Phe Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp 20 25 30 Glu Thr <210> 37 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 37 Ala Glu Gln Asn Pro Ile Tyr Phe Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp 20 25 30 Glu Gly Thr 35 <210> 38 <211> 34 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 38 Ala Glu Gln Asn Pro Ile Tyr Phe Ala Arg Tyr Ala Asp Phe Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Trp Asp Ala Asp 20 25 30 Glu Thr <210> 39 <211> 32 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 39 Ala Lys Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly 20 25 30 <210> 40 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 40 Ala Cys Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly 20 25 30 <210> 41 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 41 Ala Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Cys Gly 20 25 30 <210> 42 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 42 Ala Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Glu Cys Gly 20 25 30 <210> 43 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 43 Ala Lys Glu Asp Gln Asn Pro Tyr Trp Arg Ala Tyr Ala Asp Leu Phe 1 5 10 15 Thr Pro Leu Thr Leu Leu Asp Leu Leu Ala Leu Trp Asp Gly 20 25 30 <210> 44 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 44 Ala Cys Glu Asp Gln Asn Pro Tyr Trp Arg Ala Tyr Ala Asp Leu Phe 1 5 10 15 Thr Pro Leu Thr Leu Leu Asp Leu Leu Ala Leu Trp Asp Gly 20 25 30 <210> 45 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 45 Ala Cys Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe 1 5 10 15 Pro Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp 20 25 <210> 46 <211> 34 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 46 Thr Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu Pro 1 5 10 15 Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Pro Asn Gln Glu 20 25 30 Cys Ala <210> 47 <211> 20 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 47 Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu 20 <210> 48 <211> 21 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 48 Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Cys Leu 20 <210> 49 <211> 21 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 49 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu 20 <210> 50 <211> 20 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 50 Ala Glu Gin Asn Pro lie Tyr Phe Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu 20 <210> 51 <211> 21 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 51 Lys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Leu Leu Phe Pro 1 5 10 15 Thr Thr Leu Ala Trp 20 <210> 52 <211> 22 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Leu Leu Phe 1 5 10 15 Pro Thr Thr Leu Ala Trp 20 <210> 53 <211> 24 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 53 Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Pro Thr Thr Leu Leu Leu Leu Asp 20 <210> 54 <211> 22 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Ala Cys Glu Glu Gin Asn Pro Trp Ala Arg Tyr Ala Glu Leu Leu Phe 1 5 10 15 Pro Thr Thr Leu Ala Trp 20 <210> 55 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 55 Ala Cys Glu Glu Gin Asn Pro Trp Ala Arg Tyr Ala Glu Trp Leu Phe 1 5 10 15 Pro Thr Thr Leu Leu Leu Leu Glu 20 <210> 56 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 56 Ala Cys Glu Glu Gin Asn Pro Trp Ala Arg Tyr Leu Glu Trp Leu Phe 1 5 10 15 Pro Thr Glu Thr Leu Leu Leu Glu Leu 20 25 <210> 57 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 57 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 58 <211> 35 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 58 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Thr 35 <210> 59 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 59 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 60 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 60 Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp 20 25 30 Glu Gly Cys Thr 35 <210> 61 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 61 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 62 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 62 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 63 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 63 Ala Lys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 64 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 64 Ala Lys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Cys Thr 35 <210> 65 <211> 36 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 65 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Ala Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 66 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 66 Ala Cys Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp 1 5 10 15 Leu Phe Thr Thr Gly Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Gly Thr 35 <210> 67 <211> 34 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 67 Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Phe Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp 20 25 30 Glu Thr <210> 68 <211> 35 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 68 Ala Glu Gin Asn Pro lie Tyr Phe Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp 20 25 30 Glu Gly Thr 35 <210> 69 <211> 34 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 69 Ala Glu Gin Asn Pro lie Tyr Phe Ala Arg Tyr Ala Asp Phe Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Trp Asp Ala Asp 20 25 30 Glu Thr <210> 70 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 70 Ala Lys Glu Asp Gin Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly 20 25 30 <210> 71 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 71 Ala Cys Glu Asp Gin Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly 20 25 30 <210> 72 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 72 Ala Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly 20 25 30 <210> 73 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 73 Ala Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Glu Cys Gly 20 25 30 <210> 74 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 74 Ala Lys Glu Asp Gln Asn Pro Tyr Trp Arg Ala Tyr Ala Asp Leu Phe 1 5 10 15 Thr Pro Leu Thr Leu Leu Asp Leu Leu Ala Leu Trp Asp Gly 20 25 30 <210> 75 <211> 30 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 75 Ala Lys Glu Asp Gln Asn Pro Tyr Trp Arg Ala Tyr Ala Asp Leu Phe 1 5 10 15 Thr Pro Leu Thr Leu Leu Asp Leu Leu Ala Leu Trp Asp Gly 20 25 30 <210> 76 <211> 32 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 76 Ala Lys Glu Asp Gln Asn Asp Pro Tyr Trp Ala Arg Tyr Ala Asp Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Gly 20 25 30 <210> 77 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 77 Thr Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu Pro 1 5 10 15 Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Pro Asn Gln Glu 20 25 30 Cys Ala <210> 78 <211> 36 <212> PRT <213> Unknown <220> <223> Description of Unknown: Wild-type pH-sensitive membrane polypeptide <400> 78 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 79 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 79 Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Leu 20 <210> 80 <211> 21 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 80 Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe 1 5 10 15 Thr Thr Pro Cys Leu 20 <210> 81 <211> 21 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 81 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu 20 <210> 82 <211> 21 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 82 Ala Cys Glu Gin Asn Pro lie Tyr Phe Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu 20 <210> 83 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 83 Ala Cys Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe 1 5 10 15 Pro Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp 20 25 <210> 84 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Ala Cys Glu Glu Gln Asn Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe 1 5 10 15 Pro Thr Glu Thr Leu Leu Leu Glu Leu Leu Trp 20 25 <210> 85 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Ala Cys Asp Asp Gln Asn Pro Trp Ala Arg Tyr Leu Asp Trp Leu Phe 1 5 10 15 Pro Thr Asp Thr Leu Leu Leu Asp Leu 20 25 <210> 86 <211> 24 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 86 Cys Asp Asn Asn Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe Pro 1 5 10 15 Thr Asp Thr Leu Leu Leu Asp Trp 20 <210> 87 <211> 25 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 87 Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Leu Glu Trp Leu Phe 1 5 10 15 Pro Thr Glu Thr Leu Leu Leu Glu Leu 20 25 <210> 88 <211> 24 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 88 Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Trp Leu Phe ​1 5 10 15 Pro Thr Thr Leu Leu Leu Leu Asp 20 <210> 89 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 89 Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Ala Glu Trp Leu Phe 1 5 10 15 Pro Thr Thr Leu Leu Leu Leu Glu 20 <210> 90 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Leu Leu Phe 1 5 10 15 Pro Thr Thr Leu Ala Trp 20 <210> 91 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 91 Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Glu Leu Leu Phe 1 5 10 15 Pro Thr Thr Leu Trp 20 <210> 92 <211> 20 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 92 Lys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Leu Leu Phe Pro 1 5 10 15 Thr Thr Leu Trp 20 <210> 93 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 93 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Cys Thr 35 <210> 94 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 94 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asp Glu Cys Thr 35 <210> 95 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 95 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asp Glu Cys Thr 35 <210> 96 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 96 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asn Glu Cys Thr 35 <210> 97 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 97 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Cys Thr 35 <210> 98 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 98 Ala Cys Glu Gin Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Gly 35 <210> 99 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 99 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Thr 35 <210> 100 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 100 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 101 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 101 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Leu Asp Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 102 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 102 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Leu Asp Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 103 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 103 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Trp Asp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 104 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 104 Ala Ala Glu Gin Asn Pro He Tyr Trp Ala Arg Tyr Ala Glu Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 105 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 105 Ala Ala Glu Gin Asn Pro He Tyr Trp Ala Arg Tyr Ala Glu Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 106 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 106 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Asp Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 107 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 107 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Gin Tyr Asp Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 108 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr Cys Gly <210> 109 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 109 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr Cys Gly <210> 110 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 110 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr Cys Gly <210> 110 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide​​​Phe Thr Thr Pro Leu Leu Leu Leu Asn Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Gly 35 <210> 111 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 111 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asn Glu Cys Thr 35 <210> 112 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 112 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asp Glu Cys Thr 35 <210> 113 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 113 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Cys Thr 35 <210> 114 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 114 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Cys Thr 35 <210> 115 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 115 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Gly Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Cys Thr 35 <210> 116 <211> 36 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 116 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asn Ala 20 25 30 Asn Gln Gly Thr 35 <210> 117 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 117 Ala Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 118 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 118 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Glu Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 119 <211> 39 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 119 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Lys Cys Gly 35 <210> 120 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 120 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Gin Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 121 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 121 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Gin Tyr Asp Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 122 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequences: Synthetic Polypeptides <400> 122 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Gin Asp Tyr Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 123 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequences: Synthetic Polypeptides <400> 123 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asn Ala 20 25 30 Asn Gin Gly Thr 35 <210> 124 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequences: Synthetic Polypeptides <400> 124 Asp Asp Asp Glu Asp Asn Pro lie Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asp Glu Cys Thr 35 <210> 125 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 125 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asn Glu Cys Thr 35 <210> 126 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 126 Ala Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Lys Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Lys Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Gly 35 <210> 127 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 127 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Gln Asp Tyr Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 128 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 128 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Gln Tyr Asp Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 129 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 129 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Gin Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 130 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 130 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 131 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 131 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Asp Leu Pro Leu Leu Leu Leu Asp Leu Leu Ala Leu Leu Val 20 25 30 Asp Ala Asp Glu Gly Thr 35 <210> 132 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 132 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 133 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 133 Gly Gly Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Asp Leu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 134 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 134 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Gly Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 135 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 135 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 136 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 136 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Gin Tyr Asp Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 137 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 137 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Gin Asp Tyr Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 138 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 138 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Asp Ala Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 139 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 139 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Ala Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 140 <211> 39 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <220> <221> MOD_RES <222> (37)..(37) <223> Lys (Rhodamine) <220> <221> MOD_RES <222> (38)..(38) <223> Cys (Phalloidin) <400> 140 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Lys Cys Gly 35 <210> 141 <211> 39 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <220> <221> MOD_RES <222> (37)..(37) <223> Lys (Rhodamine) <220> <221> MOD_RES <222> (38)..(38) <223> Cys (Phalloidin) <400> 141 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Lys Cys Gly 35 <210> 142 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (37)..(37) <223> Cys (Penetratin) <400> 142 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 143 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (37)..(37) <223> Cys (Penetratin) <400> 143 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 144 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 144 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 145 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 145 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Thr 35 <210> 146 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 146 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Gly 35 <210> 147 <211> 36 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 147 Ala Cys Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr 35 <210> 148 <211> 36 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 148 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asn Ala 20 25 30 Asn Gln Gly Thr 35 <210> 149 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 149 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asp Glu Cys Thr 35 <210> 150 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 150 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asn Glu Cys Thr 35 <210> 151 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 151 Gly Gly Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 152 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <220> <221> MOD_RES <222> (37)..(37) <223> Cys (Cyclopeptide) <400> 152 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Cys Gly 35 <210> 153 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <220> <221> MOD_RES <222> (37)..(37) <223> Lys(Rhodamine) <220> <221> MOD_RES <222> (38)..(38) <223> Cys(Phalloidin) <400> 153 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Glu Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Lys Cys Gly 35 <210> 154 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <220> <221> MOD_RES <222> (37)..(37) <223> Lys(Rhodamine) <220> <221> MOD_RES <222> (38)..(38) <223> Cys(Phalloidin) <400> 154 Ala Ala Glu Gin Asn Pro lie Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala 20 25 30 Asp Glu Gly Thr Lys Cys Gly 35 <210> 155 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 155 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Cys Thr 35 <210> 156 <211> 37 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 156 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Gly Ala Leu Leu Val Asp 20 25 30 Ala Asp Glu Cys Thr 35 <210> 157 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 157 Asp Asp Asp Glu Asp Asn Pro lie Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asp Glu Cys Thr 35 <210> 158 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 158 Asp Asp Asp Glu Asp Asn Pro lie Tyr Trp Ala Arg Tyr Ala His Trp 1 5 10 15 Leu Phe Thr Thr Pro Leu Leu Leu Leu His Gly Ala Leu Leu Val Asn 20 25 30 Ala Asn Glu Cys Thr 35 <210> 159 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 159 Asp Asp Asp Glu Asp Asn Pro Ile Tyr Trp Ala Arg Tyr ...

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein: R 7 is a peptide comprising at least one of the following sequences: ADDQNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 1; Pv1); AEQNPIYWARYADWLFTTTPLLLLDLALLVDADECG (SEQ ID NO: 2; Pv2); Ac-AAEQNPIYWARYADWLFTTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4; Pv4); and AAEQNPIYWARYADWLFTTTPLLLLDLALLVDADEGTC (SEQ ID No. 5; Pv5); And where R 7 Via R 7 The cysteine ​​residue of is connected to Q; R 8 for: ； Q is: ； R 1 、R 2 、R 3 and R 4 Each independently selected from 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, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 , wherein the C 1-4 Alkyl, C 1-4 Alkenyl, C 6-10 Aryl, C 3-10 Cycloalkyl, 5-10 membered heteroaryl and 4-10 membered heterocycloalkyl are each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; or R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each of which is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C 1-4 Alkyl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; or R 1 and R 3 Together with the carbon atom to which it is attached, it forms C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each of which is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C 1-4 Alkyl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; or R 2 and R 3 Together with the carbon atom to which it is attached, it forms C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each of which is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C 1-4 Alkyl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; or R 3 and R 4 Together with the carbon atom to which it is attached, it forms C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each of which is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C 1-4 Alkyl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ;and R a1 、R b1 、R c1 and R d1 Each independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, OH, CN, NO2 and CO2CH3; wherein the C 1-6 Alkyl and C 2-6 The alkenyl groups are each optionally substituted with OH, CN, NO2 or CO2CH3.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R 7 is a peptide comprising at least one of the following sequences: ADDQNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 1; Pv1); AEQNPIYWARYADWLFTTTPLLLLDLALLVDADECG (SEQ ID NO: 2; Pv2); Ac-AAEQNPIYWARYADWLFTTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4; Pv4); and AAEQNPIYWARYADWLFTTTPLLLLDLALLVDADEGTC (SEQ ID No. 5; Pv5); And where R 7 Via R 7 The cysteine ​​residue of is connected to Q; R 8 for: ； Q is: ； R 1 、R 2 、R 3 and R 4 Each independently selected from H, C 1-4 Alkyl, C 1-4 Alkenyl, C 6-10 Aryl, 5-10 membered heteroaryl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 , wherein the C 1-4 Alkyl, C 1-4 Alkenyl, C 6-10 Aryl and 5-10 membered heteroaryl are each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; or R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl, optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; or R 1 and R 3 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl, optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; or R 2 and R 3 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl, optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; or R 3 and R 4 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl, optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ;and R a1 、R b1 、R c1 and R d1 Each independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, OH, CN, NO2 and CO2CH3; wherein the C 1-6 Alkyl and C 2-6 The alkenyl groups are each optionally substituted with OH, CN, NO2 or CO2CH.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 7 is a peptide comprising at least one of the following sequences: ADDQNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 1; Pv1), and AEQNPIYWARYADWLFTTTPLLLLDLALLVDADECG (SEQ ID NO: 2; Pv2), And where R 7 Via R 7 The cysteine ​​residue of is connected to Q.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 7 is a peptide comprising the following sequence: ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1; Pv1).

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

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R 1 、R 2 、R 3 and R 4 Each independently selected from H, C 1-4 Alkyl, C 1-4 Alkenyl, C 6-10 Aryl, 5-10 membered heteroaryl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 , wherein the C 1-4 Alkyl, C 1-4 Alkenyl, C 6-10 Aryl and 5-10 membered heteroaryl are each optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halo, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 .

7. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 1 and R 3 Together with the carbon atom to which it is attached, it forms C 3-14 cycloalkyl or 4-14 membered heterocycloalkyl, each of which is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C 1-4 Alkyl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 .

8. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 1 and R 3 Together with the carbon atom to which it is attached, it forms a cyclopentyl, cyclohexyl, cycloheptyl, 1,2,3,4-tetrahydronaphthyl, tetrahydrofuranyl or tetrahydropyranyl group.

9. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 1 and R 3 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl.

10. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 3 Together with the carbon atom to which it is attached, it forms a cyclohexyl group.

11. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein Q is: 。 12. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 are each independently selected from H and methyl.

13. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 1 and R 2 Each is H.

14. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl.

15. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cyclobutyl group.

16. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 3 and R 4 Each is H.

17. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 2 and R 4 Each is H.

18. The compound of claim 1 or a pharmaceutically acceptable salt thereof, which has formula (II): or a pharmaceutically acceptable salt thereof, wherein: Ring Z is a monocyclic C 5-7 a cycloalkyl ring or a monocyclic 5-7 membered heterocycloalkyl ring; Each R Z Independently selected from C 1-4 Alkyl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; or two adjacent R Z Together with the atoms to which it is attached, it forms a fused monocyclic ring C 5-7 Cycloalkyl ring, fused monocyclic 5-7 membered heterocycloalkyl ring, fused C 6-10 aromatic ring or fused 6-10 membered heteroaromatic ring, each of which is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C 1-4 Alkyl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 ; R a1 、R b1 、R c1 and R d1 Each independently selected from 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.

19. The compound according to claim 18 or a pharmaceutically acceptable salt thereof, wherein R 7 Via R 7 A cysteine ​​residue of is linked to the core, wherein one of the sulfur atoms of the disulfide moiety in Formula II is derived from said cysteine ​​residue.

20. The compound according to claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein ring Z is a monocyclic C 5-7 Cycloalkyl ring.

21. The compound according to claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein ring Z is a cyclopentyl ring.

22. The compound according to claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein ring Z is a cyclohexyl ring.

23. The compound according to claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein ring Z is a cycloheptyl ring.

24. The compound according to claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein ring Z is a monocyclic 5-7 membered heterocycloalkyl ring.

25. The compound according to claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein ring Z is a 5-membered heterocycloalkyl ring.

26. The compound of claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein ring Z is a 6-membered heterocycloalkyl ring.

27. The compound according to claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein ring Z is a 7-membered heterocycloalkyl ring.

28. The compound according to claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein two adjacent R Z Together with the atoms to which it is attached, it forms a fused monocyclic ring C 5-7 Cycloalkyl ring, fused monocyclic 5-7 membered heterocycloalkyl ring, fused C 6-10 aromatic ring or fused 6-10 membered heteroaromatic ring, each of which is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C 1-4 Alkyl, halogen, CN, NO2, OR a1 SR 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 NR c1 R d1 NR c1 C(O)R b1 NR c1 C(O)OR a1 and NR c1 C(O)NR c1 R d1 .

29. The compound of claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein n is 0.

30. The compound of claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein n is 1.

31. The compound of claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein n is 2.

32. The compound of claim 18 or 19, or a pharmaceutically acceptable salt thereof, wherein n is 3.

33. The compound of claim 18 or 19, wherein the compound has Formula (III), Formula (IV), or Formula (V): or a pharmaceutically acceptable salt thereof.

34. The compound of claim 1 or a pharmaceutically acceptable salt thereof, selected from: 、 、 、 、 、 、 and ; Wherein Pv2 is a peptide comprising the following sequence: AEQNPIYWARYADWLFTTTPLLLLDLALLVDADECG (SEQ ID NO: 2).

35. The compound of claim 1 or a pharmaceutically acceptable salt thereof, selected from: 、 Wherein Pv1 is a peptide comprising the following sequence: ADDQNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 1).

36. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which is: Wherein Pv1 is a peptide comprising the following sequence: ADDQNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 1).

37. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which is: Wherein Pv1 is a peptide comprising the following sequence: ADDQNPWRAYLDLLFPTDTLLLLDLLWCG (SEQ ID NO: 1).

38. A pharmaceutical composition comprising the compound of any one of claims 1-37 or a pharmaceutically acceptable salt thereof.

39. Use of a compound according to any one of claims 1 to 37, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating cancer in a patient, wherein the cancer is a solid tumor.

40. The method of claim 39, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, glioma, breast cancer, colon cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, prostate cancer, rectal cancer, skin cancer, testicular cancer, and uterine cancer.

41. The use of claim 39, wherein the cancer is selected from breast cancer, colorectal cancer, and gastric cancer.

42. The use of claim 39, wherein the cancer is breast cancer, and wherein the breast cancer is triple-negative breast cancer.

43. The use of claim 39, wherein the cancer is breast cancer.

44. The use of claim 39, wherein the cancer is ovarian cancer.

45. The use of claim 39, wherein the cancer is bowel cancer.

46. ​​The use of claim 39, wherein the cancer is a gastrointestinal tumor.

47. The use of claim 39, wherein the cancer is an epithelial cancer.

48. The use of claim 39, wherein the cancer is selected from cervical cancer, endometrial cancer, Ewing's sarcoma, Kaposi's sarcoma, laryngeal cancer, melanoma, clear cell renal carcinoma, and thyroid cancer.

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