Peptide conjugates of peptide tubulin inhibitors as therapeutic agents

Abstract

The present invention relates to peptide conjugates of peptide tubulin inhibitors (e.g., monomethyl auristatin) that are useful in the treatment of diseases such as cancer.

Description

[Technical field]

[0001] The present invention relates to peptide conjugates of peptide tubulin inhibitors, such as monomethyl auristatin, that are useful in the treatment of diseases such as cancer.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file named "43236-0020WO1_SL_ST26.XML". The XML file, created on November 15, 2022, is 436,177 bytes in size. The contents within the XML file are incorporated herein by reference in their entirety. [Background technology]

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

[0004] The treatment of cancer in humans by systemic administration of pharmaceuticals often works by slowing or terminating the uncontrolled replication that is characteristic of cancer cells. Peptide tubulin inhibitors, such as dolastatins, dolastatin-derived auristatins, monomethyl auristatins (e.g., monomethyl auristatin E and monomethyl auristatin F), and tubulysins, are a class of antimitotic agents that inhibit tubulin polymerization and can exhibit high efficacy against a wide range of cancer cells. Due to their often high cytotoxicity, peptide tubulin inhibitors, such as monomethyl auristatins, have been conjugated to tumor targeting agents, such as antibodies, to reduce off-target effects. Even so, antibody-drug conjugates of peptide tubulin inhibitors (e.g., monomethyl auristatin) can exhibit several severe side effects, including neutropenia, neuropathy, thrombocytopenia, and ocular toxicity. Thus, there is a need for more selective delivery of peptide tubulin inhibitor compounds to diseased tissues. Summary of the Invention

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

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

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

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

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

[0010] [Figure 1A] 1 shows a plot of the proliferation delay of HCT116 colorectal cells in vitro after incubation with the indicated concentrations of Compound 2 or unconjugated MMAE for 4 days. [Figure 1B] 1 shows a plot of the growth delay of PC3 prostate cells in vitro following incubation with the indicated concentrations of Compound 2 or unconjugated MMAE for 4 days. [Figure 1C] 1 shows a plot of the growth delay of NCI-H1975 NSCLC cells in vitro after incubation with the indicated concentrations of Compound 2 or unconjugated MMAE for 4 days. [Figure 1D] 1 shows a plot of the growth delay of NCI-H292 NSCLC cells in vitro after incubation with the indicated concentrations of Compound 2 or unconjugated MMAE for 4 days. [Figure 2A] FIG. 1 shows cell cycle analysis of HCT116 colorectal cells in vitro after 24 hour incubation with the indicated doses of unconjugated MMAE. [Figure 2B] 1 shows cell cycle analysis of HCT116 colorectal cells in vitro after 24 hour incubation with the indicated doses of Compound 2. [Diagram 3] 1 shows plots of plasma concentrations of Compound 2 and released MMAE following a single IV dose of 10 mg / kg Compound 2 in rats (data are presented as mean ± standard deviation). [Figure 4A]1 shows a plot of the levels of unconjugated MMAE in mouse tumors as measured by LCMS following a single intraperitoneal injection of either 0.5 mg / kg MMAE or 3 mg / kg Compound 2 in female nude mice bearing HCT116 colorectal tumors. [Figure 4B] 1 shows a plot of the levels of unconjugated MMAE in mouse muscle as measured by LCMS following a single intraperitoneal injection of either 0.5 mg / kg MMAE or 3 mg / kg Compound 2 in female nude mice bearing HCT116 colorectal tumors. [Figure 4C] 1 shows a plot of the levels of unconjugated MMAE in mouse bone marrow as measured by LCMS following a single intraperitoneal injection of either 0.5 mg / kg MMAE or 3 mg / kg Compound 2 in female nude mice bearing HCT116 colorectal tumors. [Figure 5A] Figure 1 shows a plot of the mean tumor volume resulting from dosing nude mice bearing HCT116 HER2-negative colorectal flank tumors with either 0.25 mg / kg MMAE or 40 mg / kg Compound 1 (equivalent to 7 mg / kg MMAE). Animals were dosed intraperitoneally once daily for a total of two days. [Figure 5B] 1 shows a plot of the percent change in body weight of nude mice bearing HCT116 HER2-negative colorectal flank tumors administered either 0.25 mg / kg MMAE or 40 mg / kg Compound 1 (equivalent to 7 mg / kg MMAE). [Figure 6A] 1 shows a plot of the mean tumor volume resulting from dosing nude mice bearing PC3 prostate adenocarcinoma flank tumors with 20 mg / kg of Compound 2. Animals were dosed intraperitoneally once daily, twice weekly for three weeks. [Figure 6B] Figure 1 presents the percent change in body weight of the animals in the study of Example F. Data are expressed as mean ± SEM. [Figure 7A] 1 shows a plot of the mean tumor volume resulting from dosing nude mice bearing NCI-H1975 non-small cell lung cancer flank tumors with 10 mg / kg or 20 mg / kg of Compound 2. Animals were dosed intraperitoneally once daily, twice weekly for three weeks. [Figure 7B] Figure 1 presents the percent change in body weight of the animals in the study of Example G. Data are expressed as mean ± SEM. [Figure 8] 1 shows a plot of body weight of nude mice dosed with 10 mg / kg of Compound 1 and Compound 2 once daily for four consecutive days. [Figure 9A] 1 shows a plot of peptide concentrations in tumors following a single 10 mg / kg IP dose of either Compound 7 or Compound 13 in female nude mice bearing HCT116 colorectal tumors (data are presented as mean ± standard deviation). [Figure 9B] 1 shows a plot of MMAE concentration in tumors following a single 10 mg / kg IP dose of either Compound 7 or Compound 13 in female nude mice bearing HCT116 colorectal tumors (data are presented as mean ± standard deviation). [Figure 10A] Figure 1 shows a plot of the mean tumor volume resulting from dosing nude mice bearing HT-29 colorectal flank tumors with 5 mg / kg of compound 13. Animals were dosed intraperitoneally once daily on days 0-3, 5, and 16-19. [Figure 10B] 1 presents the percent change in body weight of animals in the study of Example J. Data are expressed as mean ± SEM. [Figure 11A] Figure 1 shows a plot of the mean tumor volume resulting from dosing nude mice bearing HT-29 colorectal flank tumors with 40 mg / kg and 80 mg / kg of Compound 7. Animals were dosed parenterally once daily for 4 consecutive days per week for 2 weeks. [Figure 11B] Figure 1 presents the percent change in body weight of the animals in the study of Example K. Data are expressed as mean ± SEM. [Figure 12A] 1 shows plots of peptide concentrations in tumors following a single 10 mg / kg intraperitoneal dose of Compound 13, Compound 1, or Compound 2 in female nude mice bearing HCT116 colorectal tumors (data are presented as mean ± standard deviation). [Figure 12B]1 shows a plot of MMAE concentration in tumors following a single 10 mg / kg intraperitoneal dose of Compound 13, Compound 1, or Compound 2 in female nude mice bearing HCT116 colorectal tumors (data are presented as mean ± standard deviation). [Figure 13A] 1 shows plots of peptide levels in mouse tumors measured by ELISA and LCMS after a single 10 mg / kg intraperitoneal injection of Compound 13, Compound 7, Compound 5, or Compound 6 into female nude mice bearing HCT116 colorectal tumors (data are presented as mean ± standard deviation). [Figure 13B] 1 shows plots of the levels of unconjugated MMAE in mouse tumors measured by ELISA and LCMS following a single 10 mg / kg intraperitoneal injection of Compound 13, Compound 7, Compound 5, or Compound 6 into female nude mice bearing HCT116 colorectal tumors (data are presented as mean ± standard deviation). [Figure 14A] 1 shows a plot of the mean tumor volume resulting from dosing nude mice bearing HCT116 colorectal flank tumors with 1 mg / kg, 5 mg / kg, and 10 mg / kg of Compound 5. Animals were dosed parenterally once daily for 4 consecutive days per week. [Figure 14B] Figure 1 presents the percent change in body weight of the animals in the study of Example N. Data are expressed as mean ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Formula (I) [ka] A compound of the formula: R 1 is a peptide, R 2 is the radical of a peptide tubulin inhibitor, L is the part R 1 and part R 2 or a pharma- ceutically acceptable salt thereof.

[0012] Formula (I) [ka] A compound of the formula: R 1 is a peptide having 5 to 50 amino acids, R 2 is the radical of a peptide tubulin inhibitor, L is the part R 1 and part R 2 or a pharma- ceutically acceptable salt thereof.

[0013] Formula (I) [ka] A compound of the formula: R 1 However, R across cell membranes with acidic or hypoxic mantles 2 A peptide capable of selectively delivering L- R 2 is the radical of a peptide tubulin inhibitor, L is the part R 1 and part R 2 Also provided herein is a compound, or a pharma- ceutically acceptable salt thereof, which is a linker covalently attached to

[0014] Formula (I) [ka] A compound of the formula: R 1 is a peptide, R 2 is the radical of the auristatin compound, L is the part R 1 and part R 2 or a pharma- ceutically acceptable salt thereof.

[0015] Formula (I) [ka] A compound of the formula: R 1 is a peptide having 5 to 50 amino acids, R 2 is the radical of the auristatin compound, L is the part R 1 and part R 2 or a pharma- ceutically acceptable salt thereof.

[0016] Formula (I) [ka] A compound of the formula: R 1 However, R across cell membranes with acidic or hypoxic mantles 2 A peptide capable of selectively delivering L- R 2 is the radical of the auristatin compound, L is the part R 1 and part R 2 Also provided herein is a compound, or a pharma- ceutically acceptable salt thereof, which is a linker covalently attached to

[0017] In some embodiments, the auristatin compound is a monomethylauristatin compound. In some embodiments, L has the structure [ka] wherein an S atom of the linker is bonded to a cysteine ​​residue of the peptide to form a disulfide bond; In the formula, G 1 But, bond, C 6-10 Aryl, C 3-14cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G 1 The relevant C 6-10 Aryl, the C 3-14 The cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)2NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R d and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from 1 The relevant C 1-6 Alkyl Substituents, C 2-6 Alkenyl substituents, and the C2-6 Alkynyl substituents are CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)2NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R d and optionally substituted with 1, 2, or 3 substituents independently selected from R s and R t are each independently H, halo, C 1-6 Alkyl, and C 1-6 haloalkyl; G 2 But -NR G C(O)-, -NR G -, -O-, -S-, -C(O)O-, -OC(O)-, -NR G C(O)-, -OC(O)NR G -, and -S(O2)-; G3 But, C 6-10 Aryl, C 3-14 cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G 3 The relevant C 6-10 Aryl, the C 3-14 The cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , O.C.(O)R b1 , O-C(O)NR c1 R d1 , C(=NR e1 )NR c1 R d1 , N.R. c1 C(=NR e1 )NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)R b1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)2NR c1 R d1 , S(O)R b1 , S(O)NR c1 R d1 , S(O)2R b1 , and S(O)NR c1 R d1 wherein G is optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from 3 The relevant C1-6 Alkyl Substituents, C 2-6 Alkenyl substituents, and the C 2-6 Alkynyl substituents are CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , O.C.(O)R b1 , O-C(O)NR c1 R d1 , C(=NR e1 )NR c1 R d1 , N.R. c1 C(=NR e1 )NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)R b1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)2NR c1 R d1 , S(O)R b1 , S(O)NR c1 R d1 , S(O)2R b1 , and S(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from R u and R v are independent, H, halo, C 1-6 Alkyl, and C 1-6 haloalkyl; G 4 -C(O)-, -NR G C(O)-, -NR G -, -O-, -S-, -C(O)O-, -OC(O)-, -NR Gis selected from -C(O)-, and -S(O2)-; R G each independently represents H and C 1-4 alkyl, R a , R b , R c , R d , R a1 , R b1 , R c1 , and R d1 are each independently H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, and C 2-6 alkynyl, where R a , R b , R c , R d , R a1 , R b1 , R c1 , and R d1 The relevant C 1-6 Alkyl, the C 2-6 Alkenyl, and the C 2-6 Alkynyl is halo, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, CN, OR a2 , S.R. a2 , C(O)R b2 , C(O)NR c2 R d2 , C(O)OR a2 , O.C.(O)R b2 , O-C(O)NR c2 R d2 , N.R. c2 R d2 , N.R. c2 C(O)R b2 , N.R. c2 C(O)NR c2 R d2 , N.R. c2 C(O)OR a2 , C(=NR e2 )NR c2 R d2 , N.R. c2C(=NR e2 )NR c2 R d2 , S(O)R b2 , S(O)NR c2 R d2 , S(O)2R b2 , N.R. c2 S(O)2R b2 , N.R. c2 S(O)2NR c2 R d2 , and S(O)NR c2 R d2 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from R a2 , R b2 , R c2 , and R d2 are each independently H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, and C 2-6 alkynyl, where R a2 , R b2 , R c2 , and R d2 The relevant C 1-6 Alkyl, the C 1-6 Haloalkyl, the C 2-6 Alkenyl, and the C 2-6 Alkynyl is OH, CN, amino, halo, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl and C 1-6 optionally substituted with 1, 2, or 3 substituents independently selected from haloalkoxy; R e , R e1 , and R e2 each independently represents H and C 1-4 alkyl, m is 0, 1, 2, 3, or 4; n is 0 or 1.

[0018] Formula (I) [ka] A compound of the formula: R 1 is a peptide, R 2 is the radical of the auristatin compound, L, i) [ka] and ii) [ka] wherein the terminal S atom of the linker is bonded to a cysteine ​​residue of the peptide to form a disulfide bond; In the formula, G 1 But, bond, C 6-10 Aryl, C 3-14 cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G 1 The relevant C 6-10 Aryl, the C 3-14 The cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)Rb , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)2NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R d and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from 1 The relevant C 1-6 Alkyl Substituents, C 2-6 Alkenyl substituents, and the C 2-6 Alkynyl substituents are CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)2NR c R d, S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R d and optionally substituted with 1, 2, or 3 substituents independently selected from G 2 But -NR G C(O)-, -NR G -, -O-, -S-, -C(O)O-, -OC(O)-, -NR G C(O)-, -OC(O)NR G -, and -S(O2)-; G 3 But, C 6-10 Aryl, C 3-14 cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G 3 The relevant C 6-10 Aryl, the C 3-14 The cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , O.C.(O)R b1 , O-C(O)NR c1 R d1 , C(=NR e1 )NR c1 R d1 , N.R. c1 C(=NR e1 )NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1C(O)NR c1 R d1 , N.R. c1 S(O)R b1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)2NR c1 R d1 , S(O)R b1 , S(O)NR c1 R d1 , S(O)2R b1 , and S(O)NR c1 R d1 wherein G is optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from 3 The relevant C 1-6 Alkyl Substituents, C 2-6 Alkenyl substituents, and the C 2-6 Alkynyl substituents are CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , O.C.(O)R b1 , O-C(O)NR c1 R d1 , C(=NR e1 )NR c1 R d1 , N.R. c1 C(=NR e1 )NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)R b1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)2NR c1 R d1 , S(O)R b1 , S(O)NR c1 R d1, S(O)2R b1 , and S(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from G 4 -C(O)-, -NR G C(O)-, -NR G -, -O-, -S-, -OC(O)-, -NR G is selected from -C(O)-, and -S(O2)-; G 5 But, bond, C 6-10 Aryl, C 3-14 cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G 5 The relevant C 6-10 Aryl, C 3-14 Cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl are each selected from halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. cS(O)2R b , N.R. c S(O)2NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R d wherein G is optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from 5 The relevant C 1-6 Alkyl Substituents, C 2-6 Alkenyl substituents, and the C 2-6 Alkynyl substituents are CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)2NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R dand optionally substituted with 1, 2, or 3 substituents independently selected from G 6 But -NR G C(O)-, -NR G -, -O-, -S-, -C(O)O-, -OC(O)-, -NR G C(O)-, -OC(O)NR G -, and -S(O2)-; G 7 But -NR G C(O)-, -NR G -, -O-, -S-, -C(O)O-, -OC(O)-, -NR G C(O)-, -OC(O)NR G -, and -S(O2)-; R s and R t are each independently H, halo, C 1-6 Alkyl, and C 1-6 haloalkyl; or R s and R t together with the C atom to which they are attached, 3-6 forming a cycloalkyl ring, R u and R v are independent, H, halo, C 1-6 Alkyl, and C 1-6 haloalkyl; R G each independently represents H and C 1-4 alkyl, R a , R b , R c , R d , R a1 , R b1 , R c1 , and R d1 are each independently H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, and C 2-6 alkynyl, where R a, R b , R c , R d , R a1 , R b1 , R c1 , and R d1 The relevant C 1-6 Alkyl, the C 2-6 Alkenyl, and the C 2-6 Alkynyl is halo, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, CN, OR a2 , S.R. a2 , C(O)R b2 , C(O)NR c2 R d2 , C(O)OR a2 , O.C.(O)R b2 , O-C(O)NR c2 R d2 , N.R. c2 R d2 , N.R. c2 C(O)R b2 , N.R. c2 C(O)NR c2 R d2 , N.R. c2 C(O)OR a2 , C(=NR e2 )NR c2 R d2 , N.R. c2 C(=NR e2 )NR c2 R d2 , S(O)R b2 , S(O)NR c2 R d2 , S(O)2R b2 , N.R. c2 S(O)2R b2 , N.R. c2 S(O)2NR c2 R d2 , and S(O)NR c2 R d2 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from R a2 , R b2 , R c2 , and Rd2 are each independently H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, and C 2-6 alkynyl, where R a2 , R b2 , R c2 , and R d2 The relevant C 1-6 Alkyl, the C 1-6 Haloalkyl, the C 2-6 Alkenyl, and the C 2-6 Alkynyl is OH, CN, amino, halo, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl and C 1-6 optionally substituted with 1, 2, or 3 substituents independently selected from haloalkoxy; R e , R e1 , and R e2 each independently represents H and C 1-4 alkyl, m is 0, 1, 2, 3, or 4; n is 0 or 1; o is 0 or 1; p is 1, 2, 3, 4, 5, or 6; Provided herein are compounds, or a pharma- ceutically acceptable salt thereof, wherein q is 0 or 1.

[0019] Formula (I) [ka] A compound of the formula: R 1 is a peptide, R 2 is the radical of the auristatin compound, L is the following structure: [ka] wherein an S atom of the linker is bonded to a cysteine ​​residue of the peptide to form a disulfide bond; In the formula, G 1 But, bond, C 6-10 Aryl, C 3-14 cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G 1 The relevant C 6-10 Aryl, the C 3-14 The cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)2NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c Rd and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from 1 The relevant C 1-6 Alkyl Substituents, C 2-6 Alkenyl substituents, and the C 2-6 Alkynyl substituents are CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)2NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R d and optionally substituted with 1, 2, or 3 substituents independently selected from R s and R t are each independently H, halo, C 1-6 Alkyl, and C 1-6 haloalkyl; G 2 But -NR G C(O)-, -NRG -, -O-, -S-, -C(O)O-, -OC(O)-, -NR G C(O)-, -OC(O)NR G -, and -S(O2)-; G 3 But, C 6-10 Aryl, C 3-14 cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G 3 The relevant C 6-10 Aryl, the C 3-14 The cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , O.C.(O)R b1 , O-C(O)NR c1 R d1 , C(=NR e1 )NR c1 R d1 , N.R. c1 C(=NR e1 )NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)R b1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)2NR c1 R d1 , S(O)R b1 , S(O)NR c1 R d1, S(O)2R b1 , and S(O)NR c1 R d1 wherein G is optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from 3 The relevant C 1-6 Alkyl Substituents, C 2-6 Alkenyl substituents, and the C 2-6 Alkynyl substituents are CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , O.C.(O)R b1 , O-C(O)NR c1 R d1 , C(=NR e1 )NR c1 R d1 , N.R. c1 C(=NR e1 )NR c1 R d1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)R b1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)2NR c1 R d1 , S(O)R b1 , S(O)NR c1 R d1 , S(O)2R b1 , and S(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from R u and R v are independent, H, halo, C 1-6 Alkyl, and C 1-6 haloalkyl; G4 -C(O)-, -NR G C(O)-, -NR G -, -O-, -S-, -C(O)O-, -OC(O)-, -NR G is selected from -C(O)-, and -S(O2)-; R G each independently represents H and C 1-4 alkyl, R a , R b , R c , R d , R a1 , R b1 , R c1 , and R d1 are each independently H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, and C 2-6 alkynyl, where R a , R b , R c , R d , R a1 , R b1 , R c1 , and R d1 The relevant C 1-6 Alkyl, the C 2-6 Alkenyl, and the C 2-6 Alkynyl is halo, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, CN, OR a2 , S.R. a2 , C(O)R b2 , C(O)NR c2 R d2 , C(O)OR a2 , O.C.(O)R b2 , O-C(O)NR c2 R d2 , N.R. c2 R d2 , N.R. c2 C(O)R b2 , N.R. c2 C(O)NR c2R d2 , N.R. c2 C(O)OR a2 , C(=NR e2 )NR c2 R d2 , N.R. c2 C(=NR e2 )NR c2 R d2 , S(O)R b2 , S(O)NR c2 R d2 , S(O)2R b2 , N.R. c2 S(O)2R b2 , N.R. c2 S(O)2NR c2 R d2 , and S(O)NR c2 R d2 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from R a2 , R b2 , R c2 , and R d2 are each independently H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, and C 2-6 alkynyl, where R a2 , R b2 , R c2 , and R d2 The relevant C 1-6 Alkyl, the C 1-6 Haloalkyl, the C 2-6 Alkenyl, and the C 2-6 Alkynyl is OH, CN, amino, halo, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl and C 1-6 optionally substituted with 1, 2, or 3 substituents independently selected from haloalkoxy; R e , R e1 , and R e2 each independently represents H and C 1-4 alkyl, m is 0, 1, 2, 3, or 4; Provided herein are compounds, or pharma- ceutically acceptable salts thereof, wherein n is 0 or 1.

[0020] In some embodiments, the left side of L is R 1 and the right side of L is R 2 Bind to.

[0021] As used herein, a "peptide" refers to a targeting moiety that comprises a 10-50 amino acid sequence of naturally occurring amino acid residues and optionally one or more non-naturally occurring amino acids. In some embodiments, R 1 The peptides are peptides of 20-40, 20-30 amino acids, or 30-40 residues. Peptides suitable for use in the compounds of the invention are those that are capable of inserting across cell membranes by conformational or secondary structure changes in response to environmental pH changes. In this manner, the peptides are capable of targeting acidic tissues and selectively translocating polar cell-impermeable molecules across cell membranes in response to a drop in extracellular pH. In some embodiments, the peptides are capable of selectively translocating polar cell-impermeable molecules across cell membranes having an acidic or hypoxic mantle with a pH of less than about 6.0, in combination with a conjugated moiety (e.g., R 2 In some embodiments, the peptide can selectively deliver the conjugated moiety (e.g., R-L-) across cell membranes that have an acidic or hypoxic mantle with a pH of less than about 6.5. 2 In some embodiments, the peptide can selectively deliver the conjugated moiety (e.g., R-L-) across cell membranes that have an acidic or hypoxic mantle with a pH of less than about 5.5. 2 In some embodiments, the peptide can selectively deliver the conjugated moiety (e.g., R-L-) across cell membranes that have an acidic or hypoxic mantle with a pH of about 5.0 to about 6.0. 2 L-) can be selectively delivered.

[0022] In certain embodiments, R 1 The peptides are then coupled to a payload moiety (e.g., R2 In some embodiments, R 1 is R 1 is linked to L via a cysteine ​​residue in. In some embodiments, the sulfur atom of the cysteine ​​residue can form part of a disulfide bond of the disulfide bond-containing compound.

[0023] Suitable peptides that undergo structural changes based on pH and can be inserted across cell membranes are described, for example, in U.S. Patent Nos. 8,076,451, 9,289,508, 10,933,069, and U.S. Patent Publication Nos. 2021 / 0009536 and 2021 / 0009719, each of which is incorporated 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 herein by reference in its entirety.

[0024] In some embodiments, R 1 is the following array: ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO:1, Pv1), AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO:2, Pv2), and ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO:3, Pv3), Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4, Pv4), AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO:5, Pv5), and A peptide comprising at least one of the following: AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG (SEQ ID NO: 6, Pv6); In the formula, R 1 But R 1 is linked to L via a cysteine ​​residue in

[0025] In some embodiments, R 1 is the following array: ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO:1, Pv1), AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO:2, Pv2), and ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO:3, Pv3), and A peptide comprising at least one of the following: AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG (SEQ ID NO: 6, Pv6); In the formula, R 1 But R 1 is linked to L via a cysteine ​​residue in

[0026] In some embodiments, R 1 is a peptide containing the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1, Pv1).

[0027] In some embodiments, R 1 is a peptide containing the sequence AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO:2, Pv2).

[0028] In some embodiments, R 1 is a peptide containing the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO:3, Pv3).

[0029] In some embodiments, R 1 is an array A peptide comprising Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4, Pv4).

[0030] In some embodiments, R 1 is an array A peptide containing AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO:5, Pv5).

[0031] In some embodiments, R 1 is an array A peptide containing AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG (SEQ ID NO: 6, Pv6).

[0032] In some embodiments, R 1 is a peptide consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1, Pv1).

[0033] In some embodiments, R 1 is a peptide consisting of the sequence AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO:2, Pv2).

[0034] In some embodiments, R 1 is a peptide consisting of the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO:3, Pv3).

[0035] In some embodiments, R 1 is a peptide consisting of the sequence Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO: 4, Pv4).

[0036] In some embodiments, R 1 is an array It is a peptide consisting of AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (sequence number 5, Pv5).

[0037] In some embodiments, R 1 is an array It is a peptide consisting of AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG (sequence number 6, Pv6).

[0038] In some embodiments, R 1 is a peptide comprising at least one sequence selected from SEQ ID NO: 7 to SEQ ID NO: 311 shown in Table 1.

[0039] In some embodiments, R 1 is a peptide consisting of a sequence selected from SEQ ID NO: 7 to SEQ ID NO: 311 shown in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0040] Any of the recited peptides useful in the present invention can be modified to contain cysteine ​​residues, either by replacing a non-cysteine ​​residue with cysteine ​​or by adding a cysteine ​​residue to either the N- or C-terminus.

[0041] In some embodiments, R 1The peptides of are conformationally restricted peptides. Conformationally restricted peptides can include, for example, macrocyclic peptides and stapled peptides. Stapled peptides are peptides that are constrained by a covalent bond between two amino acid side chains to form a peptide macrocycle. Conformationally restricted peptides are described, for example, in Guerlavais et al., Annual Reports in Medicinal Chemistry 2014, 49, 331-345; Chang et al., Proceedings of the National Academy of Sciences of the United States of America (2013), 110(36), E3445-E3454; Tesauro et al., Molecules 2019, 24, 351-377; Dougherty et al., Journal of Medicinal Chemistry (2019), 62(22), 10098-10107; and Dougherty et al., Chemical Reviews (2019), 119(17), 10241-10287, each of which is incorporated herein by reference in its entirety.

[0042] In some embodiments, R 1 is a peptide having 10 to 50 amino acids. 1 is a peptide having 20 to 40 amino acids. 1 is a peptide having 20 to 40 amino acids. 1 is a peptide having 10 to 20 amino acids. 1 is a peptide having 20 to 30 amino acids. In some embodiments, R 1 is a peptide having 30 to 40 amino acids.

[0043] The term "peptide tubulin inhibitors" (e.g., R 2) refers to a compound that contains at least two amino acids and is a tubulin polymerization inhibitor. In some embodiments, the peptide tubulin inhibitor is a small molecule peptide tubulin inhibitor. In some embodiments, the peptide tubulin inhibitor is less than 1500 Da. In some embodiments, the peptide tubulin inhibitor is an auristatin compound, a dolastatin, or a tubulysin, or a derivative thereof.

[0044] Suitable auristatin compounds (e.g., R 2 ) include auristatin derivatives that exhibit anti-tubulin activity (e.g., inhibition of tubulin polymerization). Auristatin compounds are known in the art and have been used as part of antibody-drug conjugates. For example, SODoronina and PDSenter in Cytotoxic Payloads for Antibody-Drug Conjugates(Royal Society for Chemistry,2019),Chapter 4:Auristatin Payloads for Antibody-Drug Conjugates, p73-99, N. Joubert, A. Beck, C. Dumontet, C. Denevault-Sabourin, Pharmaceuticals, 2020, 13, 245, JD Bargh, A. Isid rio-Llobet, JSParker, DRSpring, Chem. Soc. Rev., 2019, 48, 4361-4374, and Kostova, V., Desos, P., Starck, J.-B., Kotschy, A, The Chemistry Behind ADCs,Pharmaceuticals 2021,14,442, Mackertish et al., Biomedicines, 2021, 9(8):872, pp. 1-25, each of which is incorporated by reference in its entirety.

[0045] In some embodiments, the auristatin is a monomethylauristatin. There are two main classes of auristatins: monomethylauristatin E type molecules and monomethylauristatin F compounds. The structure of monomethylauristatin E is shown below. [ka]

[0046] Monomethylauristatin E may also be referred to as "MMAE."

[0047] The structure of monomethylauristatin F is shown below. [ka]

[0048] Monomethylauristatin F may also be referred to as "MMAF."

[0049] In some embodiments, R 2 is the radical of a monomethyl auristatin compound.

[0050] In some embodiments, R 2 is the radical of monomethylauristatin E.

[0051] In some embodiments, R 2 is the radical of monomethylauristatin F.

[0052] In some embodiments, R 2 has the following structure: [ka] has.

[0053] In some embodiments, R 2 has the following structure: [ka] has.

[0054] In some embodiments, R 2 has the following structure: [ka]

[0055] In some embodiments, L is R 1 and R 2 is a linking moiety that covalently links 2 The compound serves to release a moiety comprising the compound into the vicinity of the acidic or hypoxic tissue, for example, into the cells of the diseased tissue.

[0056] In some embodiments, L has the structure [ka] is a linker having the formula:

[0057] In some embodiments, L is a linker having the structure: [ka]

[0058] In some embodiments, G 1 is a bond, C 6-10 Aryl, C 3-14 In some embodiments, G is selected from cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl. 1 is a bond, C 6-10 Aryl and C 3-14 In some embodiments, G 1 is C 6-10 Aryl and C 3-14 cycloalkyl.

[0059] In some embodiments, G 1 is a bond and C 3-14cycloalkyl.

[0060] In some embodiments, G 1 is a bond.

[0061] In some embodiments, G 1 is a bond, phenyl, and C 4-6 In some embodiments, G 1 is phenyl and C 4-6 cycloalkyl.

[0062] In some embodiments, G 1 is C 3-14 It is cycloalkyl.

[0063] In some embodiments, G 1 is cyclopentyl or cyclohexyl, wherein the cyclopentyl and the cyclohexyl are each optionally fused to a phenyl group.

[0064] In some embodiments, G 1 is phenyl.

[0065] In some embodiments, G 1 is cyclopentyl, cyclohexyl, or phenyl, where the cyclopentyl and the cyclohexyl are each optionally fused to a phenyl group.

[0066] In some embodiments, R s and R t are each independently H and C 1-6 is selected from alkyl.

[0067] In some embodiments, R s and R t are each independently selected from H and isopropyl. In some embodiments, R s and R t are each independently selected from H, methyl, and isopropyl.

[0068] In some embodiments, R s and R t together with the C atoms to which they are attached, 4-6 It forms a cycloalkyl group.

[0069] In some embodiments, R s and R t together with the C atom to which they are attached form a cyclobutyl ring.

[0070] In some embodiments, m is 0, 1, or 2. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2.

[0071] In some embodiments, G 2 is -OC(O)- and -OC(O)NR G - is selected.

[0072] In some embodiments, G 2 is -OC(O)-.

[0073] In some embodiments, G 3 is C 6-10 It is selected from aryl and 5-14 membered heteroaryl.

[0074] In some embodiments, G 3 is C 6-10 It is aryl.

[0075] In some embodiments, G 3 is phenyl.

[0076] In some embodiments, R u and R v are each H.

[0077] In some embodiments, G 4 is -OC(O)-.

[0078] In some embodiments, G 5 is a 4-14 membered heterocycloalkyl, where G 5 The 4-14 membered heterocycloalkyl is halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)2NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R d and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from 5 The relevant C 1-6 Alkyl Substituents, C 2-6 Alkenyl substituents, and the C 2-6Alkynyl substituents are CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)2NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R d and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0079] In some embodiments, G 5 is based on the following [ka] It is.

[0080] In some embodiments, G 6 -NR G It is C(O)-.

[0081] In some embodiments, G 7-NR G It is C(O)-.

[0082] In some embodiments, n is 0. In some embodiments, n is 1.

[0083] In some embodiments, o is 0. In some embodiments, o is 1.

[0084] In some embodiments, p is 2, 3, 4, or 5. In some embodiments, p is 3, 4, or 5. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5.

[0085] In some embodiments, q is 0. In some embodiments, q is 1.

[0086] In some embodiments, R G are each independently selected from H and methyl. In some embodiments, R G Each is H. In some embodiments, R G Each is methyl.

[0087] In some embodiments, L has the structure [ka] has.

[0088] In some embodiments, L has the structure [ka] has.

[0089] In some embodiments, L has the structure [ka] has.

[0090] In some embodiments, L has the structure [ka] has.

[0091] In some embodiments, L has the structure [ka] has.

[0092] In some embodiments, L has the structure [ka] has.

[0093] In some embodiments, L has the structure [ka] has.

[0094] In some embodiments, L has the structure [ka] has.

[0095] In some embodiments, L has the structure [ka] has.

[0096] In some embodiments, L has the structure [ka] has.

[0097] In some embodiments, L has the structure [ka] has.

[0098] In some embodiments, L has the structure [ka] has.

[0099] In some embodiments, L has the structure [ka] has.

[0100] In some embodiments, L has the structure [ka] has.

[0101] In some embodiments, L has the structure [ka] has.

[0102] In some embodiments, the compound of the present invention has the formula (II): [ka] A compound of the formula: R 1 is a peptide, R 2 is the radical of a peptide tubulin inhibitor, Ring Z is a monocyclic C 5-7 a cycloalkyl ring or a monocyclic 5- to 7-membered heterocycloalkyl ring; R Z are each independently halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a , S.R.a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , and N.R. c C(O)NR c R d or Two adjacent R Z together with the atoms to which they are attached form a fused monocyclic C 5-7 Cycloalkyl ring, fused monocyclic 5- to 7-membered heterocycloalkyl ring, fused C 6-10 aryl ring, or a condensed 6- to 10-membered heteroaryl ring, each of which is 1-6 Alkyl, halo, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , and N.R. c C(O)NR c R d and optionally substituted with 1, 2, or 3 substituents independently selected from R a , R b , R c , and R d are H and C, respectively. 1-4 Alkyl, C 2-4 Alkenyl, C 2-4alkynyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from halo, OH, CN, and NO2; p is 0, 1, 2, or 3, or a pharma- ceutically acceptable salt thereof.

[0103] In some embodiments, the compound of the present invention has the formula (II): [ka] A compound of the formula: R 1 is a peptide, R 2 is the radical of the auristatin compound, Ring Z is a monocyclic C 5-7 a cycloalkyl ring or a monocyclic 5- to 7-membered heterocycloalkyl ring; R Z are each independently halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , and N.R. c C(O)NR c R d or Two adjacent R Z together with the atoms to which they are attached form a fused monocyclic C 5-7 Cycloalkyl ring, fused monocyclic 5- to 7-membered heterocycloalkyl ring, fused C 6-10aryl ring, or a condensed 6- to 10-membered heteroaryl ring, each of which is 1-6 Alkyl, halo, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , and N.R. c C(O)NR c R d and optionally substituted with 1, 2, or 3 substituents independently selected from R a , R b , R c , and R d are H and C, respectively. 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 halo, OH, CN, and NO2; p is 0, 1, 2, or 3, or a pharma- ceutically acceptable salt thereof.

[0104] In some embodiments of the compound of Formula (II), R 1 is a peptide comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0105] In some embodiments of the compound of Formula (II), R 1 is Pv1, Pv2, or Pv3.

[0106] In some embodiments of the compound of Formula (II), R 1 is R 1wherein one of the sulfur atoms of the disulfide moiety of formula II is derived from the cysteine ​​residue.

[0107] In some embodiments of the compound of Formula (II), R 2 is the radical of a monomethyl auristatin compound.

[0108] In some embodiments of the compound of Formula (II), R 2 is the radical of monomethylauristatin E.

[0109] In some embodiments of the compound of Formula (II), R 2 is the radical of monomethylauristatin F.

[0110] In some embodiments of the compound of Formula (II), R 2 has the following structure: [ka] has.

[0111] In some embodiments of the compound of Formula (II), R 2 has the following structure: [ka] has.

[0112] In some embodiments of the compound of Formula (II), ring Z is a monocyclic C 5-7 It is a cycloalkyl ring.

[0113] In some embodiments of the compounds of Formula (II), ring Z is a cyclopentyl ring.

[0114] In some embodiments of the compounds of Formula (II), ring Z is a cyclohexyl ring.

[0115] In some embodiments of the compound of Formula (II), two adjacent R Ztogether with the atoms to which they are attached form a fused monocyclic C 5-7 Cycloalkyl ring, fused monocyclic 5- to 7-membered heterocycloalkyl ring, fused C 6-10 aryl ring, or a fused 6- to 10-membered heteroaryl ring, each of which is 1-4 Alkyl, halo, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a , O.C.(O)R b , O-C(O)NR c R d , N.R. c R d , N.R. c C(O)R b , N.R. c C(O)OR a , and N.R. c C(O)NR c R d and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0116] In some embodiments of the compound of Formula (II), p is 0.

[0117] In some embodiments of the compound of Formula (II), p is 1.

[0118] In some embodiments of the compound of Formula (II), p is 2.

[0119] In some embodiments of the compound of Formula (II), p is 3.

[0120] In some embodiments, the compound of the present invention has formula (III) or formula (IV) [ka] or a pharma- ceutically acceptable salt thereof, wherein R 1 , R 2 , R Z, and p is as defined herein.

[0121] In some embodiments of the compounds of Formula (III) and Formula (IV), R 1 is a peptide comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0122] In some embodiments of the compounds of Formula (III) and Formula (IV), R 1 is Pv1, Pv2, or Pv3.

[0123] In some embodiments of the compounds of Formula (III) and Formula (IV), R 1 is R 1 wherein one of the sulfur atoms of the disulfide moiety of formula (III) and formula (IV) is derived from the cysteine ​​residue.

[0124] In some embodiments of the compounds of Formula (III) and Formula (IV), R 2 is the radical of a monomethyl auristatin compound.

[0125] In some embodiments of the compounds of Formula (III) and Formula (IV), R 2 is the radical of monomethylauristatin E.

[0126] In some embodiments of the compounds of Formula (III) and Formula (IV), R 2 is the radical of monomethylauristatin F.

[0127] In some embodiments, the compound of formula (I) is [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt of any of the foregoing, wherein: Pv1 has the following sequence: A peptide comprising ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1), Pv2 has the following array A peptide comprising AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2), Pv3 has the following array A peptide containing ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3).

[0128] In some embodiments, the compound of formula (I) is [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt of any of the foregoing, wherein: Pv1 has the following sequence: A peptide comprising ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1), Pv2 has the following array A peptide comprising AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2), Pv3 has the following array A peptide containing ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3).

[0129] The molecules of the present invention can be tagged with a probe, such as, for example, a fluorophore, a radioisotope, etc. In some embodiments, the probe is a fluorescent probe, such as LICOR. A fluorescent probe can include any moiety (e.g., a fluorophore) that can re-emit light upon light excitation.

[0130] Amino acids are represented by their IUPAC abbreviations as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0131] The term "Pv1" refers to the peptide of SEQ ID NO:1, ADDQNPWRAYLDLLFPTDTLLLDLLWCG.

[0132] The term "Pv2" refers to the peptide of SEQ ID NO:2, AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG.

[0133] The term "Pv3" refers to the peptide of SEQ ID NO:3, ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG.

[0134] The term "Pv4" refers to the peptide of SEQ ID NO:4, Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG.

[0135] The term "Pv5" refers to the peptide of SEQ ID NO: 5, AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC. The term "Pv6" refers to the peptide of SEQ ID NO: 6, AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG. In the compounds of the invention, the peptide R 1 is linked to a disulfide linker by a cysteine ​​moiety.

[0136] The term "acidic and / or hypoxic mantle" refers to the environment of cells in the diseased tissue in question, having a pH of less than 7.0, preferably less than 6.5. The acidic or hypoxic mantle more preferably has a pH of about 5.5, most preferably about 5.0. Compounds of formula (I) insert across cell membranes having an acidic and / or hypoxic mantle in a pH-dependent manner, resulting in R 2 L is inserted into the cell, and upon insertion, the disulfide bond in the linker is cleaved to produce free R 2 L (or R 2 L*, where L* is a degradation product. Because the compounds of formula (I) are pH dependent, they preferentially insert across cell membranes only in the presence of an acidic or hypoxic mantle surrounding the cell, and do not insert across the cell membrane of "normal" cells that do not have an acidic or hypoxic mantle.

[0137] Peptide R of the compound of the present invention crosses the cell membrane 1 or its peptide R 1The term "pH-sensitive" or "pH-dependent" as used herein to refer to the mode of insertion of a peptide means that the peptide has a higher affinity for cell membrane lipid bilayers having an acidic or hypoxic mantle than for membrane lipid bilayers at neutral pH. Thus, the compounds of the present invention preferentially insert through cell membranes to mediate R activity when the cell membrane lipid bilayer has an acidic or hypoxic mantle ("disease" cells). 2 Insert L into the interior of the cell (hence R as above). 2 H), but does not insert through the cell membrane unless the mantle (the environment of the cell membrane lipid bilayer) is acidic or hypoxic ("normal" cells). This preferential insertion is due to the peptide R 1 This is thought to be achieved as a result of the formation of a helical structure that facilitates membrane insertion.

[0138] It is further understood that for clarity, certain features of the invention that are described in the context of separate embodiments can also be provided in combination in a single embodiment (although it is intended that these embodiments be combined as if described in multiple dependent forms). Conversely, for brevity, various features of the invention that are described in the context of a single embodiment can also be provided separately or in any suitable subcombination. For example, it is contemplated that the features described as embodiments of the compound of formula (I) can be combined in any suitable combination.

[0139] At various places in the present specification, certain features of the compounds are disclosed in groups or ranges. It is specifically intended that such disclosure include any and all individual subcombinations of the members of such groups and ranges. For example, "C 1-6 The term "alkyl" is specifically intended to individually disclose, including but not limited to, methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0140] The term "n-membered", where n is an integer, typically refers to the number of ring-forming atoms in a moiety where n is the number of ring-forming atoms. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.

[0141] At various places in the specification, variables defining divalent linking groups may be described. It is specifically intended that each linking substituent include both the forward and reverse forms of the linking substituent. For example, -NR(CR'R") n - is -NR(CR'R") n -and-(CR'R") n It is intended to include both NR- and NR- and to disclose each of these forms individually. When a structure requires a linking group, it is understood that the Markush variable listed for that group is the linking group. For example, when a structure requires a linking group and the Markush group definition for that variable lists "alkyl" or "aryl", it is understood that "alkyl" or "aryl" represents a linking alkylene group or a linking arylene group, respectively.

[0142] The term "substituted" means that an atom or group of atoms formally replaces hydrogen as a "substituent" attached to another group. The term "substituted" refers to any level of substitution where such substitution is permitted, e.g., mono-, di-, tri-, tetra-, or penta-substitution, unless otherwise indicated. The substituents are independently selected, and the substitutions can be at any chemically accessible position. It is understood that the substitution at a given atom is limited by the valence. It is understood that the 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 divalent substituent, e.g., oxo, can replace two hydrogen atoms.

[0143] "C n-m The term "carbon atom" refers to a range inclusive of the endpoints, where n and m are integers and indicate the number of carbons. Examples include 1-4 , C 1-6 etc.

[0144] The term "alkyl", used alone or in combination with other terms, refers to a saturated hydrocarbon group which may be straight-chained or branched. n-m The term "alkyl" refers to an alkyl group having n to m carbon atoms. An alkyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. In some embodiments, the alkyl group has 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; and higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, etc.

[0145] The term "alkenyl," used alone or in combination with other terms, refers to a straight or branched chain hydrocarbon group corresponding to an alkyl group having one or more double carbon-carbon bonds. An alkenyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound. n-m The term "alkenyl" refers to an alkenyl group having n to m carbons. In some embodiments, the alkenyl moiety has 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Exemplary alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

[0146] The term "alkynyl," used alone or in combination with other terms, refers to a straight or branched chain hydrocarbon group corresponding to an alkyl group having one or more triple carbon-carbon bonds. An alkynyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment of the alkynyl group to the remainder of the compound. n-m The term "alkynyl" refers to an alkynyl group having n to m carbons. Exemplary alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety has 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0147] The term "alkylene," used alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene group is formally equivalent to an alkane with two C-H bonds replaced by the points of attachment of the alkylene group to the remainder of the compound. n-m The term "alkylene" refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethane-1,2-diyl, ethane-1,1-diyl, propane-1,3-diyl, propane-1,2-diyl, propane-1,1-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, 2-methyl-propane-1,3-diyl, and the like.

[0148] The term "amino" refers to a group of the formula -NH2.

[0149] The term "carbonyl", used alone or in combination with other terms, refers to a -C(=O)- group, which also can be written as C(O).

[0150] The term "cyano" or "nitrile" refers to a group of formula -C≡N, which can also be written as -CN.

[0151] 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.

[0152] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced with a halogen atom. n-m The term "haloalkyl" refers to a C alkyl group having n to m carbon atoms and at least 1 and at most {2(n to m)+1} halogen atoms. n-m In some embodiments, the halogen atom is a fluoro atom. In some embodiments, the haloalkyl group has 1-6 or 1-4 carbon atoms. Exemplary haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCl3, CHCl2, C2Cl5, and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group.

[0153] The term "oxidized" with respect to a ring-forming N-atom refers to a ring-forming N-oxide.

[0154] The term "oxidized" with respect to a ring-forming S atom refers to a ring-forming sulfonyl or ring-forming sulfinyl.

[0155] 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 π (pi) electrons, where n is an integer).

[0156] The term "aryl," used alone or in combination with other terms, refers to an aromatic hydrocarbon group that can be monocyclic or polycyclic (e.g., having two fused rings). n-mThe term "aryl" refers to an aryl group having n to m ring carbon atoms. Examples of aryl groups include phenyl, naphthyl, and the like. In some embodiments, an aryl group has from 6 to about 10 carbon atoms. In some embodiments, an aryl group has 6 carbon atoms. In some embodiments, an aryl group has 10 carbon atoms. In some embodiments, an aryl group is phenyl.

[0157] The term "heteroaryl" or "heteroaromatic", used alone or in combination with other terms, refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in the heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl ring has 5-14 ring atoms including carbon atoms and 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl ring has 5-10 ring atoms including carbon atoms and 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl ring has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5- or 6-membered heteroaryl ring. In other embodiments, the heteroaryl is an 8-, 9-, or 10-membered fused bicyclic heteroaryl ring.

[0158] A 5-membered heteroaryl ring is a heteroaryl group having 5 ring atoms, in which one or more (eg, 1, 2, or 3) ring atoms are independently selected from N, O, and S.

[0159] A 6-membered heteroaryl ring is a heteroaryl group having 6 ring atoms, in which one or more (eg, 1, 2, or 3) ring atoms are independently selected from N, O, and S.

[0160] The term "cycloalkyl", used alone or in combination with other terms, refers to non-aromatic hydrocarbon ring systems (monocyclic, bicyclic, or polycyclic) including cyclized alkyl and alkenyl groups. n-m The term "cycloalkyl" refers to a cycloalkyl having n to m ring carbon atoms. Cycloalkyl groups can include monocyclic or polycyclic groups (e.g., having 2, 3, or 4 fused rings) and spirocycles. Cycloalkyl groups can have 3, 4, 5, 6, or 7 ring carbons (C 3-7 In some embodiments, the cycloalkyl group has 3 to 6 ring members, 3 to 5 ring members, or 3 to 4 ring members. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is monocyclic or bicyclic. In some embodiments, the cycloalkyl group is C 3-6 It is a monocyclic cycloalkyl group. The ring-forming carbon atoms of the cycloalkyl group can be optionally oxidized to form an oxo group or a sulfide group. The cycloalkyl group also includes cycloalkylidene. In some embodiments, the cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. The definition of cycloalkyl also includes moieties having one or more aromatic rings fused (i.e., having a common bond) to the cycloalkyl ring, such as benzo or thienyl derivatives of cyclopentane, cyclohexane, etc. Cycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, and the like. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0161] The term "heterocycloalkyl," used alone or in combination with other terms, refers to a non-aromatic ring or ring system that may optionally contain one or more alkenylene groups as part of the ring structure, has at least one heteroatom ring member independently selected from nitrogen, sulfur, oxygen, and phosphorus, and has 4 to 10 ring members, 4 to 7 ring members, or 4 to 6 ring members. The term "heterocycloalkyl" includes monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups can include mono- or bicyclic (e.g., having 2 fused or bridged rings) or spirocyclic systems. In some embodiments, heterocycloalkyl groups are monocyclic groups 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 be optionally oxidized to form oxo or sulfide groups, or other oxidized bonds (e.g., C(O), S(O), C(S), or S(O), N-oxide, etc.), or the nitrogen atom can be quaternized. A heterocycloalkyl group can be bonded through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, a heterocycloalkyl group has 0-3 double bonds. In some embodiments, a heterocycloalkyl group has 0-2 double bonds. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a common bond) to the heterocycloalkyl ring, e.g., benzo or thienyl derivatives such as piperidine, morpholine, azepine, etc. Heterocycloalkyl groups that contain fused aromatic rings can be bonded through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. Examples of heterocycloalkyl groups include 2-pyrrolidinyl, morpholinyl, azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, and piperazinyl.

[0162] In certain places, definitions or embodiments refer to certain rings (e.g., azetidine ring, pyridine ring, etc.).Unless otherwise indicated, these rings can be bonded to any ring member, as long as the valence of the atom is not exceeded.For example, the azetidine ring can be bonded to any position of the ring, while the azetidin-3-yl ring is bonded to the 3-position.

[0163] The compounds described herein can be asymmetric (e.g., have one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present invention having asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically inactive starting materials, such as by resolution of racemic mixtures or stereoselective synthesis, are known in the art. Many geometric isomers of olefins, C=N double bonds, and the like, can also exist in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can be isolated as a mixture of isomers or as separated isomeric forms.

[0164] The resolution of racemic mixtures of compounds can be carried out by any of a number of methods known in the art. One method involves fractional recrystallization using chiral resolving acids that are optically active salt-forming organic acids. Suitable resolving agents for fractional recrystallization are, for example, optically active acids, such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or the D- and L-forms of various optically active camphorsulfonic acids, such as α-camphorsulfonic acid. Other suitable resolving agents for fractional crystallization include stereoisomerically pure forms of α-methylbenzylamine (e.g., S- and R-forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

[0165] Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by one skilled in the art.

[0166] In some embodiments, the compounds of the invention have the (R) configuration. In other embodiments, the compounds have the (S) configuration. In compounds with two or more chiral centers, unless otherwise indicated, each chiral center in the compound may independently be either (R) or (S).

[0167] The compounds of the present invention also include tautomeric forms. Tautomeric forms are obtained by the exchange of a single bond with an adjacent double bond with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Exemplary prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy more than one position of the heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or sterically fixed in one form by appropriate substitution.

[0168] The compounds herein may also include all isotopes of atoms occurring in intermediates or final compounds. Isotopes include atoms with the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds of the present invention can be replaced or substituted with an isotope of the atom in 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 with 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. Synthetic methods for incorporating isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, NY, Appleton-Century-Crofts, 1971; The Renaissance of H / D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in a variety of studies, such as NMR spectroscopy, metabolic experiments, and / or assays.

[0169] Substitution with heavier isotopes, such as deuterium, may provide certain therapeutic benefits, such as increased in vivo half-life or reduced dosage requirements, resulting from higher metabolic stability and therefore may be preferred in some situations. (A. Kerekes et.al. J. Med. Chem. 2011, 54, 201-210; R. Xu et.al. J. Label Compd. Radiopharm. 2015, 58, 308-312).

[0170] The term "compound" as used herein is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. This term is intended to refer to compounds of the invention regardless of how they are prepared, for example, synthetically, by a biological process (e.g., metabolic or enzymatic conversion), or by a combination thereof.

[0171] All compounds and their pharmaceutically acceptable salts can be found or isolated with other substances such as water and solvents (e.g., hydrates and solvates). When in solid state, the compounds and their salts described herein can occur in various forms, for example, in the form of solvates, including hydrates. The compounds can be in any solid form, such as polymorphs or solvates, so unless otherwise specified, references to compounds and their salts herein should be understood to include all solid forms of the compounds.

[0172] In some embodiments, the compounds of the present invention or their salts are substantially isolated. "Substantially isolated" means that the compounds are at least partially or substantially separated from the environment in which they were formed or detected. Partial separation can include, for example, compositions enriched in the compounds of the present invention. Substantial separation can include compositions containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds of the present invention or their salts.

[0173] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0174] As used herein, the expressions "ambient temperature" and "room temperature" are understood in the art and generally refer to a temperature about the same as the temperature of the room in which the reaction is carried out, e.g., the reaction temperature, e.g., from about 20° C. to about 30° C.

[0175] The compounds of the present invention also include pharma- ceutically acceptable salts of the compounds described herein. The term "pharma- ceutically acceptable salts" refers to derivatives of the disclosed compounds, in which the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharma- ceutically 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 pharma- ceutically acceptable salts of the present invention include non-toxic salts of the parent compound, for example, formed from non-toxic inorganic or organic acids. The pharma- ceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water, an organic solvent, or in a mixture of the two (generally with non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (MeCN) being preferred). A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 th Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19, and Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). In some embodiments, the compounds described herein include N-oxide forms.

[0176] synthesis The compounds of the present invention (including salts thereof) may be prepared using known organic synthesis techniques and may be synthesised according to any of a number of possible synthetic routes, for example those in the following schemes.

[0177] The reaction for preparing the compounds of the present invention can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, which can range, for example, from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction can be carried out in one solvent or a mixture of two or more solvents. Depending on the particular reaction step, a suitable solvent for a particular reaction step can be selected by those skilled in the art.

[0178] Preparation of the compounds of the invention may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one of ordinary skill in the art. The chemistry of protecting groups can be found, for example, in Kocienski, Protecting Groups, (Thieme, 2007); Robertson, Protecting Group Chemistry, (Oxford University Press, 2000); Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6 th Ed. (Wiley, 2007), Peturssion et al., "Protective Groups in Carbohydrate Chemistry," J. Chem. Educ., 1997, 74(11), 1297, and Wuts et al., Protective Groups in Organic Synthesis, 4th Ed., (Wiley, 2006).

[0179] The reaction can be monitored according to any suitable method known in the art. For example, product formation can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), spectroscopic means such as infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0180] Compounds of formula (I) can be prepared, for example, using the processes described below.

[0181] Peptide R 1 can be prepared using the solid phase synthesis method first described by Merrifield in JACS, Vol. 85, pgs. 2149-2154 (1963), although other methods known in the art can also be used. The Merrifield technique is well understood and is a common method for preparing peptides. Techniques useful for solid phase peptide synthesis are described in several books, such as the textbook Principles of Peptide Synthesis by Bodanszky (Springer Verlag 1984). This synthesis method involves the stepwise addition of protected amino acids to a growing peptide chain that is covalently attached to a solid resin particle. This procedure allows reagents and by-products to be removed by filtration, thus eliminating the need to purify intermediates. The general concept of this method relies on the attachment of the first amino acid of the chain to a solid polymer by a covalent bond, followed by the addition of subsequent protected amino acids, one by one, stepwise until the desired sequence is assembled. Finally, the protected peptide is removed from the solid resin support and the protecting groups are cleaved.

[0182] The amino acid may be attached to any suitable polymer. The polymer must be insoluble in the solvent used, have a stable physical form that allows easy filtration, and contain a functional group to which the original protected amino acid can be firmly attached by a covalent bond. A variety of polymers are suitable for this purpose, such as cellulose, polyvinyl alcohol, polymethyl methacrylate, and polystyrene.

[0183] The preparation of the various linkers provided herein is described in U.S. Pat. No. 10,933,069, as well as U.S. Application Publication Nos. 2021 / 0009536 and 2021 / 0009719.

[0184] The compounds of the invention can be prepared according to the following general schemes. [ka]

[0185] L is a thiol-containing moiety where the S atom of compound S-1 forms a disulfide bond with L. Compound S-1 adjacent to the orthogonal leaving group is a nucleophilic R 2 Compound S-2 can then be reacted with a thiol-containing peptide (R 1 -SH) to give compounds of formula (I).

[0186] How to use Provided herein is the use of compounds of formula (I) in the treatment of diseases such as cancer or neurodegenerative diseases. Another aspect of the present invention is the use of compounds of formula (I) in the treatment of diseases involving acidic or hypoxic diseased tissues, such as cancer. Hypoxia and acidosis are physiological markers of many disease processes, including cancer. In cancer, hypoxia is one mechanism involved in the development of an acidic environment within solid tumors. As a result, hydrogen ions must be removed from the cell (e.g., by proton pumps) to maintain a normal pH within the cell. As a result of this export of hydrogen ions, cancer cells have an increased pH gradient across the cell membrane lipid bilayer and a lower pH in the extracellular environment compared to normal cells. One approach to improve the efficacy and therapeutic index of cytotoxic agents is to exploit this physiological property to selectively deliver compounds to hypoxic cells over healthy tissues.

[0187] In these treatment methods, a therapeutically effective amount of the compound of formula (I) or its pharma- ceutically acceptable salt may be administered as a single agent or in combination with other forms of therapy, such as ionizing radiation or cytotoxic agents in the case of cancer. In combination therapy, as will be understood by those skilled in the art, the compound of formula (I) may be administered before, simultaneously with, or after the other treatment modality. Any treatment method (single agent or combination with other forms of therapy) may be administered as a course of treatment with multiple doses or treatments over a period of time.

[0188] In some embodiments, examples of cancers treatable using the compounds of the present disclosure include, but are not limited to, bladder cancer, bone cancer, glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, epithelial cancer, esophageal cancer, Ewing's sarcoma, pancreatic cancer, gallbladder cancer, gastric cancer, gastrointestinal tumors, head and neck cancer, intestinal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liver cancer, lung cancer, melanoma, prostate cancer, rectal cancer, renal clear cell carcinoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer. In some embodiments, the cancer is selected from lung cancer, colorectal cancer, and prostate cancer. In some embodiments, the lung cancer is non-small cell lung cancer.

[0189] Further examples of cancers treatable using the compounds of the present disclosure include Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), diffuse large B-cell lymphoma (DLBCL), ovarian cancer, urothelial carcinoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer, squamous non-small cell lung cancer (sqNSCLC), squamous head and neck cancer, non-Hodgkin's lymphoma, pancreatic cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), fallopian tube cancer, and peritoneal cancer.

[0190] Examples of cancers treatable using the compounds of the present disclosure include colorectal cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, and the like. Further examples of cancers include, but are not limited to, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, uterine cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or urethral cancer, renal pelvic cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos induced cancer, and combinations of the foregoing cancers.

[0191] In some embodiments, cancers treatable using the compounds of the present disclosure include bladder cancer, bone cancer, glioma, breast cancer (e.g., triple negative breast cancer), cervical cancer, colon cancer, colorectal cancer, endometrial cancer, epithelial cancer, esophageal cancer, Ewing's sarcoma, pancreatic cancer, gallbladder cancer, gastric cancer, gastrointestinal tumors, head and neck cancer (upper aerodigestive cancer), intestinal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, adenocarcinoma), melanoma, prostate cancer, rectal cancer, renal clear cell carcinoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer.

[0192] In some embodiments, cancers treatable using the compounds of the present disclosure include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, triple-negative breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer and small cell lung cancer). In addition, the present disclosure includes refractory or recurrent malignancies whose growth may be inhibited using the compounds of the present disclosure.

[0193] In some embodiments, cancers treatable using the compounds of the present disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, renal cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., leukemias such as lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic 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 foregoing cancers.

[0194] In certain embodiments, the compound of formula (I) or a pharma- ceutically acceptable salt thereof may be used in combination with a chemotherapeutic agent, a targeted cancer therapy, an immunotherapy, or a radiation therapy. These agents may be combined with the compound in a single dosage form, or these agents may be administered simultaneously or sequentially as separate dosage forms. In some embodiments, the chemotherapeutic agent, the targeted cancer therapy, the immunotherapy, or the radiation therapy may be administered in combination with a corresponding microtubule targeting agent (e.g., R 2 -H), the compound of formula (I) or a pharma- ceutical acceptable salt thereof is less toxic to the patient by exhibiting reduced bone marrow toxicity.

[0195] Suitable chemotherapeutic or other anti-cancer agents include, for example, alkylating agents (including, but not limited to, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, and triazenes) such as uracil mustard, chlormethine, cyclophosphamide (Cytoxan™), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

[0196] Other drugs suitable for use in combination with the compounds of the invention include dacarbazine (DTIC), optionally with other chemotherapeutic agents such as carmustine (BCNU) and cisplatin; the "Dartmouth regimen" consisting of DTIC, BCNU, cisplatin, and tamoxifen; a combination of cisplatin, vinblastine, and DTIC; or temozolomide. The compounds according to the invention may also be combined with immunotherapeutic agents, including cytokines such as interferon alpha, interleukin 2, and tumor necrosis factor (TNF).

[0197] Suitable chemotherapeutic or other anti-cancer agents include, for example, antimetabolites (including but not limited to, antifolates, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors), such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

[0198] Suitable chemotherapeutic or other anti-cancer agents further include, for example, certain natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), such as vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel (TAXOL™), mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, interferons (especially IFN-a), etoposide, and teniposide.

[0199] Other cytotoxic agents which may be administered in combination with the compounds of the invention include, for example, navelbene, CPT-11, anastrozole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, and droloxafine.

[0200] Also suitable are cytotoxic agents such as, for example, epidophyllotoxins; anti-tumor enzymes; topoisomerase inhibitors; procarbazine; mitoxantrone; platinum coordination complexes, such as cisplatin and carboplatin; biological response modifiers; growth inhibitory agents; anti-hormonal therapeutics; leucovorin; tegafur; and hematopoietic growth factors.

[0201] Other anti-cancer drug(s) include antibody therapeutics such as trastuzumab (Herceptin), antibodies against costimulatory molecules such as CTLA-4, 4-1BB, and PD-1, or antibodies against cytokines (IL-10, TGF-α, etc.).

[0202] Other anti-cancer drugs include those that block immune cell migration, such as antagonists against chemokine receptors, including CCR2 and CCR4.

[0203] Other anti-cancer drugs include those that boost the immune system, such as adjuvants or adoptive T-cell transfer.

[0204] Anti-cancer vaccines that can be administered in combination with the compounds of the invention include, for example, dendritic cells, synthetic peptides, DNA vaccines, and recombinant viruses.

[0205] Other suitable agents for use in combination with the compounds of the invention include chemotherapy combinations such as platinum-based doublets (cisplatin or carboplatin + gemcitabine; cisplatin or carboplatin + docetaxel; cisplatin or carboplatin + paclitaxel; cisplatin or carboplatin + pemetrexed) used in lung cancer and other solid tumors, or gemcitabine + paclitaxel conjugated particles (Abraxane®).

[0206] The compounds of the present invention can be effective in combination with antihormonal agents for the treatment of breast cancer and other tumors.Suitable examples are antiestrogens, including but not limited to tamoxifen and toremifene, aromatase inhibitors, including but not limited to letrozole, anastrozole and exemestane, adrenergic corticosteroids (e.g., prednisone), progestins (e.g., megastrol acetate), and estrogen receptor antagonists (e.g., fulvestrant).Suitable antihormonal agents used in the treatment of prostate cancer and other cancers can also be combined with the compounds of the present invention. These include antiandrogens, including but not limited to flutamide, bicalutamide, and nilutamide, luteinizing hormone-releasing hormone (LHRH) analogues, 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).

[0207] The compounds of the present invention may be combined or administered in sequence with other agents against membrane receptor kinases, especially for patients who have developed primary or acquired resistance to targeted therapy. These therapeutic agents include inhibitors or antibodies against EGFR, Her2, VEGFR, c-Met, Ret, IGFR1, or Flt-3, as well as inhibitors or antibodies against cancer-related fusion protein kinases such as Bcr-Abl and EML4-Alk. Inhibitors against EGFR include gefitinib and erlotinib, and inhibitors 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. c-Met inhibitors may be used in combination with the compounds of the present invention. These include onartumzumab, tivantnib, and INC-280. Agents directed against Abl (or Bcr-Abl) include imatinib, dasatinib, nilotinib, and ponatinib, and agents directed against Alk (or EML4-ALK) include crizotinib.

[0208] Angiogenesis inhibitors may be effective in some tumors in combination with the compounds of the present invention.These include antibodies against VEGF or VEGFR, or VEGFR kinase inhibitors.Antibodies against VEGF or other therapeutic proteins include bevacizumab and aflibercept.VEGFR kinase inhibitors and other antiangiogenesis inhibitors include, but are not limited to, sunitinib, sorafenib, axitinib, cediranib, pazopanib, regorafenib, brivanib, and vandetanib.

[0209] Activation of intracellular signaling pathways occurs frequently in cancer, and drugs that target the components of these pathways are combined with receptor targeting agents to enhance efficacy and reduce resistance.Examples of drugs that can be combined with the compounds of the present invention include PI3K-AKT-mTOR pathway inhibitors, Raf-MAPK pathway inhibitors, JAK-STAT pathway inhibitors, and protein chaperone and cell cycle progression inhibitors.

[0210] Drugs against PI3 kinase include, but are not limited to, piralalisib, idelalisib, buparlisib. mTOR inhibitors such as rapamycin, sirolimus, temsirolimus, and everolimus can be combined with the compounds of the present invention. Other suitable examples include, but are not limited to, vemurafenib and dabrafenib (Raf inhibitors), and trametinib, selumetinib, and GDC-0973 (MEK inhibitors). Inhibitors of one or more JAK (e.g., ruxolitinib, baricitinib, tofacitinib), Hsp90 (e.g., tanespimycin), cyclin-dependent kinase (e.g., palbociclib), HDAC (e.g., panobinostat), PARP (e.g., olaparib), and proteasome (e.g., bortezomib, carfilzomib) can also be combined with the compounds of the present invention. A further example of a PARP inhibitor that can be combined with the compounds of the invention is talazoparib.

[0211] Methods for safely and effectively administering most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the "Physicians' Desk Reference" (PDR, e.g., 1996 edition, Medical Economics Company, Montvale, NJ), the disclosure of which is incorporated herein by reference as if set forth in its entirety.

[0212] The phrase "therapeutically effective amount" of a compound (therapeutic agent, active ingredient, drug, etc.) refers to the amount of the compound administered to a subject in need of therapy or treatment that relieves symptoms, improves a condition, or delays the onset of a pathology according to clinically accepted criteria for the disorder or condition being treated. For example, a therapeutically effective amount can be an amount that has been demonstrated to have the desired therapeutic effect in an in vitro assay, an in vivo animal assay, or a clinical trial. A therapeutically effective amount can vary based on a number of factors, including the particular dosage form, method of administration, treatment protocol, the particular disease or condition being treated, the benefit / risk ratio, etc.

[0213] The therapeutically effective amount can be obtained from clinical trials, animal models, or in vitro cell culture assays. It is known in the art that the effective amount suitable for human use can be calculated from the effective amount determined from the animal model or in vitro cell culture assay. For example, as reported by Reagan-Shaw et al., FASEB J. 2008:22(3)659-61, "μg / ml" (effective amount based on in vitro cell culture assay) is equivalent to "mg / kg body weight / day" (effective amount for mice). Furthermore, based on the fact that the metabolic rate of mice is six times faster than that of humans, the effective amount for humans can be calculated from the effective amount for mice.

[0214] As an example of the treatment using the compound of formula (I) in combination with a cytotoxic agent, a therapeutically effective amount of the compound of formula (I) may be administered to a patient suffering from cancer as part of a treatment regimen that also includes a therapeutically effective amount of ionizing radiation or a cytotoxic agent.In the context of this treatment regimen, the term "therapeutically effective" amount should be understood to mean effective in combination therapy.Those skilled in the art of cancer treatment will understand how to adjust dosage to achieve optimal therapeutic results.

[0215] Similarly, appropriate dosages of the compounds of the invention for the treatment of non-cancerous diseases or conditions (such as cardiovascular disease) can be readily determined by those skilled in the medical arts.

[0216] The term "treat" as used herein includes administration of a compound or composition that reduces the frequency, delays the onset, or reduces the progression of diseases involving acidic or hypoxic diseased tissues, such as cancer, stroke, myocardial infarction, or long-term neurodegenerative diseases, in a subject compared to subjects to which the compound or composition is not administered. This includes reversing, reducing, or arresting the symptoms, clinical signs, or underlying pathology of a condition in a manner that improves or stabilizes the condition of the subject (e.g., in the case of cancer, regression of tumor growth, or reduction or amelioration of myocardial ischemia-reperfusion injury in myocardial infarction, stroke, or similar cardiovascular disease). The terms "inhibit" or "reduce" are used in cancer in reference to methods of inhibiting or reducing tumor growth (e.g., reducing tumor size) in a population compared to an untreated control population.

[0217] All publications (including patents) mentioned herein are incorporated by reference 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 disclosure set forth herein. The publications discussed throughout the specification are provided solely for their disclosure prior to the filing date of the present application.

[0218] Several types of ranges are disclosed herein. When any type of range is disclosed or claimed, the intention is to separately disclose or claim each possible number that such range (including the endpoints of the range, and any subranges and subrange combinations contained therein) can reasonably encompass. For example, when a range of therapeutically effective amounts of an active ingredient is disclosed or claimed, the intention is to separately disclose or claim all possible numbers that such range can encompass, consistent with the present disclosure herein. For example, it is disclosed or claimed by disclosure that a therapeutically effective amount of a compound can range from about 1 mg / kg to about 50 mg / kg (body weight of a subject).

[0219] Formulation, Dosage Forms, and Administration To prepare the pharmaceutical composition of the present invention, the compound of formula (I) or its pharma- ceutically acceptable salt is combined as an active ingredient intimately mixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, and the carrier can take various forms depending on the form of preparation desired for administration, such as oral or parenteral.When preparing the composition in oral dosage form, any of the usual pharmaceutical media, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc., can be used for oral liquid preparations, such as suspensions, elixirs, and solutions, or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, etc., can be used for oral solid preparations, such as powders, capsules, and tablets.Because of ease of administration, tablets and capsules are the most advantageous oral dosage unit forms, in which case, obviously, solid pharmaceutical carriers are used.If desired, tablets can be sugar-coated or enteric-coated by standard techniques. For parenteral preparations, the carrier usually comprises sterile water, but other ingredients, for example, ingredients to aid solubility or for preservation purposes, may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, and the like may be used. Those skilled in the pharmaceutical and medical fields will be able to easily determine the dosage of the pharmaceutical composition of the present invention suitable for the particular disease or condition being treated. EXAMPLES

[0220] mass spectrometry Mass spectrometry was performed on an Agilent 1260 Infinity II with a 6130B quadrupole MS and an Agilent 1290 Infinity II with a 6125B quadrupole MS.

[0221] Alternatively, Maldi-TOF (Matrix-Assisted Laser Desorption / Ionization-Time of Flight) mass spectrometry was measured on an Applied Biosystems Voyager System 6268. Samples were prepared as a matrix of α-cyanohydroxycinnamic acid on AB Science plates (part number V700666).

[0222] ESI (electrospray ionization) mass spectrometry was measured on either an Agilent 1100 series LC-MS equipped with a 1946 MSD or a Waters Xevo Qtof high resolution MS, both providing mass / charge species (m / z=3).

[0223] HPLC method HPLC were recorded on an Agilent 1260 Infinity II machine. HPLC methods are described in more detail in each example below, where appropriate.

[0224] Preparation of the linker The preparation of various linkers provided herein is described in U.S. Patent No. 10,933,069, as well as U.S. Application Publication Nos. 2021 / 0009536 and 2021 / 0009719. For example, the synthesis of the following linkers is described in U.S. Application Publication No. 2021 / 0009719: [Table 2]

[0225] [ka] Step 1: Synthesis of S-(2-hydroxycyclopentyl)ethanethioate To a stirred solution of 6-oxabicyclo[3.1.0]hexane (5 g, 59.4 mmol) in water (50 ml) was added thioacetic acid (4.98 g, 65.4 mmol) at room temperature. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with ethyl acetate. The organic layer was dried over Na2SO4 and evaporated to give S-(2-hydroxycyclopentyl)ethanethioate (6.1 g, 38.1 mmol, 64.0% yield) as a colorless liquid. The crude product was carried on to the next step without purification.

[0226] Step 2: Synthesis of 2-mercaptocyclopentan-1-ol To a stirred solution of S-(2-hydroxycyclopentyl)ethanethioate (6.1 g, 38.1 mmol) in THF (60 ml) was added dropwise 2.0 M LAH / THF solution (28.6 ml, 57.1 mmol) at 0° C. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was cooled to 0° C., quenched with 1.5 N aqueous HCl, and extracted with DCM. The organic layer was dried over Na2SO4 and evaporated to give 2-mercaptocyclopentan-1-ol (5 g, 42.3 mmol, 111% yield) as a colorless liquid. The crude product was carried on to the next step without purification. 1 H NMR(400MHz,DMSO-d6):δ 4.90(s,1H),3.78(s,1H),2.93-2.87(m,1H),2.44-2.42(m,1H),2.19- 2.05(m,1H),1.96-1.88(m,1H),1.69-1.67(m,2H),1.50-1.35(m,2H).

[0227] Step 3: 2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol To a stirred solution of 2-mercaptocyclopentan-1-ol (5 g, 42.3 mmol) in methanol (60 ml) was added 1,2-di(pyridin-2-yl)disulfane (13.98 g, 63.5 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was evaporated to dryness. Ice cold water was added and extracted with ethyl acetate. The organic layer was separated, washed with brine, dried over Na2SO4 and evaporated to give the crude residue. The crude residue was purified twice by flash column chromatography using 10% ethyl acetate / petroleum ether to give 2-(pyridin-2-yldisulfaneyl)cyclopentan-1-ol as a racemic mixture. LCMS: [M+H] + C 10 H 13Calculated for NOS2 227.04; Found 228.1 (M+H).SFC Chiral Purity: Column: Lux A1; Co-solvent: 40% MeOH; Flow rate: 4 mL / min; RT(min): 2.98; Area%: 49.92; RT(min): 4.26; Area%: 48.79.HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT(min): 5.76; Purity (max): 99.41%

[0228] SFC separation of isomeric (1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentan-1-ols (L-50 alcohols) and isomeric (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentan-1-ols (L-51 alcohols) The isomers were separated by SFC purification of racemic 2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol. The resulting SFC fraction isomer-1 (first eluting peak) was concentrated under reduced pressure at 30° C. to give (1R,2R)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol (L-50 alcohol) (1.2 g, 5.18 mmol, 12.25% yield) as a colorless oil. The absolute stereochemistry was assigned as described in Yamashita H., Bull. Chem. Soc. Jpn., 61, 1213-1220 (1988). LCMS: [M+H] + C 10 H 13 NOS2 calculated 227.04; found 228.1 (M+H).HPLC: Column: X-Bridge C8 (50×4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 2.0 mL / min; RT(min): 2.71; Purity(max): 98.19%.SFC Chiral Purity: Column: Lux A1; Co-solvent: 40% MeOH; Flow rate: 40 mL / min; RT(min): 2.94; Area%: 100.0. 1H NMR(400 MHz,CDCl3):δ 8.55(s,1H),7.65-7.61(m,1H),7.54-7.51(m,1H),7.21-7.17(m,1H),4.05-4.04(m,1H),3.03(t,J=8.00 Hz,1H),2.12-2.05(m,2H),1.72-1.64(m,5H).

[0229] Synthesis of precursor to linker L50 To a stirred solution of (1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentan-1-ol (1.1 g, 4.84 mmol) in DMF (10 ml) was added bis(4-nitrophenyl)carbonate (2.94 g, 9.68 mmol) and DIPEA (2.51 ml, 14.52 mmol) at room temperature. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with ice-cold water and extracted with ethyl acetate. The organic layer was washed with brine and dried over Na2SO4 to give the crude product. The crude product was purified by reverse phase chromatography using 0.1% HCOOH / H2O and ACN. The product fractions were concentrated under reduced pressure to give the pure product, which was lyophilized to give 4-nitrophenyl((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)carbonate (1.7 g, 4.32 mmol, 89% yield) as a pale yellow gum. LCMS: [M+H] + C 17 H 16 Calculated for N2O5S2 392.05; Found 392.9 (M+H).HPLC: Column: X-Bridge C8 (50×4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 2.0 mL / min; RT(min): 5.01; Purity (max): 99.73%.SFC Chiral Purity: Column: YMC Amylose-SA; Co-solvent: 30% IPA; Flow rate: 3 mL / min; RT(min): 4.26; Area%: 99.95. 1H NMR(400 MHz,CDCl3):δ 400 MHz,CDCl3:δ 8.50(s,1H),8.29-8.27(m,2H),7.71-7.65(m,2H),7.39-7.36(m,2H),7.14-7.11(m,1H),5.25(t,J=3.20 Hz,1H),3.60-3.55(m,1H),2.30-2.27(m,2H),2.03-1.79(m,3H),1.70-1.69(m,1H).

[0230] Synthesis of precursor to linker L51 To a stirred solution of (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol (1.1 g, 4.84 mmol) in DMF (10 ml) was added bis(4-nitrophenyl)carbonate (2.94 g, 9.68 mmol) and DIPEA (2.51 ml, 14.52 mmol) at room temperature. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with ice-cold water and extracted with ethyl acetate. The organic layer was washed with brine and dried over Na2SO4 to give the crude product. The crude product was purified by reverse phase chromatography using 0.1% HCOOH / H2O and ACN. The product fractions were concentrated under reduced pressure to give the pure product, which was lyophilized to give 4-nitrophenyl((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)carbonate (1.7 g, 4.24 mmol, 88% yield) as a pale yellow gum. LCMS: [M+H] + C 17 H 16 Calculated for N2O5S2 392.05; Found 392.8 (M+H).HPLC: Column: X-Bridge C8 (50×4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 2.0 mL / min; RT(min): 5.01; Purity(max): 97.86%.SFC Chiral Purity: Column: YMC Amylose-SA; Co-solvent: 30% IPA; Flow rate: 3 mL / min; RT(min): 3.56; Area%: 99.74. 1H NMR(400 MHz,CDCl3):δ 400 MHz,CDCl3:δ 8.50(s,1H),8.29-8.27(m,2H),7.71-7.65(m,2H),7.39-7.36(m,2H),7.14-7.11(m,1H),5.25(t,J=3.20 Hz,1H),3.60-3.55(m,1H),2.30-2.27(m,2H),2.03-1.79(m,3H),1.70-1.69(m,1H).

[0231] Alternative synthesis of linker L51 Linker L51 can be prepared according to the enzymatic chiral resolution process disclosed in International Application No. WO2022 / 150596 (see, for example, Example 11 of International Application No. WO2022 / 150596), which is incorporated herein in its entirety.

[0232] Synthesis of Compounds of the Disclosure Example 1: Synthesis of Compound 1 [ka] Step 1: Synthesis of (1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3) To a stirred solution of (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide (150 mg, 0.209 mmol) in DMF (1 mL) was added 4-nitrophenyl((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)carbamate (L50) (98 mg, 0.251 mmol) at 0° C. Then, 1-hydroxy-7-azabenzotriazole / 1M DMA solution (0.104 ml, 0.104 mmol) and DIPEA (0.054 ml, 0.313 mmol) were added and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by fractionation using 0.1% HCOOH / HO and ACN. The product fractions were lyophilized to give (1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (138 mg, 0.140 mmol, 67.1% yield) as a white solid. LCMS: [M+H] + C 50 H 78 Calculated for N6O9S2 970.53; Found 971.3 (M+H).HPLC: Column: X-Bridge C8 (50×4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 2.0 mL / min; RT(min): 5.49; Purity(max): 98.69%.

[0233] Step 2: Synthesis of Compound 1 (1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxopropyl To a stirred solution of (3-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (115 mg, 0.118 mmol) in DMF (1.5 ml) was added Pv1 peptide (425.6 mg, 0.130 mmol) and triethylamine (0.02 ml, 0.141 mmol) at 0° C. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The product fractions were lyophilized to give compound 1 (205 mg, 0.049 mmol, 41.5% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 197 H 299 F6N 41 O 52 S2 calculated 4135.15; found 1380.3 (M+3) / 3. HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT(min): 12.29; Purity(max): 99.69%

[0234] Example 2: Synthesis of Compound 2 [ka] Step 1: (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate To a stirred solution of (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide (180 mg, 0.251 mmol) in DMF (1 mL) was added 4-nitrophenyl((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)carbamate (L51) (118 mg, 0.301 mmol) at 0 °C. Then, 1-hydroxy-7-azabenzotriazole / 1M DMA solution (0.125 ml, 0.125 mmol) and DIPEA (0.066 ml, 0.376 mmol) were added and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was purified by fractionation using 0.1% HCOOH / HO and ACN. The product fractions were lyophilized to give (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (200 mg, 0.251 mmol, 79% yield) as a white solid. LCMS: [M+H] + C 50 H 78 Calculated for N6O9S2 970.53; Found 971.4 (M+H).Column: X-Bridge C8 (50×4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 2.0 mL / min; RT(min): 5.48; Purity(max): 95.59%.

[0235] Step 2: Synthesis of compound 2 (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxo To a stirred solution of 2-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (200 mg, 0.206 mmol) in DMF (2 ml) was added Pv1 peptide (742.9 mg, 0.226 mmol) and triethylamine (0.035 ml, 0.247 mmol) at 0° C. The reaction mixture was stirred at room temperature for 1 h 30 min. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The product fractions were lyophilized to give compound 2 (410 mg, 0.099 mmol, 48% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 197 H 299 F6N 41 O 52 S2 calculated 4135.15; found 1380.0 (M+3) / 3. HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT (min): 11.94; Purity (max): 99.66%

[0236] Example 3: Synthesis of Compound 3 [ka] Step 1: Synthesis of (1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl(4-hydroxymethyl)phenyl)carbamate A stirred solution of (4-aminophenyl)methanol (120 mg, 0.974 mmol) and 4-nitrophenyl((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)carbamate (L50) (382 mg, 0.974 mmol) in DMF (1 ml) was cooled on ice. To the above solution was added HOBt (65.8 mg, 0.487 mmol) and DIPEA (0.338 ml, 1.949 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ice-cold water, extracted with ethyl acetate and washed with brine. The organic layer was concentrated to give a crude residue. The crude residue was purified by flash column chromatography using 40% ethyl acetate / petroleum ether to give (1R,2R)-2-(pyridin-2-yldisulfanyl)cyclopentyl(4-(hydroxymethyl)phenyl)carbamate (350 mg, 0.876 mmol, 90% yield) as a brown gum. LCMS: [M+H] + C 18 H 20 Calculated for F6N2O3S2: 376.09; Found: 377.6 (M+H). 1 H NMR(400 MHz,DMSO-d6):δ 9.61(s,1H),8.45(d,J=4.80 Hz,1H),7.79-7.77(m,2H),7.39-7.22(m,2H),7.19-7.14(m,3H),5.08(t,J=5.60 Hz,1H),5.00(s,1H),4.41(d,J=5.60 Hz,2H),3.51-3.34(m,1H),2.17-2.00(m,2H),1.78-1.67(m,4H).

[0237] Step 2: Synthesis of (1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl (4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate To a stirred solution of (1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl(4-(hydroxymethyl)phenyl)carbamate (0.35 g, 0.930 mmol) in DMF (5 ml) was added bis(4-nitrophenyl)carbamate (1.131 g, 3.72 mmol) and DIPEA (0.242 ml, 1.394 mmol) at room temperature. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with ice-cold water and extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure to give a crude residue. The crude residue was purified by flash column chromatography using 20% ​​ethyl acetate / petroleum ether to give (1R,2R)-2-(pyridin-2-yldisulfanyl)cyclopentyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (420 mg, 0.740 mmol, 80% yield) as a brown gum. LCMS: [M+H] + C 25 H 23 Calculated for N3O7S2: 541.10; Found: 542.1 (M+H). 1 H NMR(400 MHz,DMSO-d6):δ 9.78(s,1H),8.45(d,J=5.20 Hz,1H),8.33-8.31(m,2H),7.79-7.77(m,2H),7.59-7.56(m,2H),7.49-7.4 7(m,2H),7.39-7.37(m,2H),7.24-7.21(m,1H),5.23(s,2H),5.03(t,J=2.40 Hz,1H),3.52-3.51(m,1H),2.20-2.10(m,2H),1.78-1.68(m,4H).

[0238] Step 3: Synthesis of 4-(((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino) To a stirred solution of (1R,2R)-2-(pyridin-2-yldisulfanyl)cyclopentyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (113 mg, 0.209 mmol), 1-hydroxy-7-azabenzotriazole / 1M DMA solution (0.104 ml, 0.104 mmol), and DIPEA (0.054 ml, 0.313 mmol) were added at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The product fractions were lyophilized to give 4-(((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (150 mg, 0.133 mmol, 63.6% yield) as a white solid. LCMS: [M+H] + C 58 H 85 N7O 11S2 calculated 1119.57; found 1121.4 (M+H). HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT (min): 13.61; Purity (max): 99.26%.

[0239] Step 4: Synthesis of compound 3 4-(((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3 To an ice-cold solution of 3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (60 mg, 0.054 mmol) in DMF (0.5 ml) was added Pv1 peptide (176 mg, 0.054 mmol) and triethylamine (8.96 μl, 0.064 mmol). The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The product fractions were lyophilized to give compound 3 (65 mg, 0.015 mmol, 27.8% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 205 H 306 N 42 O 54 S2 calculated 4284.19; found 1430.2 (M+3) / 3. HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT(min): 12.20; Purity(max): 99.84%.

[0240] Example 4: Synthesis of Compound 4 [ka] Step 1: Synthesis of (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl(4-(hydroxymethyl)phenyl)carbamate A stirred solution of (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl 2-(4-nitrophenyl)acetate (L-51) (380 mg, 0.974 mmol) and (4-aminophenyl)methanol (120 mg, 0.974 mmol) in DMF (2.5 ml) was cooled to 0° C. Then, DIPEA (0.339 ml, 1.949 mmol) was added at 0° C., followed by HOBt (74.6 mg, 0.487 mmol). The reaction mixture was stirred at room temperature for 18 h. Ice-cold water was added to the reaction mixture and extracted with ethyl acetate. The ethyl acetate layer was dried over Na2SO4 and concentrated under reduced pressure to give the crude product. The crude product was purified by flash column chromatography using 50% EtOAc / petroleum ether as eluent. The product fractions were evaporated under reduced pressure to give (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclopentyl(4-(hydroxymethyl)phenyl)carbamate (304 mg, 0.798 mmol, 82% yield) as a brown gum. LCMS: [M+H] + C 18 H 20 Calculated for F6N2O3S2: 376.09; Found: 377.1 (M+H). 1 H NMR(400 MHz,DMSO-d6):δ 9.61(s,1H),8.45(d,J=4.80 Hz,1H),7.79-7.77(m,2H),7.39-7.22(m,2H),7.19-7.14(m,3H),5.08(t,J=5.60 Hz,1H),5.00(s,1H),4.41(d,J=5.60 Hz,2H),3.51-3.34(m,1H),2.17-2.00(m,2H),1.78-1.67(m,4H).

[0241] Step 2: Synthesis of (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl (4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate To a solution of (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl(4-(hydroxymethyl)phenyl)carbamate (300 mg, 0.797 mmol) and bis(4-nitrophenyl)carbamate (970 mg, 3.19 mmol) in DMF (5 ml) was added DIPEA (0.208 ml, 1.195 mmol) at 0° C. and the reaction mixture was stirred at room temperature for 18 h. Ice-cold water was added to the reaction mixture and extracted with ethyl acetate. The ethyl acetate layer was washed with cold water, brine, dried over Na2SO4 and concentrated under reduced pressure to give the crude product. The crude product was purified by flash column chromatography using 25% ethyl acetate / petroleum ether as eluent. The product fractions were concentrated under reduced pressure to give (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclopentyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (330 mg, 0.599 mmol, 75% yield) as a yellow gummy solid. LCMS: [M+H] + C 25 H 23 Calculated for N3O7S2: 541.10; Found: 542.0 (M+H). 1 H NMR(400 MHz,DMSO-d6):δ 9.78(s,1H),8.45(d,J=5.20 Hz,1H),8.31-8.33(m,2H),7.81-7.77(m,1H),7.57(d,J=8.80 Hz,2H),7.48(d,J=8.40 Hz,2H),7.38(d,J=8.40 Hz,2H),7.24-7.21(m,1H),5.23(s,2H),5.03(t,J=2.40 Hz,1H),3.54-3.49(m,1H),2.20-2.10(m,2H),1.80-1.67(m,4H).

[0242] Step 3: Synthesis of 4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino) To a stirred solution of (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (113 mg, 0.209 mmol) in DMF (1 ml) was added 1-hydroxy-7-azabenzotriazole / 1M DMA solution (0.104 ml, 0.104 mmol) and DIPEA (0.055 ml, 0.313 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The product fractions were lyophilized to give 4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (150 mg, 0.129 mmol, 61.9% yield) as a white solid. LCMS: [M+H] + C 58 H 85 N7O 11 S2 calculated 1119.57; found 1120.6 (M+H). HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT (min): 13.60; Purity (max): 96.55%.

[0243] Step 4: Synthesis of compound 4 4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-meth To a stirred solution of 5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (70 mg, 0.062 mmol) and Pv1 peptide (203.2 mg, 0.062 mmol) in DMF (0.75 ml) was added triethylamine (10.45 μl, 0.074 mmol) at 0° C. The reaction mixture was stirred at room temperature for 26 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The product fractions were lyophilized to give compound 4 (41 mg, 9.56 μmol, 15.4% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 205 H 306 N 42 O 54 S2 calculated 4284.19; found 1430.1 (M+3) / 3. HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT(min): 12.33; Purity(max): 99.61%.

[0244] Example 5: Synthesis of Compound 5 [ka] Step 1: Synthesis of (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl(4-(hydroxymethyl)phenyl)carbamate A stirred solution of (4-aminophenyl)methanol (20 mg, 0.162 mmol) and 4-nitrophenyl((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl)carbamate (79 mg, 0.195 mmol) in DMF (1 ml) was cooled on ice. DIPEA (0.057 ml, 0.325 mmol) and 1H-benzo[d][1,2,3]triazol-1-ol (10.97 mg, 0.081 mmol) were added and the reaction mixture was stirred at room temperature for 36 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4 and concentrated to give a crude residue. The crude residue was purified by flash column chromatography using 75-80% ethyl acetate / petroleum ether as eluent. The product fractions were evaporated to give (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl(4-(hydroxymethyl)phenyl)carbamate (40 mg, 0.090 mmol, 55.4% yield) as a gummy solid. LCMS: [M+H] + C 19 H 22 Calculated for N2O3S2 390.11; Found 391.1 (M+H). HPLC: Column: X-Bridge C8 (50×4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 2.0 mL / min; RT (min): 3.80; Purity (max): 87.78%.

[0245] Step 2: Synthesis of (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate To a stirred solution of (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl(4-(hydroxymethyl)phenyl)carbamate (20 mg, 0.051 mmol) in DMF (1 ml) was added bis(4-nitrophenyl)carbamate (62.3 mg, 0.205 mmol) and DIPEA (0.013 ml, 0.077 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. Ice-cold water was added to the reaction mixture and extracted with ethyl acetate. The ethyl acetate layer was dried over Na2SO4 and concentrated under reduced pressure to give the crude product. The crude product was purified by flash column chromatography using 15% ethyl acetate and petroleum ether as eluent. The product fractions were concentrated under reduced pressure to give (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (12 mg, 0.021 mmol, 40.3% yield) as a gummy solid. LCMS: [M+H] + C 26 H 25 Calculated for N3O7S2: 555.11; Found: 555.9 (M+H).

[0246] Step 3: Synthesis of (4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl)oxy)carbonyl)amino)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate A solution of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide (15.51 mg, 0.022 mmol) and (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (12 mg, 0.022 mmol) in DMF (0.8 ml) was cooled on ice. To this was added DIPEA (5.66 μl, 0.032 mmol) and 1-hydroxy-7-azabenzotriazole / 1M DMA solution (10.80 μl, 10.80 μmol) and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The product fractions were lyophilized to give 4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl)oxy)carbonyl)amino)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (15 mg, 0.013 mmol, 60.5% yield) as a white solid. LCMS: [M+H] + C 59 H 87 N7O 11 S2 calculated 1133.59; found 1135.1 (M+H). HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT (min): 15.62; Purity (max): 98.83%.

[0247] Step 4: Synthesis of compound 5 4-(((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclohexyl)oxy)carbonyl)amino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl A solution of )pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (15 mg, 0.013 mmol) and Pv1 peptide (47.7 mg, 0.015 mmol) in DMF (0.5 ml) was cooled on ice. To this was added triethylamine (2.211 μl, 0.016 mmol) and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The product fraction was lyophilized to give compound 5 (42 mg, 9.58 μmol, 72.5% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 206 H 308 N 42 O 54 S2 calculated 4298.21; found 1435.0 (M+3) / 3. HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT (min): 12.93; Purity (max): 98.12%.

[0248] Example 6: Synthesis of Compound 6 [ka] Step 1: Synthesis of (4-(methylamino)phenyl)methanol To a stirred solution of methyl 4-(methylamino)benzoate (0.2 g, 1.211 mmol) in THF (2 ml) was added LAH 2M in THF (0.726 ml, 1.453 mmol) at 0° C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NH4Cl solution. The ethyl acetate layer was separated, concentrated and purified by flash column chromatography using 20% ​​ethyl acetate / petroleum ether. The product fractions were evaporated to give (4-(methylamino)phenyl)methanol (150 mg, 0.847 mmol, 70.0% yield) as a yellow liquid. LCMS: [M+H] + C8H 11 Calculated NO 137.08; Found 138.2 (M+H). 1 H NMR(400 MHz,DMSO-d6):δ 7.03(d,J=8.40 Hz,2H),6.50-6.50(m,2H),5.49-5.48(m,1H),4.33(t,J=5.60 Hz,1H),4.04(d,J=6.80 Hz,2H),2.66(s,3H).

[0249] Step 2: Synthesis of (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl(4-(hydroxymethyl)phenyl)(methyl)carbamate To a stirred solution of (4-(methylamino)phenyl)methanol (30 mg, 0.219 mmol) in DMF (2 ml) was added 4-nitrophenyl((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl)carbamate (107 mg, 0.262 mmol), DIPEA (0.076 ml, 0.437 mmol) and 1H-benzo[d][1,2,3]triazol-1-ol (14.78 mg, 0.109 mmol) at 0° C. The reaction mixture was stirred at 80° C. for 18 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over Na2SO4 and concentrated to give a crude residue. The crude residue was purified by flash column chromatography. The product was eluted with 35% ethyl acetate / petroleum ether. The product fractions were evaporated to give (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl(4-(hydroxymethyl)phenyl)(methyl)carbamate (40 mg, 0.057 mmol, 26.1% yield) as a yellow liquid. LCMS: [M+H] + C 20 H 24 Calculated for N2O3S2: 404.12; Found: 405.1 (M+H).

[0250] Step 3: Synthesis of (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl methyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate To a stirred solution of (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl(4-(hydroxymethyl)phenyl)(methyl)carbamate (40 mg, 0.099 mmol) in DMF (1 ml) was added bis(4-nitrophenyl)carbamate (120 mg, 0.396 mmol) and DIPEA (0.035 ml, 0.198 mmol) at 0° C. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was diluted with ice-cold water and extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure to give a crude residue. The crude residue was purified by flash column chromatography. The product was eluted with 20% ethyl acetate / petroleum ether. The product fractions were evaporated to give (1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl methyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (25 mg, 0.044 mmol, 44.3% yield) as a colorless gummy solid. LCMS: [M+H] + C 27 H 27 Calculated for N3O7S2: 569.13; Found: 570.1 (M+H).

[0251] Step 4: Synthesis of 4-(methyl((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl)oxy)carbonyl)amino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide (25 mg, 0.035 mmol) and (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl To a stirred solution of methyl (4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (19.83 mg, 0.035 mmol) in DMF (1 ml) was added DIPEA (9.12 μl, 0.052 mmol) and 1-hydroxy-7-azabenzotriazole / 1M DMA solution (0.017 ml, 0.017 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The crude reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The product fractions were lyophilized to give 4-(methyl((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl)oxy)carbonyl)amino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)-2-propane-4-yl)-2-methyl-2-propane-2-yl)-2-propane-2-yl)-2-methyl ... yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (33 mg, 0.026 mmol, 74.8% yield). LCMS: [M+H] + C 60 H 89 N7O 11 S2 calculated 1147.61; found 1149.6 (M+H).

[0252] Step 5: Synthesis of compound 6 A solution of (1R,2R)-2-(pyridin-2-yldisulfanayl)cyclohexyl(4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)(methyl)carbamate (23 mg, 0.020 mmol) and Pv1 peptide (72.2 mg, 0.022 mmol) in DMF (1 ml) was cooled on ice. To this was added triethylamine (2.432 mg, 0.024 mmol). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The product fractions were lyophilized to give compound 6 (45 mg, 10.03 μmol, 50.1% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 207 H 310 N 42 O 54 S2 calculated 4312.22; found 1437.7 (M-3) / 3. HPLC: Column: Atlantis dC18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.1% TFA / H2O; Mobile phase: B: 0.1% TFA / ACN; Flow rate: 1.0 mL / min; RT(min): 12.66; Purity(max): 96.18%.

[0253] The following compounds in Table 2 were prepared using the procedures described in the Examples above. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0254] Example 21: Synthesis of Compound 21 [ka] Step 1: Synthesis of trans-4-(pyridin-2-yldisulfanayl)cyclohexyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide To a stirred solution of 1,3-dihydro-2,4-triphenyltrimethylsilyl (20 mg, 0.028 mmol) and 4-nitrophenyl(trans-4-(pyridin-2-yldisulfanayl)cyclohexyl)carbamate (11.32 mg, 0.028 mmol) in DMF (0.5 ml) was added DIPEA (7.3 μL, 0.042 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (13.92 μL, 13.92 μmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give trans-4-(pyridin-2-yldisulfanyl)cyclohexyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (19 mg, 0.019 mmol, 69.2% yield) as a white solid. LCMS: [M+H] + C 51 H 80 Calculated value of N6O9S2: 985.34; Found value: 985.3.

[0255] Step 2: Synthesis of compound 21 A solution of trans-4-(pyridin-2-yldisulfanyl)cyclohexyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (19 mg, 0.019 mmol) in DMF (0.5 ml) was cooled to 0° C. Pv1 peptide (69.54 mg, 0.021 mmol) and triethylamine (3.22 μl, 0.023 mmol) were added and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The fractions were lyophilized to give compound 21 (80 mg, 0.019 mmol, 99.9% yield) as a white solid. The product obtained was the di-TFA salt. LCMS: [M+H] + C 198 H 301 N 41 O 52 S2 calculated 4151.941; found 1384.8 [(M+3) / 3]; HPLC: Column Atlantis dC18 (250×4.6) mm, 5 μm, Mobile phase A: 0.1% TFA / milli-Q water, Mobile phase B: ACN; Flow rate: 1.0 mL / min; RT (min): 11.826; Purity (max): 99.71%.

[0256] Example 22: Synthesis of Compound 22 [ka] Step 1: 4-(pyridin-2-yldisulfanayl)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide To a stirred solution of tanamide (50 mg, 0.069 mmol) and 4-nitrophenyl(4-(pyridin-2-yldisulfanayl)benzyl)carbamate (37.52 mg, 0.090 mmol) in DMF (1 ml) was added DIPEA (24.13 μl, 0.014 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (0.47 ml, 0.035 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give 4-(pyridin-2-yldisulfanayl)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (22 mg, 0.022 mmol, 31.80% yield) as a white solid. LCMS: [M+H] + C52 H 76 Calculated value of N6O9S2: 993.333; Found value: 994.5.

[0257] Step 2: Synthesis of compound 22 4-(Pyridin-2-yldisulfanyl)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl) A solution of (methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (22 mg, 0.022 mmol) in DMF (1 ml) was cooled to 0° C., Pv1 peptide (80 mg, 0.024 mmol) and triethylamine (12.34 μl, 0.088 mmol) were added, and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The fractions were lyophilized to give compound 22 (40 mg, 0.022 mmol, 43.41% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 199 H 297 N 41 O 52 S2 calculated value 4159.920; Found value 1385.1 [(M-3) / 3]; HPLC: Column X-Bridge C8 (50×4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA / water, Mobile phase: B: 0.1% TFA / ACN, Flow rate: 2.0 mL / min; RT(min): 5.52; Purity(max): 99.147%.

[0258] Example 23: Synthesis of Compound 23 [ka] Step 1: (S)-2-(pyridin-2-yldisulfanayl)propyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butane To a stirred solution of amide (50 mg, 0.069 mmol) and (S)-4-nitrophenyl(2-(pyridin-2-yldisulfanayl)propyl)carbamate (30.61 mg, 0.090 mmol) in DMF (1 ml) was added DIPEA (24.13 μl, 0.014 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (0.47 ml, 0.035 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give (S)-2-(pyridin-2-yldisulfanyl)propyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (40 mg, 0.041 mmol, 60.77% yield) as a white solid. LCMS: [M+H]+ C 48 H 76 Calculated value of N6O9S2: 945.289; Found value: 945.5.

[0259] Step 2: Synthesis of compound 23 A solution of (S)-2-(pyridin-2-yldisulfanyl)propyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (40 mg, 0.042 mmol) in DMF (1 ml) was cooled to 0° C. Pv1 peptide (152 mg, 0.046 mmol) and triethylamine (23.59 μl, 0.169 mmol) were added and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The fractions were lyophilized to give compound 23 (126.3 mg, 0.030 mmol, 72.59% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 195 H 297 N 41 O 52 S2 calculated value 4111.876; found value 1371.1 [(M+3) / 3]; HPLC: Column X-Bridge C8 (50×4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA / water, Mobile phase: B: 0.1% TFA / ACN, Flow rate: 2.0 mL / min; RT(min): 5.43; Purity(max): 98.857%.

[0260] Example 24: Synthesis of Compound 24 [ka] Step 1: (R)-2-(pyridin-2-yldisulfanayl)propyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide To a stirred solution of (R)-4-nitrophenyl(2-(pyridin-2-yldisulfanayl)propyl)carbamate (30.619 mg, 0.083 mmol) in DMF (1 ml) was added DIPEA (24.13 μL, 0.014 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (0.47 ml, 0.035 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give (R)-2-(pyridin-2-yldisulfanyl)propyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (40 mg, 0.042 mmol, 60.77% yield) as a white solid. LCMS: [M+H]+ C 48 H 76 Calculated value of N6O9S2: 945.289; Found value: 946.4.

[0261] Step 2: Synthesis of compound 24 A solution of (R)-2-(pyridin-2-yldisulfanyl)propyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (40 mg, 0.042 mmol) in DMF (1 ml) was cooled to 0° C. Pv1 peptide (138.73 mg, 0.042 mmol) and triethylamine (11.79 μl, 0.084 mmol) were added and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The fractions were lyophilized to give compound 24 (40 mg, 0.01 mmol, 22.98% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 195 H 297 N 41 O 52 S2 calculated value 4111.876; found value 1369.1 [(M-3) / 3]; HPLC: Column X-Bridge C8 (50×4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA / water, Mobile phase: B: 0.1% TFA / ACN, Flow rate: 2.0 mL / min; RT(min): 5.43; Purity(max): 98.413%.

[0262] Example 25: Synthesis of Compound 25 [ka] Step 1: ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (100 mg, 0.137 mmol) and (1S,2S)-2-(pyridine To a stirred solution of 1-(2-yldisulfanyl)cyclopentyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (74 mg, 0.137 mmol) in DMF (1 ml) was added DIPEA (0.036 ml, 0.205 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (0.068 ml, 0.068 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (80 mg, 0.066 mmol, 51.61% yield) as a white solid. LCMS: [M+H] + C 58 H 83 N7O 12Calculated S2 value 1134.459; measured value 1134.5.

[0263] Step 2: Synthesis of compound 25 A solution of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (50 mg, 0.044 mmol) in DMF (1 ml) was cooled to 0° C. PV1 peptide (145 mg, 0.044 mmol) and triethylamine (7.37 μl, 0.053 mmol) were added and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The fractions were lyophilized to give compound 25 (40 mg, 0.01 mmol, 21.10% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 205 H 304 N 42 O 55 S2 calculated 4301.046; found 1434.8 [(M+3) / 3]; HPLC: Column Atlantis dC18 (250×4.6) mm, 5 μm, Mobile phase A: 0.1% TFA / milli-Q water, Mobile phase B: ACN; Flow rate: 1.0 mL / min; RT (min): 12.056; Purity (max): 99.47%.

[0264] Example 26: Synthesis of Compound 26 [ka] Step 1: ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-(((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (40 mg, 0.054 mmol) and (1R,2R)-2-(pyridin-2-yl To a stirred solution of (4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (29.59 mg, 0.054 mmol) in DMF (1 ml) was added DIPEA (14.20 μl, 0.082 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (2.73 μl, 0.027 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-(((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (35 mg, 0.031 mmol, 56.46% yield) as a white solid. LCMS: [M+H] + C 58 H 83 N7O 12S2 calculated value 1134.459; measured value 1133.8.

[0265] Step 2: Synthesis of compound 26 A solution of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-(((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (35 mg, 0.031 mmol) in DMF (1 ml) was cooled to 0° C. Pv1 peptide (101.15 mg, 0.031 mmol) and triethylamine (5.16 μl, 0.037 mmol) were added and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The fractions were lyophilized to give compound 26 (15 mg, 0.003 mmol, 11.30% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 205 H 304 N 42 O 55 S2 calculated 4301.046; found 1434.5 [(M+3) / 3]; HPLC: Column Atlantis dC18 (250×4.6) mm, 5 μm, Mobile phase A: 0.1% TFA / milli-Q water, Mobile phase B: ACN; Flow rate: 1.0 mL / min; RT (min): 12.243; Purity (max): 99.10%.

[0266] Example 27: Synthesis of Compound 27 [ka] Step 1: ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-((((R)-3-methyl-2-(pyridin-2-yldisulfanayl)butoxy)carbonyl)amino)￥benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (40 mg, 0.054 mmol) and (R)-3-methyl-2-(pyridin-2-yl To a stirred solution of (disulfanyl)butyl (4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (1) (29.70 mg, 0.054 mmol) in DMF (1 ml) was added DIPEA (10.59 μl, 0.082 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (3.71 μl, 0.027 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-((((R)-3-methyl-2-(pyridin-2-yldisulfanayl)butoxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (35 mg, 0.029 mmol, 56.36% yield) as a white solid. LCMS: [M+H] + C 58 H 85 N7O 12S2 calculated value 1136.475; measured value 1136.5.

[0267] Step 2: Synthesis of compound 27 A solution of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-((((R)-3-methyl-2-(pyridin-2-yldisulfanayl)butoxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (35 mg, 0.031 mmol) in DMF (1 ml) was cooled to 0° C. Pv1 peptide (100.9 mg, 0.031 mmol) and triethylamine (5.15 μl, 0.037 mmol) were added and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The fractions were lyophilized to give compound 27 (64 mg, 0.014 mmol, 47.22% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 205 H 306 N 42 O 55 S2 calculated 4303.062; found 1435.4 [(M+3) / 3]; HPLC: Column X-Bridge C8 (50×4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA / water, Mobile phase: B: 0.1% TFA / ACN, Flow rate: 2.0 mL / min; RT(min): 5.83; Purity(max): 96.842%.

[0268] Example 28: Synthesis of Compound 28 [ka] Step 1: ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl To a stirred solution of (1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (38.71 mg, 0.069 mmol) in DMF (1 ml) was added DIPEA (18.10 μl, 0.104 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (3.48 μl, 0.034 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (33 mg, 0.029 mmol, 41.23% yield) as a white solid. LCMS: [M+H] + C 59 H 85 N7O 12S2 calculated value 1148.486; measured value 1148.5.

[0269] Step 2: Synthesis of compound 28 A solution of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-(((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclohexyl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (33 mg, 0.029 mmol) in DMF (1 ml) was cooled to 0° C. Pv1 peptide (103.63 mg, 0.031 mmol) and triethylamine (4.80 μl, 0.034 mmol) were added and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The fractions were lyophilized to give compound 28 (75 mg, 0.017 mmol, 60.49% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 206 H 306 N 42 O 55 S2 calculated 4315.073; found 1439.3 [(M+3) / 3]; HPLC: Column Atlantis dC18 (250×4.6) mm, 5 μm, Mobile phase A: 0.1% TFA / milli-Q water, Mobile phase B: ACN; Flow rate: 1.0 mL / min; RT (min): 12.516; Purity (max): 99.59%.

[0270] Example 29: Synthesis of Compound 29 [ka] Step 1: ((2R,3R)-3-((R)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((R)-3-methyl-2-(pyridin-2-yldisulfanayl)butoxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine To a stirred solution of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (230 mg, 0.314 mmol) and (R)-3-methyl-2-(pyridin-2-yldisulfanayl)butyl(4-nitrophenyl)carbamate (124 mg, 0.314 mmol) in DMF (1 ml) was added DIPEA (0.11 ml, 0.628 mmol) followed by the 1-hydroxy-7-azabenzotriazole / DMA solution (0.157 ml, 0.157 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give ((2R,3R)-3-((R)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((R)-3-methyl-2-(pyridin-2-yl disulfanayl)butoxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (150 mg, 0.148 mmol, 48.35% yield) as a white solid. LCMS: [M+H] + C 50 H 78 NO 10 S2 calculated value 987.326; measured value 986.4 (MH)

[0271] Step 2: Synthesis of compound 29 A solution of ((2R,3R)-3-((R)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((R)-3-methyl-2-(pyridin-2-yldisulfanayl)butoxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (150 mg, 0.152 mmol) in DMF (1 ml) was cooled to 0° C. Pv1 peptide (498 mg, 0.152 mmol) and triethylamine (41.8 μl, 0.304 mmol) were added and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / HO and ACN. The fractions were lyophilized to give compound 29 (425 mg, 0.101 mmol, 67.34% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 197 H 299 N 41 O 53 S2 calculated 4153.913; found 1385.7 [(M+3) / 3]; HPLC: Column Atlantis dC18 (250×4.6) mm, 5 μm, Mobile phase A: 0.1% TFA / milli-Q water, Mobile phase B: ACN; Flow rate: 1.0 mL / min; RT (min): 12.257; Purity (max): 98.793%.

[0272] Example 30: Synthesis of Compound 30 [ka] Step 1: ((2R,3R)-3-((R)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine To a stirred solution of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (56 mg, 0.076 mmol) and 4-nitrophenyl((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)carbamate (35.84 mg, 0.092 mmol) in DMF (1 ml) was added DIPEA (20.42 μl, 0.115 mmol) followed by the 1-hydroxy-7-azabenzotriazole / DMA solution (5.20 μl, 0.038 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give ((2R,3R)-3-((R)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (2) (45 mg, 0.041 mmol, 59.69% yield) as a white solid. LCMS: [M+H] + C 50 H 76 NO 10 S2 calculated value 985.310; measured value 983.4 (MH)

[0273] Step 2: Synthesis of compound 30 A solution of ((2R,3R)-3-((R)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl((((1S,2S)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (43 mg, 0.044 mmol) in DMF (1 ml) was cooled to 0° C. Pv1 peptide (143 mg, 0.044 mmol) and triethylamine (12.16 μl, 0.087 mmol) were added and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / HO and ACN. The fractions were lyophilized to give compound 30 (102 mg, 0.024 mmol, 56.29% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 197 H 297 N 41 O 53 S2 calculated value 4151.897; Found value 1383.1 [(M-3) / 3]; HPLC: Column X-Bridge C8 (50×4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA / water, Mobile phase: B: 0.1% TFA / ACN, Flow rate: 2.0 mL / min; RT (min): 5.596; Purity (max): 98.55%

[0274] Example 31: Synthesis of Compound 31 [ka] Step 1: ((2R,3R)-3-((R)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine To a stirred solution of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (56 mg, 0.076 mmol) and (4-nitrophenyl((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)carbamate (35.84 mg, 0.092 mmol) in DMF (1 mL) was added DIPEA (20.42 μl, 0.115 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (5.20 μl, 0.038 mmol). was added at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give ((2R,3R)-3-((R)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (28 mg, 0.028 mmol, 37.14% yield) as a white solid. LCMS: [M+H] + C 50 H 76 NO 10 S2 calculated value 985.310; measured value 984.4 (MH)

[0275] Step 2: Synthesis of compound 31 A solution of ((2R,3R)-3-((R)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl((((1R,2R)-2-(pyridin-2-yldisulfanayl)cyclopentyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (25 mg, 0.025 mmol) in DMF (1 ml) was cooled to 0° C. Pv1 peptide (83 mg, 0.025 mmol) and triethylamine (2.56 μl, 0.025 mmol) were added and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / HO and ACN. The fractions were lyophilized to give compound 31 (70 mg, 0.017 mmol, 66.44% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 197 H 297 N 41 O 53 S2 calculated value 4151.897; found value 1384.9 [(M+3) / 3]; HPLC: Column Atlantis dC18 (250×4.6) mm, 5 μm, Mobile phase A: 0.1% TFA / milli-Q water, Mobile phase B: ACN; Flow rate: 1.0 mL / min; RT (min): 12.134; Purity (max): 98.998%

[0276] Example 32: Synthesis of Compound 32 [ka] Step 1: (1-(pyridin-2-yldisulfanayl)cyclobutyl)methyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (S)-N-((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide To a stirred solution of 4-nitrophenyl((1-(pyridin-2-yldisulfanayl)cyclobutyl)methyl)carbamate (2) (50 mg, 0.069 mmol) and 4-nitrophenyl((1-(pyridin-2-yldisulfanayl)cyclobutyl)methyl)carbamate (2) (36.55 mg, 0.083 mmol) in DMF (1 ml) was added DIPEA (24.13 μL, 0.014 mmol) followed by 1-hydroxy-7-azabenzotriazole / DMA solution (0.47 ml, 0.035 mmol) at 0° C. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by preparative HPLC using 0.1% HCOOH / HO and ACN. The fractions were lyophilized to give (1-(pyridin-2-yldisulfanyl)cyclobutyl)methyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (35 mg, 0.034 mmol, 49.45% yield) as a white solid. LCMS: [M+H] + C 50 H 77 N7O 11 Calculated value of S2 is 1016.324; measured value is 1015.0 (MH)

[0277] Step 2: Synthesis of compound 32 A solution of (1-(pyridin-2-yldisulfanyl)cyclobutyl)methyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((R)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (35 mg, 0.034 mmol) in DMF (1 ml) was cooled to 0° C. Pv1 peptide (112.9 mg, 0.034 mmol) and triethylamine (9.6 μl, 0.068 mmol) were added and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was purified by preparative HPLC using 0.1% TFA / H2O and ACN. The fractions were lyophilized to give compound 32 (82 mg, 0.020 mmol, 57.45% yield) as a white solid. The product obtained is a di-TFA salt. LCMS: [M+H] + C 197 H 299 N 41 O 52 S2 calculated value 4137.914; Found value 1378.1 [(M-3) / 3]; HPLC: Column X-Bridge C8 (50×4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA / water, Mobile phase: B: 0.1% TFA / ACN, Flow rate: 2.0 mL / min; RT (min): 5.53; Purity (max): 98.659%

[0278] The following compounds in Table 3 were prepared using the procedures described in the Examples above. [Table 4-1] [Table 4-2] Example 38: Synthesis of Compound 38 [ka]

[0279] Step 1: Synthesis of allyl 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azatricosane-23-oate (38-2) [ka] To a solution of 38-1 (2.00 g, 1.0 equiv, 4.88 mmol) in acetonitrile (50 mL) was added cesium carbonate (3.18 g, 2.0 equiv, 9.77 mmol) and allyl bromide (630 μL, 1.50 equiv, 7.33 mmol). The reaction mixture was stirred at room temperature for 18 h. The remaining cesium carbonate was filtered off and the solvent was removed in vacuo. Purification by flash chromatography (EtOAc / cyclohexane, 0% over 2 CV, 0%-100% over 10 CV) afforded the title compound (1.80 g, 82%) as a white solid. 1 H NMR(400 MHz,DMSO-d6)6.75(t,J=5.7 Hz,1H),5.95-5.85(m,1H),5.34-5.24(m,1H),5.22-5.16(m,1H),4.55(dt,J=5.3,1.6 Hz,2H),3.64(t,J=6.2 Hz,2H),3.54-3.50(m,16H)3.4(t,J=6.1 Hz,2H),3.05(q,J=6.0 Hz,2H),2.57(t,J=6.2 Hz,2H),1.37(s,9H).

[0280] Step 2: Synthesis of allyl 1-amino-3,6,9,12,15-pentaoxaoctadecane-18-oate hydrochloride (38-3) [ka] To a solution of 38-2 (1.80 g, 1.0 equiv, 4.00 mmol) in dioxane (20 mL) was added 4N HCl / dioxane (20.0 mL, 20.0 equiv, 80.0 mmol) and the reaction was stirred at room temperature for 18 h. The reaction was concentrated in vacuo and the residue was triturated with diethyl ether to give the title compound (1.55 g, 99%) as a colorless oil. 1H NMR(400 MHz,MeOD-d4)δ 5.95(ddt,J=17.2,10.5,5.6 Hz,1H),5.37-5.27(m,1H),5.24-5.20(m,1H),4.61(dt,J=5.6,1.5 Hz,2H),3.68-3.62(m,20H),3.17-3.11(m,2H),2.64(t,J=6.0 Hz,2H).

[0281] Step 3: Synthesis of allyl 1-(((1S,2S)-2-(((4-(hydroxymethyl)phenyl)carbamoyl)oxy)cyclohexyl)disulfanayl)-3-oxo-7,10,13,16,19-pentaoxa-4-azadocosane-22-oate (38-4) [ka] To a solution of 38-3' (500 mg, 1.0 equiv, 1.30 mmol) in anhydrous DMF (10 mL) was added 1H-benzo[d][1,2,3]triazol-1-ol (228 mg, 1.3 equiv, 1.69 mmol), N,N'-diisopropylcarbodiimide (262 μL, 1.3 equiv, 1.69 mmol), and N-ethyl-N-isopropylpropan-2-amine (838 mg, 5.0 equiv, 6.49 mmol). The mixture was stirred for 10 min. Then, a solution of allyl 1-amino-3,6,9,12,15-pentaoxaoctadecane-18-oate hydrochloride 38-3 (651 mg, 1.3 equiv, 1.69 mmol) in DMF (10 mL) was added and the solution was continued to stir at room temperature for 18 h. The mixture was purified by reverse phase chromatography (methanol / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (545 mg, 59%) as a white solid. 1H NMR(400 MHz,DMSO-d6)δ 9.58(s,1H),7.97(t,J=5.7 Hz,1H),7.41(d,J=8.3 Hz,2H),7.24-7.16(m,2H),5.90(ddt,J=17.3,10.6,5.4 Hz,1H),5.38-5.12(m,2H),4.67-4.58(m,1H),4.55(dt,J=5.4,1.5 Hz,2H),4.43-4.37(m,2H),3.64(t,J=6.2 Hz,2H),3.52-3.44(m,16H),3.38(t,J=5.9 Hz,2H),3.21-3.13(m,2H),2.92-2.81(m,3H),2.59-2.53(m,2H),2.44(t,J=7.2 Hz,2H),2.16-2.00(m,2H),1.77-1.26(m,6H).LC-MS(ESI+)[C 33 H 53 N2O 11 S2] + [M+H] + Accurate mass calculation: 717, measured mass: 717.

[0282] Step 4: Synthesis of allyl 1-(((1S,2S)-2-(((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)oxy)cyclohexyl)disulfanayl)-3-oxo-7,10,13,16,19-pentaoxa-4-azadocosane-22-oate (38-5) [ka] To a solution of 38-4 (540 mg, 1.0 equiv, 753 μmol) in anhydrous DMF (15 mL) was added bis(4-nitrophenyl)carbamate (458 mg, 2.0 equiv, 1.51 mmol) and diisopropylethylamine (388 μL, 3.0 equiv, 2.26 mmol) at 4° C. The mixture was allowed to warm to room temperature and stirred for 18 h. The mixture was purified by reverse phase chromatography (methanol / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (444 mg, 67%) as a white solid. 1H NMR(400 MHz,MeOD-d4)δ 8.37-8.26(m,2H),7.55-7.43(m,4H),7.42-7.34(m,2H),5.93(ddt,J=17.2,10.8,5.5 Hz,1H),5.34-5.27(m,1H),5.26-5.23(m,2H),5.22-5.18(m,1H),4.74-4.64(m,1H),4.60-4.56(m,2H),3.73(t,J=6.2 Hz,2H),3.61-3.56(m,16H),3.49(t,J=5.3 Hz,2H),2.95(td,J=7.2,1.8 Hz, 2H), 2.88-2.79 (m, 1H), 2.61-2.55 (m, 4H), 2.23-2.12 (m, 2H), 1.82-1.38 (m, 6H), three protons are likely covered by methanol signals. LC-MS (ESI+) [C 40 H 56 N3O 15 S2] + [M+H] + Accurate mass calculation: 882, measured mass: 882.

[0283] Step 5: Synthesis of compound 38-6 [ka] To a solution of 38-5 (400 mg, 1.0 equiv, 454 μmol) in DMF (4 mL) was added HOBt (93.1 mg, 1.2 equiv, 544 μmol), DIPEA (234 μL, 3.0 equiv, 1.36 mmol), MMAE (391 mg, 1.2 equiv, 544 μmol), and 3 Å molecular sieves. The reaction was stirred at room temperature for 18 h. The mixture was purified by reverse phase chromatography (methanol / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (313 mg, 47%) as a fluffy white solid. LC-MS (ESI+) [C 73 H 118 N7O 19 S2] + [M+H] + Accurate mass calculation: 1461, measured mass: 1461.

[0284] Step 7: Synthesis of compound 38-8 [ka] To a solution of 38-7 (60 mg, 1.0 equiv, 42 μmol) in DMF (5 mL) was added HATU (21 mg, 1.3 equiv, 55 μmol) and diisopropylethylamine (29 μL, 4.0 equiv, 0.17 mmol). After stirring at room temperature for 15 min, a solution of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (9.7 mg, 1.3 equiv, 55 μmol) in DMF (5 mL) was added and the mixture was stirred at room temperature for 18 h. The reaction was purified by reverse phase chromatography (acetonitrile / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (65 mg, 99%) as a white solid. LC-MS (ESI+) [C 76 H 120 N9O 20 S2] + [M+H] + Accurate mass calculation: 1542.8, measured value: 1543.3

[0285] Step 8: Synthesis of compound 38 [ka] To a solution of 38-8 (65 mg, 1.0 equiv, 42 μmol) in DMF (3 mL) was added Pv1 (150 mg, 1.1 equiv, 46 μmol) and diisopropylethylamine (51 μL, 7.0 equiv, 0.29 mmol). The mixture was stirred at room temperature for 18 h and purified by reverse phase chromatography (acetonitrile / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give compound 38 (16 mg, 8%) as a white solid. HPLC: 96% at 220 nm. LC-MS (ESI-) [C 228 H 341 N 44 O 64 S3] 3- [M-3H] 3-Accurate mass calculation: 1605.8, measured value: 1605.9. [C 228 H 340 N 44 O 64 S3] 4- [M-4H] 4- Accurate mass calculation: 1204.1, measured value: 1204.1

[0286] Example 39: Synthesis of Compound 39 [ka] Step 1: Synthesis of allyl 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oate (39-2) [ka] To a solution of 39-1 (2.00 g, 1.0 equiv, 6.23 mmol) in acetonitrile (50 mL) was added cesium carbonate (4.06 g, 2.0 equiv, 12.5 mmol) and allyl bromide (803 μL, 1.5 equiv, 9.35 mmol). The reaction mixture was stirred at room temperature for 18 h. The remaining cesium carbonate was filtered off and the solvent was removed in vacuo. Purification by flash chromatography (EtOAc / cyclohexane, 0% over 2 CV, 0%-100% over 10 CV) afforded the title compound (1.60 g, 71%) as a white powder. 1 H NMR(400 MHz,CDCl3)δ 5.91(ddt,J=17.2,10.4,5.7 Hz,1H),5.37-5.18(m,2H),5.11-4.73(br s,1H),4.59(dt,J=5.7,1.4 Hz,2H),3.77(t,J=6.5 Hz,2H),3.68-3.58(m,8H),3.53(dd,J=5.5,4.7 Hz,2H),3.30(t,J=5.1 Hz,2H),2.63(t,J=6.5 Hz,2H),1.43(s,9H).

[0287] Step 2: Synthesis of allyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate hydrochloride (39-3) [ka] To a solution of 39-2 (1.60 g, 1.00 equiv, 4.23 mmol) in dioxane (20 mL) was added 4N HCl / dioxane (22.1 mL, 20.0 equiv, 88.5 mmol) and the reaction was stirred at room temperature for 18 h. The reaction was concentrated in vacuo and the residue was triturated with diethyl ether to give the title compound (1.32 g, 99%) as a colorless oil. 1 H NMR(400 MHz,MeOD-d4)5.99-5.89(m,1H),5.32(dq,J=17.2,1.6 Hz,1H),5.22(dq,J=10.5,1.4 Hz,1H),4.60(dt,J=5.6,1.5 Hz,2H),3.78-3.74(m,2H),3.72-3.69(m,2H)3.67-3.65(m,6H),3.64-3.62(m,4H),3.14-3.11(m,2H),2.63(t,J=6.0 Hz,2H).

[0288] Step 3: Synthesis of allyl 1-(((1S,2S)-2-(((4-(hydroxymethyl)phenyl)carbamoyl)oxy)cyclohexyl)disulfanayl)-3-oxo-7,10,13-trioxa-4-azahexadecan-16-oate (39-4) [ka] To a solution of 39-3' (500 mg, 1.0 equiv, 1.30 mmol) in anhydrous DMF (10 mL) was added 1H-benzo[d][1,2,3]triazol-1-ol (228 mg, 1.3 equiv, 1.69 mmol), N,N'-diisopropylcarbodiimide (262 μL, 1.3 equiv, 1.69 mmol), and diisopropylethylamine (838 mg, 5.0 equiv, 6.49 mmol). The mixture was stirred for 10 min. Allyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate hydrochloride (502 mg, 1.3 equiv, 1.69 mmol) in DMF (10 mL) was then added and the solution was continued to stir at room temperature for 18 h. The mixture was purified by reverse phase chromatography (methanol / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (747 mg, 92%) as a white solid. 1 H NMR(400 MHz,DMSO-d6)δ 7.97(t,J=5.7 Hz,1H),7.41(m,2H),7.21-7.19(m,2H),5.90(ddt,J=17.2,10.6,5.3 Hz,1H),5.34-5.17(m,2H),5.07-5.02(m,1H),4.65-4.57(m,1H),4.55(dt,J=5.3,1.6 Hz,2H),4.43-4.48(m,2H),3.63(t,J=6.2 Hz,2H),3.53-3.43(m,8H),3.38(t,J=5.9 Hz,2H),3.22-3.13(m,3H),2.92-2.83(m,3H),2.57(t,J=6.2 Hz,2H),2.44(t,J=7.2 Hz,2H),2.17-1.99(m,2H),1.77-1.28(m,6H).LC-MS(ESI+)[C 29 H 45 N2O9S2] + [M+H] + Accurate mass calculation: 629, measured mass: 629.

[0289] Step 4: Synthesis of allyl 1-(((1S,2S)-2-(((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)oxy)cyclohexyl)disulfanayl)-3-oxo-7,10,13-trioxa-4-azahexadecan-16-oate (5) [ka] To a solution of 39-4 (740 mg, 1.0 equiv, 1.18 mmol) in anhydrous DMF (15 mL) was added bis(4-nitrophenyl)carbamate (716 mg, 2.0 equiv, 2.35 mmol) and diisopropylethylamine (607 μL, 3.0 equiv, 3.53 mmol) at 4° C. The mixture was allowed to warm to room temperature and stirred for 18 h. The mixture was purified by reverse phase chromatography (methanol / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (520 mg, 56%) as a white solid. 1 H NMR(400 MHz,DMSO-d6)δ 8.35-8.27(m,2H),7.97(t,J=5.6 Hz,1H),7.60-7.54(m,2H),7.53-7.48(m,2H),7.40-7.36(m,2H),5.89(ddt,J=17.3,10.6,5.3 Hz,1H),5.32-5.25(m,1H),5.24-5.21(m,2H),5.21-5.17(m,1H),4.69-4.59(m,1H),4.55(dt,J=5.3,1.6 Hz,2H),3.63(t,J=6.2 Hz,2H),3.51-3.43(m,8H),3.37(t,J=5.9 Hz,2H),3.22-3.12(m,3H),2.88(t,J=6.9 Hz,3H),2.56(t,J=6.2 Hz,2H),2.44(t,J=7.2 Hz,2H),2.17-2.02(m,2H),1.75-1.31(m,6H).LC-MS(ESI+)[C 36 H 48 N3O 13 S2] + [M+H] + Accurate mass calculation: 794, measured mass: 794.

[0290] Step 5: Synthesis of 39-6 [ka] To a solution of 39-5 (420 mg, 1.0 equiv, 529 μmol) in DMF (4 mL) was added HOBt (109 mg, 1.2 equiv, 635 μmol), diisopropylethylamine (273 μL, 3.0 equiv, 1.59 mmol), MMAE (456 mg, 1.2 equiv, 635 μmol), and 3 Å molecular sieves. The reaction was stirred at room temperature for 18 h. The mixture was purified by reverse phase chromatography (methanol / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (313 mg, 28%) as a fluffy white solid. LC-MS (ESI+) [C 69 H 110 N7O 17 S2] + [M+H] + The calculated mass is 1372.7, and the measured mass is 1372.9.

[0291] Step 6: Synthesis of 39-7 [ka] To a solution of 39-6 (200 mg, 1.0 equiv, 146 μmol) in dry CHCl (2 mL) was added triphenylphosphine (3.8 mg, 10 mol%, 15 μmol). The solution was purged with nitrogen for 2 min, then Pd(PPh) (33.7 mg, 20 mol%, 29.1 μmol) and pyrrolidine (14 μL, 1.2 equiv, 175 μmol) were added. The mixture was allowed to stir at room temperature for 18 h. The reaction was concentrated in vacuo and the residue was purified by reverse phase chromatography (methanol / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (64 mg, 33%) as a fluffy yellow solid. 1 The H NMR spectrum is too complex to interpret. LC-MS(ESI+)[C 66 H 106 N7O17 S2] + [M+H] + The calculated mass is 1332.7, and the measured mass is 1332.5.

[0292] Step 7: Synthesis of 39-8 [ka] To a solution of 39-7 (60 mg, 1.0 equiv, 45 μmol) in DMF (4 mL) was added HATU (22 mg, 1.3 equiv, 59 μmol) and diisopropylethylamine (31 μL, 4.0 equiv, 0.18 mmol). After stirring at room temperature for 15 min, a solution of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (10 mg, 1.3 equiv, 59 μmol) in DMF (4 mL) was added and the mixture was stirred at room temperature for 18 h. The reaction was purified by reverse phase chromatography (acetonitrile / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (60 mg, 92%) as a white solid. LC-MS (ESI+) [C 72 H 112 N9O 18 S2] + [M+H] + Accurate mass calculation: 1454.7, measured mass: 1454.8.

[0293] Step 8: Synthesis of compound 39 [ka] To a solution of 39-8 (60 mg, 1.0 equiv, 41 μmol) in DMF (3 mL) was added Pv1 (150 mg, 1.1 equiv, 45 μmol) and diisopropylethylamine (50 μL, 7.0 equiv, 0.29 mmol). The mixture was stirred at room temperature for 18 h and purified by reverse phase chromatography (acetonitrile / water (0.1% formic acid), 5% over 2 CV, 5%-95% over 12 CV, 95% over 2 CV) to give the title compound (60 mg, 31%) as a white solid. HPLC: 99% at 220 nm. LC-MS (ESI+) [C 224 H333 N 44 O 62 S3] 3- [MH] - Accurate mass calculation: 1576.5, measured value: 1576.2. [C 224 H 332 N 44 O 62 S3] 4- [MH] - Accurate mass calculation: 1182.1, measured value: 1182.8

[0294] Example A: In vitro growth delay assay in cancer cells Cells (HCT116 colorectal, PC3 prostate, NCI-H1975 NSCLC, and NCI-H292 NSCLC) were plated at 3000 cells / well in 96-well black-walled, clear-bottom plates (Griener) in growth medium containing 10% FBS. Cells were allowed to attach for 60 minutes at room temperature and then returned to a 37°C, 5% CO2 incubator. After 24 hours, medium was removed and replaced with fresh growth medium containing various drug concentrations. Each drug concentration was added in triplicate. Drug-untreated controls contained growth medium only. Cells were returned to the incubator. 96 hours after drug addition, cells were fixed with 4% paraformaldehyde for 20 minutes and stained with Hoechst at 1 μg / mL. Plates were imaged on a Cytation 5 autoimager (BioTek) and cells were counted using CellProfiler (http: / / cellprofiler.org). Percent cell growth delay was calculated and data plotted using GraphPad Prism.

[0295] FIG. 1A shows a plot of the proliferation delay of HCT116 colorectal cells in vitro after incubation with the indicated concentrations of Compound 2 or unconjugated MMAE for 4 days.

[0296] FIG. 1B shows a plot of the growth delay of PC3 prostate cells in vitro after incubation with the indicated concentrations of Compound 2 or unconjugated MMAE for 4 days.

[0297] FIG. 1C shows a plot of the growth delay of NCI-H1975 NSCLC cells in vitro after incubation with the indicated concentrations of Compound 2 or unconjugated MMAE for 4 days.

[0298] FIG. 1D shows a plot of the growth delay of NCI-H292 NSCLC cells in vitro after incubation with the indicated concentrations of compound 2 or unconjugated MMAE for 4 days.

[0299] The following table shows the 4-day proliferation inhibition (IC) of HCT116 colorectal cells following treatment with the indicated exemplary compounds. 50 ) is shown. [Table 5]

[0300] Example B: In vitro cell cycle arrest functional assay in cancer cells Incubation of cells with MMAE and Compound 2 and staining with propidium iodide HCT116 cells were seeded at 500,000 cells / well in 2 mL of DMEM in 6-well tissue culture plates and incubated overnight at 37° C. in a 5% CO2 incubator. 200 μL dilutions of MMAE and Compound 2 made at 10x concentration in DMEM + 4% DMSO were added to the appropriate wells of the 6-well plate and the plate was incubated for 24 hours. After exposure of HCT116 cells to either MMAE or Compound 2, cells were harvested for propidium iodide staining and flow cytometry. Media was collected from each well and transferred to a 15 mL conical centrifuge tube to collect non-adherent cells. PBS (1 mL) was added to wash the wells and then transferred to a 15 mL tube. Tryp-LE (1 mL) was added to each well and the plate was incubated for 5 minutes in a 37° C., 5% CO2 incubator until the cells lifted off the well surface. DMEM + 10% fetal bovine serum solution (1 mL) was added to each well. The wells were triturated and the cells were transferred to a test tube. DMEM + 10% fetal bovine serum solution (1 mL) was added to the wells to ensure collection of the cells. These were transferred again to a 15 mL tube. The cell number and viability of each sample was assessed by trypan blue exclusion on a Bio-Rad TC20 cell counter. The cells were centrifuged at 1200 rpm for 5 minutes. The supernatant was decanted and the cells were diluted to 1 x 10 6 Cells / mL were resuspended in PBS and stained with propidium iodide. [Table 6]

[0301] An aliquot (1 mL) of each cell suspension was transferred to a deep-well polypropylene plate. The plate was centrifuged at 1200 rpm for 5 min. The supernatant was decanted and the cells were resuspended in 330 μL of cold PBS. A volume of 670 μL of cold ethanol was slowly added to the side of each well. The cells were gently triturated to achieve a uniform 67% ethanol for cell fixation on ice for 3 h before staining. After fixation, the plate was centrifuged at 1200 rpm for 5 min and the ethanol:PBS was decanted. The cells were resuspended in a solution of RNase (300 μg / mL) and propidium iodide (50 μg / mL) in PBS (200 μL). The plate was sealed and incubated in the dark at 37° C. for 30 min or at room temperature overnight, after which the cells were resuspended and transferred to a small volume polypropylene plate for flow cytometry. The propidium iodide stained cells were analyzed using a BD Acuri flow cytometer. Three plots were made per sample.

[0302] FIG. 2A shows cell cycle analysis of HCT116 colorectal cells in vitro after 24 h incubation with the indicated doses of unconjugated MMAE.

[0303] FIG. 2B shows cell cycle analysis of HCT116 colorectal cells in vitro after incubation with the indicated doses of Compound 2 for 24 hours.

[0304] Cells exhibited a dose-responsive accumulation in G2 / M and IC 50 is 2.6 nM, and the IC 50 is 19.6nM.

[0305] Example C: Plasma pharmacokinetics of Compound 2 in a rat model Animal Medication Female Sprague Dawley rats underwent jugular vein cannulation and insertion of vascular access buttons (VAB, Instech Labs catalog number VABR1B / 22) at Envigo Labs prior to shipping. A magnetic aluminum cap (Instech Labs catalog number VABRC) was used to protect the jugular vein catheter access port, and animals were housed 2 / cage in corncob bedding for 4-5 days prior to the study. Rats were administered a single dose of 10 mg / kg compound 2 intravenously prepared in 5% mannitol / citrate buffer vehicle. Blood (250 μL) was collected from fed rats into K2EDTA-filled microtainers at 2 min, 30 min, 1 h, 2 h, 4 h, 7 h, and 24 h after compound administration. Plasma was separated by centrifugation, and 100 μL aliquots were transferred to 96-well polypropylene plates on dry ice. Samples were stored at -80 °C until processed for quantification by LC-MS / MS.

[0306] LC-MS / MS measurement of plasma conjugate concentrations A volume of 20 μL of each sample (double blank (D-BLK), blank (BLK), standard (STD), quality control (QC), or matrix sample) was added to a clean 1 mL 96-well protein precipitation plate containing 20 μL of 4% aqueous phosphoric acid. The fortified samples were vortexed at 700 rpm for 2 min and then centrifuged at 1500 rpm for 1 min to solidify all liquid to the bottom of the plate. A volume of 20 μL of working internal standard (WIS) was added to each matrix sample, followed by the addition of 180 μL of acetonitrile:methanol:formic acid (500:500:1, v:v:v). Samples were vortexed at 700 rpm for 2 min and centrifuged at 3000 rpm for 10 min at 4 °C. A volume of 50 μL of the supernatant was transferred to a clean LoBind 0.700 mL 96-well polypropylene collection plate followed by the addition of 100 μL of water:acetonitrile:formic acid (900:100:1, v:v:v). The final sample was vortexed at 700 rpm for 2 min and 5 μL was injected into the LC-MS / MS system for analysis.

[0307] LC-MS / MS measurement of plasma MMAE concentrations A volume of 25 μL of each matrix sample was added to a well of a 96-well polypropylene plate, followed by the addition of 150 μL of ammonium formate buffer (pH 6.9) and 25 μL of working internal standard (WIS). For double blank controls, the WIS was replaced with 25 μL of water:acetonitrile:formic acid (500:500:1, v:v:v). The fortified samples were vortexed at 700 rpm for 2 min. Working with a negative pressure manifold, a volume of 200 μL of fortified matrix sample was added to the supporting liquid extraction plate and the sample was passed through the plate frit under a negative pressure of 650-700 Torr for up to 1 min. The sample was allowed to fully absorb into the plate over a period of 5 min. Prior to elution, a 2 mL 96-well TrueTaper plate was placed in the vacuum manifold to serve as a collection plate. A volume of 1000 μL of MTBE was added to the original sample plate and the solvent was allowed to flow under gravity for 5 min. Negative pressure of approximately 650 Torr was applied to 10-30 sections or until the sample was completely expelled from the well. The collected eluate was evaporated under a heated nitrogen stream at 40 °C. Samples were reconstituted in 100 μL of acetonitrile:water:200 mM ammonium formate (90:5:5, v:v:v) and covered with a silicone cap mat. The final sample was vortexed at 900 rpm for 2 min and then centrifuged at 3000 rpm for 5 min at 4 °C. Analysis was accomplished by injecting 10 μL of sample into the LC-MS / MS system.

[0308] Figure 3 shows a plot of the plasma concentrations of Compound 2 and released MMAE after a single IV dose of 10 mg / kg Compound 2 in rats (data are expressed as mean ± standard deviation). As shown in Figure 3, 0.02% of the MMAE payload was released after 24 hours of circulation. Figure 3 shows that Compound 2 is stable in plasma for at least 24 hours.

[0309] Example D: Tissue Pharmacokinetics of Compound 2 in a Mouse Model Animal Medication Six-week-old female athymic nude Fox nuMice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system (Innovive). Human HCT116 cancer cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and cultured at 2.5 × 10 cells / 100 μL. 6 The cells were then subcutaneously implanted into the left flank of each mouse at a density of 100 x 100 mm. 3 When the minimum volume of MMAE was reached, mice were administered a single intraperitoneal injection of 0.5 mg / kg MMAE or 3 mg / kg Compound 2 prepared in a 5% mannitol / citric acid vehicle. Tumor, quadriceps, and bone marrow samples were collected from anesthetized fed mice at 4, 24, and 48 hours after compound administration. MMAE concentrations in tissues were measured by LCMS.

[0310] LC-MS / MS measurement of plasma and tissue MMAE concentrations plasma MMAE A volume of 75 μL of acetonitrile:formic acid (1000:1, v:v) containing internal standard (MMAE-D8) was added to wells of a 96-well protein precipitation plate located on a 0.700 mL 96-well LoBind polypropylene plate. Double blank sample wells and carryover sample wells received 75 μL of acetonitrile:formic acid (1000:1, v:v) without internal standard. A volume of 25 μL of each matrix sample was added to the plate wells containing the internal standard. The fortified samples were vortexed at 700 rpm for 1 min and centrifuged at 3000 rpm for 2 min at 4° C. The protein precipitation plate was discarded. A volume of 50 μL of mobile phase A (acetonitrile:water:200 mM ammonium formate (90:5:5, v:v:v)) was added to a 96-well polypropylene collection plate covered with a silicone cap mat. The final sample was vortexed at 700 rpm for 2 min and analysis was achieved by injecting 2 μL of sample into the LC-MS / MS system.

[0311] Tumor and muscle MMAE Thawed tissue samples kept on wet ice were adjusted to 100 mg / mL with PBS based on tissue weight. Samples were homogenized in a Precellys Evolution machine at 7200 rpm for 2 x 30 sec cycles (with 10 sec pause between cycles). Homogenates were centrifuged at 14,000 rpm for 5 min at 4°C and the supernatants were transferred to 2 mL clean LoBind Eppendorf tubes. A volume of 100 μL of homogenate was added to 2 mL of a clean 96-well polypropylene plate, followed by 75 μL of ammonium formate buffer (pH 6.9) and 25 μL of working internal standard (WIS). Double blank controls received 75 μL of water:acetonitrile:formic acid (1:1:0.001, v:v:v) without internal standard. Fortified samples were covered with a silicone cap mat and vortexed at 700 rpm for 2 min. Working with a negative pressure manifold, 200 μL of enriched matrix sample was added to a supported liquid extraction (SLE) plate and the sample was passed through the plate frit at a negative pressure of approximately 650-700 Torr for up to 1 min. The sample was allowed to fully absorb into the SLE plate for 5 min. Prior to sample elution, a 2 mL 96-well TrueTaper collection plate was placed in the vacuum manifold as a collection vessel. Samples were evaporated under a heated nitrogen stream at 40 °C and reconstituted in 150 μL of acetonitrile:water:200 mM ammonium formate (90:5:5, v:v:v). The collection plate was covered with a silicone cap mat and vortexed at 900 rpm for 2 min. The final sample was centrifuged at 3000 rpm for 5 min at 4 °C and analysis was achieved by injecting 2 μL of sample into the LC-MS / MS system.

[0312] Bone marrow MMAE Thawed bone marrow sample pellets kept on wet ice were diluted to 1.0 x 10 with ice-cold RIPA buffer. 7The final concentration of 100 μL was adjusted to 0.05%. Samples were homogenized in a Precellys Evolution machine at 7200 rpm for 2 × 30 sec cycles (with 10 sec pauses between cycles). Bone marrow cell homogenates were centrifuged at 14,000 rpm for 5 min at 4 °C and the supernatant was transferred to a 2 mL clean LoBind Eppendorf tube. A volume of 200 μL of each bone marrow cell homogenate was added to a 2 mL well of a clean 96-well polypropylene plate, followed by 175 μL of ammonium formate buffer (pH 6.9) and 25 μL of working internal standard (WIS). Double blank controls received 175 μL of water:acetonitrile (1:1, v:v:v) without internal standard. Fortified samples were covered with a silicone cap mat and vortexed at 700 rpm for 2 min. Working with a negative pressure manifold, 400 μL of enriched matrix sample was added to a supported liquid extraction (SLE) plate and the sample was passed through the plate frit at approximately 650-700 Torr negative pressure for up to 1 min. The sample was allowed to fully absorb into the SLE plate for 5 min. Prior to sample elution, a 2 mL 96-well TrueTaper collection plate was placed in the vacuum manifold as a collection vessel. Elution was achieved by applying 900 μL of MTBE:Ethyl acetate (1:1, v:v) to the system and allowing the solvent to flow under gravity for 5 min. Approximately 650 Torr negative pressure was applied for 10-30 s or until the wells were completely drained. The elution process was repeated. The samples were evaporated under a heated nitrogen stream at 40 °C and reconstituted in 25 μL of water:acetonitrile:formic acid (900:100:1, v:v:v). The collection plate was covered with a silicone cap mat and vortexed at 900 rpm for 2 min. The final samples were centrifuged at 3000 rpm for 5 min at 4° C. and analysis was achieved by injecting 2 μL into the LC-MS / MS system.

[0313] FIG. 4A shows a plot of the levels of unconjugated MMAE in mouse tumors measured by LCMS following a single intraperitoneal injection of either 0.5 mg / kg MMAE or 3 mg / kg Compound 2 in female nude mice bearing HCT116 colorectal tumors.

[0314] FIG. 4B shows a plot of the levels of unconjugated MMAE in mouse muscle as measured by LCMS following a single intraperitoneal injection of either 0.5 mg / kg MMAE or 3 mg / kg Compound 2 in female nude mice bearing HCT116 colorectal tumors.

[0315] FIG. 4C shows a plot of the levels of unconjugated MMAE in mouse bone marrow as measured by LCMS following a single intraperitoneal injection of either 0.5 mg / kg MMAE or 3 mg / kg Compound 2 in female nude mice bearing HCT116 colorectal tumors.

[0316] Administration of the unconjugated MMAE warhead results in indiscriminate distribution of MMAE across all tissues. In contrast, administration of compound 2 results in tumor-selective delivery of the MMAE warhead, efficiently delivering MMAE to tumors but not to healthy tissues.

[0317] Example E: Efficacy of Compound 1 in the HCT116 colorectal xenograft model Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system. Human HCT116 cells derived from colorectal cancer were diluted 1:1 in phenol red-free Matrigel and cultured at 2.5 × 10 cells / 100 μL. 6 The cells were then subcutaneously implanted into the left flank of each mouse at a cell density of 100–200 mm. 3Upon reaching an average volume of 1000 mg / kg, mice were randomized into groups and treated as detailed in the table below. Mice were administered intraperitoneally (IP) with a dose of vehicle, 0.25 mg / kg MMAE, or 40 mg / kg Compound 1 (equivalent to 7 mg / kg unconjugated MMAE). Doses were prepared by diluting a 0.1 mg / μL DMSO stock in 5% mannitol / citrate buffer and administered in two doses at a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured with calipers and volumes were calculated using the equation for ellipsoid volume: volume = π / 6 × (length) × (width). 2 Animals were killed when their tumor size reached 2000 mm 3 Rats were removed from the study if their body weight loss exceeded 100% or if their body weight loss exceeded 20%. The table below shows the dosing schedule for the various treatment groups. [Table 7]

[0318] 5A shows a plot of the mean tumor volume resulting from dosing nude mice bearing HCT116 HER2-negative colorectal flank tumors with either 0.25 mg / kg MMAE or 40 mg / kg Compound 1 (equivalent to 7 mg / kg MMAE). Animals were dosed intraperitoneally once daily for a total of two days.

[0319] FIG. 5B shows a plot of the percent change in body weight of nude mice bearing HCT116 HER2-negative colorectal flank tumors administered either 0.25 mg / kg MMAE or 40 mg / kg Compound 1 (equivalent to 7 mg / kg MMAE).

[0320] Animals administered unconjugated MMAE experienced a rapid loss of body weight and were removed from the study by day 6. In contrast, animals administered Compound 1 did not experience any change in body weight. These data demonstrate that Compound 1 exhibits potent antitumor activity and safety in preclinical colorectal cancer models.

[0321] Example F: Efficacy of Compound 2 in a PC3 Prostate Xenograft Model (shown with FIG. 6) Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system. Human PC3 cells derived from prostate cancer were diluted 1:1 in phenol red-free Matrigel and 2.5 × 10 cells / 100 μL. 6 The cells were then subcutaneously implanted into the left flank of each mouse at a cell density of 100–200 mm. 3 Upon reaching an average volume of 100 μL, mice were randomized into groups and treated as detailed in the table below. Mice were administered vehicle or a dose of 20 mg / kg compound 2 intraperitoneally (IP). Doses were prepared by diluting a 0.1 mg / μL DMSO stock in 5% mannitol / citrate buffer and administered QD×2 / week for 3 weeks in a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured with calipers and volumes were calculated using the equation for ellipsoid volume: volume=π / 6×(length)×(width). 2 Animals were killed when their tumor size reached 2000 mm 3 Rats were removed from the study if their body weight loss exceeded 100% or if their body weight loss exceeded 20%. The table below shows the dosing schedule for the various treatment groups. [Table 8]

[0322] 6A shows a plot of the mean tumor volume resulting from dosing nude mice bearing PC3 prostate adenocarcinoma flank tumors with 20 mg / kg of Compound 2. Animals were dosed intraperitoneally once daily, twice weekly for three weeks.

[0323] Figure 6B presents the percent change in body weight of the animals in this study. Data are expressed as mean ± SEM.

[0324] These data demonstrate that Compound 2 exhibits potent antitumor activity and safety in a preclinical prostate cancer model. Animals administered Compound 2 did not experience any changes in body weight.

[0325] Example G: Efficacy of Compound 2 in the NCI-H1975 Non-Small Cell Lung Xenograft Model Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system. Human NCI-H1975 cells derived from non-small cell lung carcinoma were diluted 1:1 in phenol red-free Matrigel and 5 × 10 cells / 100 μL. 6 The cells were then subcutaneously implanted into the left flank of each mouse at a cell density of 100–200 mm. 3 Upon reaching an average volume of 1000 mg / kg, mice were randomized into groups and treated as detailed in the table below. Mice were administered intraperitoneally (IP) with a dose of vehicle, 10 mg / kg compound 2, or 20 mg / kg compound 2. Doses were prepared by diluting a 0.1 mg / μL DMSO stock in 5% mannitol / citrate buffer and administered QD×2 / week for 3 weeks at a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured with calipers and volumes were calculated using the equation for ellipsoid volume: volume=π / 6×(length)×(width). 2 Animals were killed when their tumor size reached 2000 mm 3 Rats were removed from the study if their body weight loss exceeded 100% or if their body weight loss exceeded 20%. The table below shows the dosing schedule for the various treatment groups. [Table 9]

[0326] 7A shows a plot of the mean tumor volume resulting from dosing nude mice bearing NCI-H1975 non-small cell lung cancer flank tumors with 10 mg / kg or 20 mg / kg of Compound 2. Animals were dosed intraperitoneally once daily, twice weekly for three weeks.

[0327] Figure 7B presents the percent change in body weight of the animals in this study. Data are expressed as mean ± SEM.

[0328] These data demonstrate that Compound 2 exhibits potent antitumor activity and safety in a preclinical non-small cell lung cancer model. Animals administered Compound 2 did not experience any changes in body weight.

[0329] Example H: Safety of Compound 2 in Nude Mice Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog number NCRNU-F) and housed 3 / cage on Alpha-Dri bedding in a disposable cage system. Mice were administered intraperitoneally (IP) with a dose of vehicle, 10 mg / kg compound 2, or 20 mg / kg compound 2. Doses were prepared by diluting 0.1 mg / μL DMSO stock in 5% mannitol / citrate buffer and administered once daily for 4 consecutive days in a volume of 12 mL / kg (300 μL per 25 g mouse). The following table shows the dosing schedule for the various treatment groups. [Table 10]

[0330] FIG. 8 shows plots of body weight of nude mice dosed with 10 mg / kg of Compound 1 and Compound 2 once daily for four consecutive days.

[0331] Animals administered Compound 1 and Compound 2 showed no change in body weight, demonstrating the safety of these conjugates in mice.

[0332] Example I: Tissue pharmacokinetics of Compound 13 and Compound 7 in a mouse model Animal Medication Six-week-old female athymic nude Fox nuMice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system (Innovive). Human HCT116 cancer cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and cultured at 2.5 × 10 cells / 100 μL. 6 The cells were then subcutaneously implanted into the left flank of each mouse at a density of 100 x 100 mm. 3 When the tumor reached a minimum volume of 100 μg / kg, mice were administered a single intraperitoneal injection of 10 mg / kg compound 13 or compound 7 in a 5% mannitol / citric acid vehicle. Tumors were harvested from anesthetized fed mice at 2, 4, 8, and 24 hours after compound administration. MMAE concentrations in tumors were measured by LCMS and peptide concentrations by ELISA.

[0333] LC-MS / MS measurement of tissue MMAE concentrations Thawed tissue samples kept on wet ice were adjusted to 100 mg / mL with PBS based on tissue weight. Samples were homogenized in a Precellys Evolution machine at 7200 rpm for 2 x 30 sec cycles (with 10 sec pause between cycles). Homogenates were centrifuged at 14,000 rpm for 5 min at 4°C and the supernatants were transferred to 2 mL clean LoBind Eppendorf tubes. A volume of 100 μL of homogenate was added to 2 mL of a clean 96-well polypropylene plate, followed by 75 μL of ammonium formate buffer (pH 6.9) and 25 μL of working internal standard (WIS). Double blank controls received 75 μL of water:acetonitrile:formic acid (1:1:0.001, v:v:v) without internal standard. Fortified samples were covered with a silicone cap mat and vortexed at 700 rpm for 2 min. Working with a negative pressure manifold, 200 μL of enriched matrix sample was added to a supported liquid extraction (SLE) plate and the sample was passed through the plate frit at a negative pressure of approximately 650-700 Torr for up to 1 min. Samples were allowed to fully absorb into the SLE plate for 5 min. Prior to sample elution, a 2 mL 96-well TrueTaper collection plate was placed in the vacuum manifold as a collection vessel. Samples were evaporated under a heated nitrogen stream at 40 °C and reconstituted in 150 μL of acetonitrile:water:200 mM ammonium formate (90:5:5, v:v:v). The collection plate was covered with a silicone cap mat and vortexed at 900 rpm for 2 min. Final samples were centrifuged at 3000 rpm for 5 min at 4 °C and analysis was achieved by injecting 2 μL into the LC-MS / MS system.

[0334] ELISA measurement of total peptide tissue concentrations 96-well plates were coated with 0.1 μM BSA-labeled peptides prepared in 0.2 M carbonate / bicarbonate buffer (pH 9.4), 100 μL / well, and incubated overnight at 4 °C. Plates were washed 4 times with ELISA wash buffer (PBS + 0.05% Tween 20), incubated with blocking buffer (PBS + 5% milk powder + 0.05% Tween 20) (300 μL / well) for 2 h at room temperature, and washed 4 times with ELISA wash buffer. Concurrently, 2x Compound 7 / Compound 13 standards (in their respective tissue matrices) or sample tumor homogenates diluted in antibody diluent (PBS + 2% milk powder + 0.05% Tween 20) were preincubated with 1–10 ng / mL primary antibody specific for Pv1 peptide for 30 min at room temperature. Preincubated samples were added, 100 μL / well, to the precoated and preblocked assay plates and incubated for 1 h at room temperature. Plates were washed 4 times with ELISA wash buffer and incubated with 100 μL / well of secondary goat anti-mouse IgG HRP antibody (1:5,000 in antibody diluent) for 1 hour at room temperature. Plates were washed 4 times with ELISA wash buffer and incubated with 100 μL / well of SuperSignal substrate for 1 minute at room temperature with gentle shaking. Luminescence was read from plates on a BioTek Cytation 5 plate reader.

[0335] FIG. 9A shows plots of peptide concentrations in tumors following a single 10 mg / kg IP dose of either compound 7 or compound 13 in female nude mice bearing HCT116 colorectal tumors (data presented as mean±standard deviation).

[0336] FIG. 9B shows a plot of MMAE concentration in tumors following a single 10 mg / kg IP dose of either compound 7 or compound 13 in female nude mice bearing HCT116 colorectal tumors (data presented as mean±standard deviation).

[0337] The data demonstrate that while both conjugates insert similarly into the tumor, compound 13 is more labile, releasing 30-40 times more warheads into the tumor compared to compound 7.

[0338] Example J: Efficacy of Compound 13 in the HT-29 Colorectal Xenograft Model Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system. Human HT-29 cells derived from colorectal cancer were diluted 1:1 in phenol red-free Matrigel and cultured at 2.5 × 10 cells / 100 μL. 6 The cells were subcutaneously implanted into the left flank of each mouse at a density of 100–200 mm. 3 Upon reaching an average volume of 100 μL, mice were randomized into groups and treated as detailed in the table below. Mice were administered vehicle or a dose of 5 mg / kg compound 13 intraperitoneally (IP). Doses were prepared by diluting a 0.1 mg / μL DMSO stock in 5% mannitol / citrate buffer and administered at a volume of 12 mL / kg (300 μL per 25 g mouse) on days 0-3, 5, and 16-19. Xenograft tumors were measured with calipers and volumes were calculated using the equation for ellipsoid volume: volume = π / 6 x (length) x (width). 2 Animals were killed when their tumor size reached 2000 mm 3 Rats were removed from the study if their body weight loss exceeded 100% or if their body weight loss exceeded 20%. The table below shows the dosing schedule for the various treatment groups. [Table 11]

[0339] 10A shows a plot of the mean tumor volume resulting from dosing nude mice bearing HT-29 colorectal flank tumors with 5 mg / kg of compound 13. Animals were dosed intraperitoneally once daily on days 0-3, 5, and 16-19.

[0340] Figure 10B presents the percent change in body weight of the animals in this study. Data are expressed as mean ± SEM.

[0341] Animals administered compound 13 experienced no change in body weight. These data demonstrate that compound 13 exhibits potent antitumor activity and safety in a preclinical colorectal cancer model.

[0342] Example K: Efficacy of Compound 7 in the HT-29 Colorectal Xenograft Model Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system. Human HT-29 cells derived from colorectal cancer were diluted 1:1 in phenol red-free Matrigel and cultured at 2.5 × 10 cells / 100 μL. 6 The cells were then subcutaneously implanted into the left flank of each mouse at a cell density of 100–200 mm. 3 Upon reaching an average volume of 1000 mg / kg, mice were randomized into groups and treated as detailed in the table below. Mice were administered intraperitoneally (IP) with a dose of vehicle, 40 mg / kg compound 7, or 80 mg / kg compound 7. Doses were prepared by diluting a 0.1 mg / μL DMSO stock in 5% mannitol / citrate buffer and administered QD×4 / week for 2 weeks at a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured with calipers and volumes were calculated using the equation for ellipsoid volume: volume=π / 6×(length)×(width). 2 Animals were killed when their tumor size reached 2000 mm 3 Rats were removed from the study if their body weight loss exceeded 100% or if their body weight loss exceeded 20%. The table below shows the dosing schedule for the various treatment groups. [Table 12]

[0343] 11A shows a plot of the mean tumor volume resulting from dosing nude mice bearing HT-29 colorectal flank tumors with 40 mg / kg and 80 mg / kg of Compound 7. Animals were dosed parenterally once daily for 4 consecutive days per week for 2 weeks.

[0344] Figure 11B presents the percent change in body weight of the animals in this study. Data are expressed as mean ± SEM.

[0345] These data demonstrate that compound 7 demonstrates efficacy and safety in the HT-29 model at higher doses compared to compound 13, consistent with the different release profiles of the two conjugates.

[0346] Example L: Tissue Pharmacokinetics of Compound 13, Compound 1, and Compound 2 in a Mouse Model Animal Medication Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system (Innovive). Human HCT116 cancer cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and cultured at 2.5 × 10 cells / 100 μL. 6 The cells were then subcutaneously implanted into the left flank of each mouse at a density of 100 x 100 mm. 3 When the tumor reached a minimum volume of 100 μg / kg, the mice were administered a single intraperitoneal injection of 10 mg / kg of Compound 13, Compound 1, or Compound 2 in a 5% mannitol / citric acid vehicle. Tumors were harvested at 4 and 24 hours after compound administration. MMAE concentrations in the tumors were measured by LCMS and peptide concentrations were measured by ELISA.

[0347] LC-MS / MS measurement of tissue MMAE concentrations Thawed tissue samples kept on wet ice were adjusted to 100 mg / mL with PBS based on tissue weight. Samples were homogenized in a Precellys Evolution machine at 7200 rpm for 2 x 30 sec cycles (with 10 sec pause between cycles). Homogenates were centrifuged at 14,000 rpm for 5 min at 4°C and the supernatants were transferred to 2 mL clean LoBind Eppendorf tubes. A volume of 100 μL of homogenate was added to 2 mL of a clean 96-well polypropylene plate, followed by 75 μL of ammonium formate buffer (pH 6.9) and 25 μL of working internal standard (WIS). Double blank controls received 75 μL of water:acetonitrile:formic acid (1:1:0.001, v:v:v) without internal standard. Fortified samples were covered with a silicone cap mat and vortexed at 700 rpm for 2 min. Working with a negative pressure manifold, 200 μL of enriched matrix sample was added to a supported liquid extraction (SLE) plate and the sample was passed through the plate frit at a negative pressure of approximately 650-700 Torr for up to 1 min. Samples were allowed to fully absorb into the SLE plate for 5 min. Prior to sample elution, a 2 mL 96-well TrueTaper collection plate was placed in the vacuum manifold as a collection vessel. Samples were evaporated under a heated nitrogen stream at 40 °C and reconstituted in 150 μL of acetonitrile:water:200 mM ammonium formate (90:5:5, v:v:v). The collection plate was covered with a silicone cap mat and vortexed at 900 rpm for 2 min. Final samples were centrifuged at 3000 rpm for 5 min at 4 °C and analysis was achieved by injecting 2 μL into the LC-MS / MS system.

[0348] ELISA measurement of total peptide tissue concentrations 96-well plates were coated with 0.1 μM BSA-labeled peptides prepared in 0.2 M carbonate / bicarbonate buffer (pH 9.4), 100 μL / well, and incubated overnight at 4 °C. Plates were washed 4 times with ELISA wash buffer (PBS + 0.05% Tween 20), incubated with blocking buffer (PBS + 5% milk powder + 0.05% Tween 20) (300 μL / well) for 2 h at room temperature, and washed 4 times with ELISA wash buffer. Concurrently, 2x Compound 7 / Compound 13 standards (in their respective tissue matrices) or sample tumor homogenates diluted in antibody diluent (PBS + 2% milk powder + 0.05% Tween 20) were preincubated with 1–10 ng / mL primary antibody specific for Pv1 peptide for 30 min at room temperature. Preincubated samples were added, 100 μL / well, to the precoated and preblocked assay plates and incubated for 1 h at room temperature. Plates were washed 4 times with ELISA wash buffer and incubated with 100 μL / well of secondary goat anti-mouse IgG HRP antibody (1:5,000 in antibody diluent) for 1 hour at room temperature. Plates were washed 4 times with ELISA wash buffer and incubated with 100 μL / well of SuperSignal substrate for 1 minute at room temperature with gentle shaking. Luminescence was read from plates on a BioTek Cytation 5 plate reader.

[0349] FIG. 12A shows plots of peptide concentrations in tumors following a single 10 mg / kg intraperitoneal administration of Compound 13, Compound 1, or Compound 2 to female nude mice bearing HCT116 colorectal tumors (data are presented as mean±standard deviation).

[0350] FIG. 12B shows a plot of MMAE concentration in tumors following a single 10 mg / kg intraperitoneal administration of Compound 13, Compound 1, or Compound 2 to female nude mice bearing HCT116 colorectal tumors (data are presented as mean±standard deviation).

[0351] The data demonstrate that while the conjugates insert similarly into the tumor, Compound 1 and Compound 2 release intermediate levels of MMAE compared to Compound 13.

[0352] Example M: ​​Tissue Pharmacokinetics of Compound 13, Compound 7, Compound 5, and Compound 6 in a Mouse Model Animal Medication Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system (Innovive). Human HCT116 cancer cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and cultured at 2.5 × 10 cells / 100 μL. 6 The cells were then subcutaneously implanted into the left flank of each mouse at a density of 100 x 100 mm. 3 When the tumor reached a minimum volume of 100 μg / kg, the mice were administered a single intraperitoneal injection of 10 mg / kg of Compound 13, Compound 7, Compound 5, or Compound 6 in a 5% mannitol / citric acid vehicle. Tumors were harvested at 4 and 24 hours after compound administration. MMAE concentrations in the tumors were measured by LCMS and peptide concentrations were measured by ELISA.

[0353] LC-MS / MS measurement of tissue MMAE concentrations Thawed tissue samples kept on wet ice were adjusted to 100 mg / mL with PBS based on tissue weight. Samples were homogenized in a Precellys Evolution machine at 7200 rpm for 2 x 30 sec cycles (with 10 sec pause between cycles). Homogenates were centrifuged at 14,000 rpm for 5 min at 4°C and the supernatants were transferred to 2 mL clean LoBind Eppendorf tubes. A volume of 100 μL of homogenate was added to 2 mL of a clean 96-well polypropylene plate, followed by 75 μL of ammonium formate buffer (pH 6.9) and 25 μL of working internal standard (WIS). Double blank controls received 75 μL of water:acetonitrile:formic acid (1:1:0.001, v:v:v) without internal standard. Fortified samples were covered with a silicone cap mat and vortexed at 700 rpm for 2 min. Working with a negative pressure manifold, 200 μL of enriched matrix sample was added to a supported liquid extraction (SLE) plate and the sample was passed through the plate frit at a negative pressure of approximately 650-700 Torr for up to 1 min. The sample was allowed to fully absorb into the SLE plate for 5 min. Prior to sample elution, a 2 mL 96-well TrueTaper collection plate was placed in the vacuum manifold as a collection vessel. Samples were evaporated under a heated nitrogen stream at 40 °C and reconstituted in 150 μL of acetonitrile:water:200 mM ammonium formate (90:5:5, v:v:v). The collection plate was covered with a silicone cap mat and vortexed at 900 rpm for 2 min. The final sample was centrifuged at 3000 rpm for 5 min at 4 °C and analysis was achieved by injecting 2 μL of sample into the LC-MS / MS system.

[0354] ELISA measurement of total peptide tissue concentrations 96-well plates were coated with 0.1 μM BSA-labeled peptides prepared in 0.2 M carbonate / bicarbonate buffer (pH 9.4), 100 μL / well, and incubated overnight at 4 °C. Plates were washed 4 times with ELISA wash buffer (PBS + 0.05% Tween 20), incubated with blocking buffer (PBS + 5% milk powder + 0.05% Tween 20) (300 μL / well) for 2 h at room temperature, and washed 4 times with ELISA wash buffer. Concurrently, 2x Compound 7 / Compound 13 standards (in their respective tissue matrices) or sample tumor homogenates diluted in antibody diluent (PBS + 2% milk powder + 0.05% Tween 20) were preincubated with 1–10 ng / mL primary antibody specific for Pv1 peptide for 30 min at room temperature. Preincubated samples were added, 100 μL / well, to the precoated and preblocked assay plates and incubated for 1 h at room temperature. Plates were washed 4 times with ELISA wash buffer and incubated with 100 μL / well of secondary goat anti-mouse IgG HRP antibody (1:5,000 in antibody diluent) for 1 hour at room temperature. Plates were washed 4 times with ELISA wash buffer and incubated with 100 μL / well of SuperSignal substrate for 1 minute at room temperature with gentle shaking. Luminescence was read from plates on a BioTek Cytation 5 plate reader.

[0355] FIG. 13A shows plots of peptide levels in mouse tumors measured by ELISA and LCMS after a single 10 mg / kg intraperitoneal injection of Compound 13, Compound 7, Compound 5, or Compound 6 into female nude mice bearing HCT116 colorectal tumors (data are presented as mean ± standard deviation).

[0356] FIG. 13B shows plots of the levels of unconjugated MMAE in mouse tumors measured by ELISA and LCMS following a single 10 mg / kg intraperitoneal injection of Compound 13, Compound 7, Compound 5, or Compound 6 into female nude mice bearing HCT116 colorectal tumors (data are presented as mean ± standard deviation).

[0357] The data demonstrate that while the conjugates insert similarly into tumors, they release their payloads within the tumor with a wide range of kinetics.

[0358] Example N: Efficacy of Compound 5 in the HCT116 colorectal xenograft model Six-week-old female athymic nude Fox nu Mice were obtained from Taconic Labs (catalog no. NCRNU-F) and housed 5 / cage on Alpha-Dri bedding in a disposable cage system. Human HCT116 cells derived from colorectal cancer were diluted 1:1 in phenol red-free Matrigel and cultured at 2.5 × 10 cells / 100 μL. 6 The cells were then subcutaneously implanted into the left flank of each mouse at a cell density of 100–200 mm. 3 Upon reaching an average volume of 1000 mg / kg, mice were randomized into groups and treated as detailed in the table below. Mice were administered intraperitoneally (IP) with doses of vehicle, 1 mg / kg compound 5, 5 mg / kg compound 5, or 10 mg / kg compound 5. Doses were prepared by diluting 0.1 mg / μL DMSO stock in 5% mannitol / citrate buffer and administered QD×2 / week for 3 weeks at a volume of 12 mL / kg (300 μL per 25 g mouse). Xenograft tumors were measured with calipers and volumes were calculated using the equation for ellipsoid volume: volume=π / 6×(length)×(width). 2 Animals were killed when their tumor size reached 2000 mm 3 Rats were removed from the study if their body weight loss exceeded 100% or if their body weight loss exceeded 20%. The table below shows the dosing schedule for the various treatment groups. [Table 13]

[0359] 14A shows a plot of the mean tumor volume resulting from dosing nude mice bearing HCT116 colorectal carcinoma flank tumors with 1 mg / kg, 5 mg / kg, and 10 mg / kg of Compound 5. Animals were dosed parenterally once daily for 4 consecutive days per week.

[0360] Figure 14B presents the percent change in body weight of the animals in this study. Data are expressed as mean ± SEM.

[0361] These data demonstrate that compound 5 exhibits dose-responsive efficacy in the HCT116 model.

[0362] In addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each of the references cited in this application, including but not limited to all patents, patent applications, and publications, is hereby incorporated by reference in its entirety.

Claims

1. Formula (I) 【Chemistry 1】 [During the ceremony, R １ is a peptide, R ２ is the radical of the auristatin compound, L, i) 【Chemistry 2】 and ii) 【Transformation 3】 wherein the terminal S atom of the linker is bonded to a cysteine ​​residue of the peptide to form a disulfide bond; In the formula, G １ is bond, C ６－１０ Aryl, C ３－１４ cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G １ The above C ６－１０ aryl, the C ３－１４ cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Haloalkyl, CN, NO ２ , OR ａ , S.R. ａ , C(O)R ｂ , C(O)NR ｃ R ｄ , C(O)OR ａ , O.C.(O.)R ｂ , OC(O)NR ｃ R ｄ , C(=NR ｅ ) NR ｃ R ｄ , N.R. ｃ C (=NR ｅ ) NR ｃ R ｄ , N.R. ｃ R ｄ , N.R. ｃ C(O)R ｂ , N.R. ｃ C(O)OR ａ , N.R. ｃ C(O)NR ｃ R ｄ , N.R. ｃ S(O)R ｂ , N.R. ｃ S (O) ２ R ｂ , N.R. ｃ S (O) ２ NR ｃ R ｄ , S(O)R ｂ , S(O)NR ｃ R ｄ , S(O) ２ R ｂ , and S(O) ２ NR ｃ R ｄ and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from G １ The above C １－６ Alkyl substituents, the C ２－６ Alkenyl substituents, and the C ２－６ The alkynyl substituent is CN, NO ２ , OR ａ , S.R. ａ , C(O)R ｂ , C(O)NR ｃ R ｄ , C(O)OR ａ , O.C.(O.)R ｂ , OC(O)NR ｃ R ｄ , C(=NR ｅ ) NR ｃ R ｄ , N.R. ｃ C (=NR ｅ ) NR ｃ R ｄ , N.R. ｃ R ｄ , N.R. ｃ C(O)R ｂ , N.R. ｃ C(O)OR ａ , N.R. ｃ C(O)NR ｃ R ｄ , N.R. ｃ S(O)R ｂ , N.R. ｃ S (O) ２ R ｂ , N.R. ｃ S (O) ２ NR ｃ R ｄ , S(O)R ｂ , S(O)NR ｃ R ｄ , S(O) ２ R ｂ , and S(O) ２ NR ｃ R ｄ optionally substituted with 1, 2, or 3 substituents independently selected from G ２ が、-NR Ｇ C(O)-、-NR Ｇ -、-O-、-S-、-C(O)O-、-OC(O)-、 -NR Ｇ C(O)-, -OC(O)NR Ｇ -, and -S(O ２ ) - is selected from G ３ But C ６－１０ Aryl, C ３－１４ cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G ３ The above C ６－１０ aryl, the C ３－１４ cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Haloalkyl, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , C(=NR ｅ１ ) NR ｃ１ R ｄ１ , N.R. ｃ１ C (=NR ｅ１ ) NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , N.R. ｃ１ C(O)NR ｃ１ R ｄ１ , N.R. ｃ１ S(O)R ｂ１ , N.R. ｃ１ S (O) ２ R ｂ１ , N.R. ｃ１ S (O) ２ NR ｃ１ R ｄ１ , S(O)R ｂ１ , S(O)NR ｃ１ R ｄ１ , S(O) ２ R ｂ１ , and S(O) ２ NR ｃ１ R ｄ１ and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from ３ The above C １－６ Alkyl substituents, the C ２－６ Alkenyl substituents, and the C ２－６ The alkynyl substituent is CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , C(=NR ｅ１ ) NR ｃ１ R ｄ１ , N.R. ｃ１ C (=NR ｅ１ ) NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , N.R. ｃ１ C(O)NR ｃ１ R ｄ１ , N.R. ｃ１ S(O)R ｂ１ , N.R. ｃ１ S (O) ２ R ｂ１ , N.R. ｃ１ S (O) ２ NR ｃ１ R ｄ１ , S(O)R ｂ１ , S(O)NR ｃ１ R ｄ１ , S(O) ２ R ｂ１ , and S(O) ２ NR ｃ１ R ｄ１ optionally substituted with 1, 2, or 3 substituents independently selected from G ４ is -C(O)-, -NR Ｇ C(O)-, -NR Ｇ -, -O-, -S-, -C(O)O-, -OC(O)-, -NR Ｇ C(O)- and -S(O ２ ) - is selected from G ５ is bond, C ６－１０ Aryl, C ３－１４ cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G ５ The above C ６－１０ Aryl, C ３－１４ cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl are each selected from halo, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Haloalkyl, CN, NO ２ , OR ａ , S.R. ａ , C(O)R ｂ , C(O)NR ｃ R ｄ , C(O)OR ａ , O.C.(O.)R ｂ , OC(O)NR ｃ R ｄ , C(=NR ｅ ) NR ｃ R ｄ , N.R. ｃ C (=NR ｅ ) NR ｃ R ｄ , N.R. ｃ R ｄ , N.R. ｃ C(O)R ｂ , N.R. ｃ C(O)OR ａ , N.R. ｃ C(O)NR ｃ R ｄ , N.R. ｃ S(O)R ｂ , N.R. ｃ S (O) ２ R ｂ , N.R. ｃ S (O) ２ NR ｃ R ｄ , S(O)R ｂ , S(O)NR ｃ R ｄ , S(O) ２ R ｂ , and S(O) ２ NR ｃ R ｄ and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from ５ The above C １－６ Alkyl substituents, the C ２－６ Alkenyl substituents, and the C ２－６ The alkynyl substituent is CN, NO ２ , OR ａ , S.R. ａ , C(O)R ｂ , C(O)NR ｃ R ｄ , C(O)OR ａ , O.C.(O.)R ｂ , OC(O)NR ｃ R ｄ , C(=NR ｅ ) NR ｃ R ｄ , N.R. ｃ C (=NR ｅ ) NR ｃ R ｄ , N.R. ｃ R ｄ , N.R. ｃ C(O)R ｂ , N.R. ｃ C(O)OR ａ , N.R. ｃ C(O)NR ｃ R ｄ , N.R. ｃ S(O)R ｂ , N.R. ｃ S (O) ２ R ｂ , N.R. ｃ S (O) ２ NR ｃ R ｄ , S(O)R ｂ , S(O)NR ｃ R ｄ , S(O) ２ R ｂ , and S(O) ２ NR ｃ R ｄ optionally substituted with 1, 2, or 3 substituents independently selected from G ６ が、-NR Ｇ C(O)-、-NR Ｇ -、-O-、-S-、-C(O)O-、-OC(O)-、 -NR Ｇ C(O)-, -OC(O)NR Ｇ -, and -S(O ２ ) - is selected from G ７ が、-NR Ｇ C(O)-、-NR Ｇ -、-O-、-S-、-C(O)O-、-OC(O)-、 -NR Ｇ C(O)-, -OC(O)NR Ｇ -, and -S(O ２ ) - is selected from R ｓ and R ｔ each independently represents H, halo, C １－６ Alkyl, and C １－６ haloalkyl; or R ｓ and R ｔ each together with the C atom to which they are attached to form C ３－６ forming a cycloalkyl ring, R ｕ and R ｖ are independent, H, halo, C １－６ Alkyl, and C １－６ haloalkyl; R Ｇ are each independently H and C １－４ alkyl, R ａ , R ｂ , R ｃ , R ｄ , R ａ１ , R ｂ１ , R ｃ１ , and R ｄ１ are each independently H, C １－６ Alkyl, C １－６ Haloalkyl, C ２－６ Alkenyl, and C ２－６ alkynyl, wherein R ａ , R ｂ , R ｃ , R ｄ , R ａ１ , R ｂ１ , R ｃ１ , and R ｄ１ The above C １－６ alkyl, the C ２－６ alkenyl, and the C ２－６ Alkynyl is halo, C １－４ Alkyl, C １－４ Haloalkyl, C １－６ Haloalkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, CN, OR ａ２ , S.R. ａ２ , C(O)R ｂ２ , C(O)NR ｃ２ R ｄ２ , C(O)OR ａ２ , O.C.(O.)R ｂ２ , OC(O)NR ｃ２ R ｄ２ , N.R. ｃ２ R ｄ２ , N.R. ｃ２ C(O)R ｂ２ , N.R. ｃ２ C(O)NR ｃ２ R ｄ２ , N.R. ｃ２ C(O)OR ａ２ , C(=NR ｅ２ ) NR ｃ２ R ｄ２ , N.R. ｃ２ C (=NR ｅ２ ) NR ｃ２ R ｄ２ , S(O)R ｂ２ , S(O)NR ｃ２ R ｄ２ , S(O) ２ R ｂ２ , N.R. ｃ２ S (O) ２ R ｂ２ , N.R. ｃ２ S (O) ２ NR ｃ２ R ｄ２ , and S(O) ２ NR ｃ２ R ｄ２ optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from R ａ２ , R ｂ２ , R ｃ２ , and R ｄ２ are each independently H, C １－６ Alkyl, C １－６ Haloalkyl, C ２－６ Alkenyl, and C ２－６ alkynyl, wherein R ａ２ , R ｂ２ , R ｃ２ , and R ｄ２ The above C １－６ alkyl, the C １－６ haloalkyl, the C ２－６ alkenyl, and the C ２－６ Alkynyl is independently OH, CN, amino, halo, C １－６ Alkyl, C １－６ Alkoxy, C １－６ Haloalkyl, and C １－６ optionally substituted with 1, 2, or 3 substituents independently selected from haloalkoxy; R ｅ , R ｅ１ , and R ｅ２ are each independently H and C １－４ alkyl, m is 0, 1, 2, 3, or 4; n is 0 or 1; o is 0 or 1; p is 1, 2, 3, 4, 5, or 6; q is 0 or 1. or a pharmaceutically acceptable salt thereof.

2. Formula (I) 【Chemistry 4】 [During the ceremony, R １ is a peptide, R ２ is the radical of the auristatin compound, L is the following structure: 【Transformation 5】 wherein an S atom of the linker is bonded to a cysteine ​​residue of the peptide to form a disulfide bond; In the formula, G １ is bond, C ６－１０ Aryl, C ３－１４ cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G １ The above C ６－１０ aryl, the C ３－１４ cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Haloalkyl, CN, NO ２ , OR ａ , S.R. ａ , C(O)R ｂ , C(O)NR ｃ R ｄ , C(O)OR ａ , O.C.(O.)R ｂ , OC(O)NR ｃ R ｄ , C(=NR ｅ ) NR ｃ R ｄ , N.R. ｃ C (=NR ｅ ) NR ｃ R ｄ , N.R. ｃ R ｄ , N.R. ｃ C(O)R ｂ , N.R. ｃ C(O)OR ａ , N.R. ｃ C(O)NR ｃ R ｄ , N.R. ｃ S(O)R ｂ , N.R. ｃ S (O) ２ R ｂ , N.R. ｃ S (O) ２ NR ｃ R ｄ , S(O)R ｂ , S(O)NR ｃ R ｄ , S(O) ２ R ｂ , and S(O) ２ NR ｃ R ｄ and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from G １ The above C １－６ Alkyl substituents, the C ２－６ Alkenyl substituents, and the C ２－６ The alkynyl substituent is CN, NO ２ , OR ａ , S.R. ａ , C(O)R ｂ , C(O)NR ｃ R ｄ , C(O)OR ａ , O.C.(O.)R ｂ , OC(O)NR ｃ R ｄ , C(=NR ｅ ) NR ｃ R ｄ , N.R. ｃ C (=NR ｅ ) NR ｃ R ｄ , N.R. ｃ R ｄ , N.R. ｃ C(O)R ｂ , N.R. ｃ C(O)OR ａ , N.R. ｃ C(O)NR ｃ R ｄ , N.R. ｃ S(O)R ｂ , N.R. ｃ S (O) ２ R ｂ , N.R. ｃ S (O) ２ NR ｃ R ｄ , S(O)R ｂ , S(O)NR ｃ R ｄ , S(O) ２ R ｂ , and S(O) ２ NR ｃ R ｄ optionally substituted with 1, 2, or 3 substituents independently selected from R ｓ and R ｔ each independently represents H, halo, C １－６ Alkyl, and C １－６ haloalkyl; G ２ が、-NR Ｇ C(O)-、-NR Ｇ -、-O-、-S-、-C(O)O-、-OC(O)-、 -NR Ｇ C(O)-, -OC(O)NR Ｇ -, and -S(O ２ ) - is selected from G ３ But C ６－１０ Aryl, C ３－１４ cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl, wherein G ３ The above C ６－１０ aryl, the C ３－１４ cycloalkyl, the 5- to 14-membered heteroaryl, and the 4- to 14-membered heterocycloalkyl are each selected from halo, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Haloalkyl, CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , C(=NR ｅ１ ) NR ｃ１ R ｄ１ , N.R. ｃ１ C (=NR ｅ１ ) NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , N.R. ｃ１ C(O)NR ｃ１ R ｄ１ , N.R. ｃ１ S(O)R ｂ１ , N.R. ｃ１ S (O) ２ R ｂ１ , N.R. ｃ１ S (O) ２ NR ｃ１ R ｄ１ , S(O)R ｂ１ , S(O)NR ｃ１ R ｄ１ , S(O) ２ R ｂ１ , and S(O) ２ NR ｃ１ R ｄ１ and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from ３ The above C １－６ Alkyl substituents, the C ２－６ Alkenyl substituents, and the C ２－６ The alkynyl substituent is CN, NO ２ , OR ａ１ , S.R. ａ１ , C(O)R ｂ１ , C(O)NR ｃ１ R ｄ１ , C(O)OR ａ１ , O.C.(O.)R ｂ１ , OC(O)NR ｃ１ R ｄ１ , C(=NR ｅ１ ) NR ｃ１ R ｄ１ , N.R. ｃ１ C (=NR ｅ１ ) NR ｃ１ R ｄ１ , N.R. ｃ１ R ｄ１ , N.R. ｃ１ C(O)R ｂ１ , N.R. ｃ１ C(O)OR ａ１ , N.R. ｃ１ C(O)NR ｃ１ R ｄ１ , N.R. ｃ１ S(O)R ｂ１ , N.R. ｃ１ S (O) ２ R ｂ１ , N.R. ｃ１ S (O) ２ NR ｃ１ R ｄ１ , S(O)R ｂ１ , S(O)NR ｃ１ R ｄ１ , S(O) ２ R ｂ１ , and S(O) ２ NR ｃ１ R ｄ１ optionally substituted with 1, 2, or 3 substituents independently selected from R ｕ and R ｖ are independent, H, halo, C １－６ Alkyl, and C １－６ haloalkyl; G ４ が、-C(O)-、-NR Ｇ C(O)-、-NR Ｇ -、-O-、-S-、-C(O)O-、-OC(O)-、 -NR Ｇ C(O)- and -S(O ２ ) - is selected from R Ｇ are each independently H and C １－４ alkyl, R ａ , R ｂ , R ｃ , R ｄ , R ａ１ , R ｂ１ , R ｃ１ , and R ｄ１ are each independently H, C １－６ Alkyl, C １－６ Haloalkyl, C ２－６ Alkenyl, and C ２－６ alkynyl, wherein R ａ , R ｂ , R ｃ , R ｄ , R ａ１ , R ｂ１ , R ｃ１ , and R ｄ１ The above C １－６ alkyl, the C ２－６ alkenyl, and the C ２－６ Alkynyl is halo, C １－４ Alkyl, C １－４ Haloalkyl, C １－６ Haloalkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, CN, OR ａ２ , S.R. ａ２ , C(O)R ｂ２ , C(O)NR ｃ２ R ｄ２ , C(O)OR ａ２ , O.C.(O.)R ｂ２ , OC(O)NR ｃ２ R ｄ２ , N.R. ｃ２ R ｄ２ , N.R. ｃ２ C(O)R ｂ２ , N.R. ｃ２ C(O)NR ｃ２ R ｄ２ , N.R. ｃ２ C(O)OR ａ２ , C(=NR ｅ２ ) NR ｃ２ R ｄ２ , N.R. ｃ２ C (=NR ｅ２ ) NR ｃ２ R ｄ２ , S(O)R ｂ２ , S(O)NR ｃ２ R ｄ２ , S(O) ２ R ｂ２ , N.R. ｃ２ S (O) ２ R ｂ２ , N.R. ｃ２ S (O) ２ NR ｃ２ R ｄ２ , and S(O) ２ NR ｃ２ R ｄ２ optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from R ａ２ , R ｂ２ , R ｃ２ , and R ｄ２ are each independently H, C １－６ Alkyl, C １－６ Haloalkyl, C ２－６ Alkenyl, and C ２－６ alkynyl, wherein R ａ２ , R ｂ２ , R ｃ２ , and R ｄ２ The above C １－６ alkyl, the C １－６ haloalkyl, the C ２－６ alkenyl, and the C ２－６ Alkynyl is independently OH, CN, amino, halo, C １－６ Alkyl, C １－６ Alkoxy, C １－６ Haloalkyl, and C １－６ optionally substituted with 1, 2, or 3 substituents independently selected from haloalkoxy; R ｅ , R ｅ１ , and R ｅ２ are each independently H and C １－４ alkyl, m is 0, 1, 2, 3, or 4; n is 0 or 1. or a pharmaceutically acceptable salt thereof.

3. R １ The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein is a peptide having 5 to 50 amino acids.

4. R １ crosses the cell membrane with an acidic or hypoxic mantle ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, which is a peptide capable of selectively delivering L-.

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

6. R １ But the following array ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1, Pv1), AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2, Pv2), and ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3, Pv3) 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, which is a peptide comprising at least one of:

7. R １ But at least the following array 2. The compound of claim 1, which is a peptide comprising ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1, Pv1), or a pharmaceutically acceptable salt thereof.

8. R １ But at least the following array 2. The compound of claim 1, which is a peptide comprising AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2, Pv2), or a pharmaceutically acceptable salt thereof.

9. R １ But at least the following array 2. The compound of claim 1, which is a peptide comprising ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3, Pv3), or a pharmaceutically acceptable salt thereof.

10. R ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: is a radical of a monomethyl auristatin compound.

11. R ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: is a radical of monomethylauristatin E.

12. R ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: is a radical of monomethylauristatin F.

13. R ２ But the following structure 【Transformation 6】 2. The compound of claim 1, wherein:

14. R ２ But the following structure 【Transformation 7】 2. The compound of claim 1, wherein:

15. R ２ But the following structure 【Transformation 8】 2. The compound of claim 1, wherein:

16. L is the following structure: 【Chemistry 9】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

17. L is the following structure: 【Chemistry 10】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

18. G １ is bond, C ６－１０ Aryl, C ３－１４ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the heteroaryl is selected from cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl.

19. G １ is a bond, phenyl, and C ４－６ 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: R is selected from the group consisting of cycloalkyl;

20. G １ is a bond and C ３－１４ 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: R is selected from the group consisting of cycloalkyl;

21. G １ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: is a bond.

22. G １ But C ３－１４ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

23. G １ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: is cyclopentyl or cyclohexyl, wherein said cyclopentyl and said cyclohexyl are each optionally fused to a phenyl group.

24. G １ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: is phenyl.

25. R ｓ and R ｔ are each independently H and C １－６ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

26. R ｓ and R ｔ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each is independently selected from H and isopropyl.

27. R ｓ and R ｔ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each is independently selected from H, methyl, and isopropyl.

28. R ｓ and R ｔ The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, together with the C atom to which they are attached, form a cyclobutyl ring.

29. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, or 2.

30. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 0.

31. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 2.

32. G ２ is —OC(O)— and —OC(O)NR Ｇ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

33. G ２ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is -OC(O)-.

34. G ３ But C ６－１０ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from aryl and 5- to 14-membered heteroaryl.

35. G ３ But C ６－１０ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

36. G ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: is phenyl.

37. R ｕ and R ｖ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each is H.

38. G ４ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is -OC(O)-.

39. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein n is 0.

40. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein n is 1.

41. G ５ However, the following base 【Chemistry 11】 2. The compound of claim 1, wherein:

42. G ６ But, -NR Ｇ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, which is C(O)-.

43. G ７ But, -NR Ｇ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, which is C(O)-.

44. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein o is 1.

45. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein p is 3.

46. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein p is 5.

47. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein q is 1.

48. R Ｇ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each is independently selected from H and methyl.

49. R Ｇ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each is H.

50. L is the following structure: 【Chemistry 12-1】 【Chemistry 12-2】 or a pharmaceutically acceptable salt thereof.

51. L is the following structure: 【Chemistry 13】 or a pharmaceutically acceptable salt thereof.

52. Formula (II) 【Chemistry 14】 [During the ceremony, R １ is a peptide, R ２ is the radical of the auristatin compound, Ring Z is a monocyclic C ５－７ a cycloalkyl ring or a monocyclic 5- to 7-membered heterocycloalkyl ring; R Ｚ each independently represents a halo, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Haloalkyl, CN, NO ２ , OR ａ , S.R. ａ , C(O)R ｂ , C(O)NR ｃ R ｄ , C(O)OR ａ , O.C.(O.)R ｂ , OC(O)NR ｃ R ｄ , N.R. ｃ R ｄ , N.R. ｃ C(O)R ｂ , N.R. ｃ C(O)OR ａ , and N.R. ｃ C(O)NR ｃ R ｄ Selected from or Two adjacent R Ｚ together with the atoms to which they are attached form a fused monocyclic C ５－７ Cycloalkyl ring, fused monocyclic 5- to 7-membered heterocycloalkyl ring, fused C ６－１０ aryl ring, or a fused 6- to 10-membered heteroaryl ring, each of which is C １－６ Alkyl, halo, CN, NO ２ , OR ａ , S.R. ａ , C(O)R ｂ , C(O)NR ｃ R ｄ , C(O)OR ａ , O.C.(O.)R ｂ , OC(O)NR ｃ R ｄ , N.R. ｃ R ｄ , N.R. ｃ C(O)R ｂ , N.R. ｃ C(O)OR ａ , and N.R. ｃ C(O)NR ｃ R ｄ optionally substituted with 1, 2, or 3 substituents independently selected from R ａ , R ｂ , R ｃ , and R ｄ are H and C, respectively. １－４ Alkyl, C ２－４ Alkenyl, C ２－４ alkynyl, each of which is independently selected from halo, OH, CN, and NO ２ optionally substituted with 1, 2, or 3 substituents independently selected from p is 0, 1, 2, or 3.

2. The compound of claim 1, wherein:

53. R １ 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein is a peptide comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

54. R １ 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein: is Pv1, Pv2, or Pv3.

55. R １ But, R １ 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein the disulfide moiety of formula II is linked to the core through a cysteine ​​residue of formula II, wherein one of the sulfur atoms of the disulfide moiety of formula II is derived from the cysteine ​​residue.

56. R ２ But the following structure 【Chemistry 15】 53. The compound of claim 52, having the formula: or a pharmaceutically acceptable salt thereof.

57. R ２ But the following structure 【Chemistry 16】 53. The compound of claim 52, having the formula: or a pharmaceutically acceptable salt thereof.

58. R ２ is attached to the core via an N atom, or a pharmaceutically acceptable salt thereof.

59. Ring Z is a monocyclic C ５－７ 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, which is a cycloalkyl ring.

60. 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein ring Z is a cyclopentyl ring.

61. 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein ring Z is a cyclohexyl ring.

62. Two adjacent R Ｚ together with the atoms to which they are attached form a fused monocyclic C ５－７ Cycloalkyl ring, fused monocyclic 5- to 7-membered heterocycloalkyl ring, fused C ６－１０ aryl ring, or a fused 6- to 10-membered heteroaryl ring, each of which is C １－４ Alkyl, halo, CN, NO ２ , OR ａ , S.R. ａ , C(O)R ｂ , C(O)NR ｃ R ｄ , C(O)OR ａ , O.C.(O.)R ｂ , OC(O)NR ｃ R ｄ , N.R. ｃ R ｄ , N.R. ｃ C(O)R ｂ , N.R. ｃ C(O)OR ａ , and N.R. ｃ C(O)NR ｃ R ｄ 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, optionally substituted with 1, 2, or 3 substituents independently selected from:

63. 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein p is 0.

64. 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein p is 1.

65. 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein p is 2.

66. 53. The compound of claim 52, or a pharmaceutically acceptable salt thereof, wherein p is 3.

67. Formula (III) or Formula (IV) 【Chemistry 17】 53. The compound of claim 52, having the formula: or a pharmaceutically acceptable salt thereof. 【Request Item 68】 【Chemistry 18-1】 【Chemistry 18-2】 【Chemistry 18-3】 【Chemistry 18-4】 【Chemistry 18-5】 【Chemistry 18-6】 【Chemistry 18-7】 and pharmaceutically acceptable salts thereof, wherein Pv1 is the following array ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1), Pv2 is the following array A peptide comprising: AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2); Pv3 is the following array ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3) 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof. 【Request Item 69】 【Chemistry 19-1】 【Chemistry 19-2】 【Chemistry 19-3】 【Chemistry 19-4】 【Chemistry 19-5】 【Chemistry 19-6】 【Chemistry 19-7】 and pharmaceutically acceptable salts thereof, wherein Pv1 is the following array ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO: 1), Pv2 is the following array A peptide comprising: AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO: 2); Pv3 is the following array ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO: 3) 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof.

70. 70. A pharmaceutical composition comprising a compound according to any one of claims 1 to 69 or a pharmaceutically acceptable salt thereof.

71. A pharmaceutical for treating cancer in a patient in need thereof, comprising the compound according to any one of claims 1 to 69 or a pharmaceutically acceptable salt thereof.

72. 72. The pharmaceutical composition of claim 71, wherein the cancer is selected from bladder cancer, bone cancer, glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, epithelial cancer, esophageal cancer, Ewing's sarcoma, pancreatic cancer, gallbladder cancer, gastric cancer, gastrointestinal tumors, head and neck cancer, intestinal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liver cancer, lung cancer, melanoma, prostate cancer, rectal cancer, renal clear cell carcinoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer.

73. 72. The method of claim 71, wherein the cancer is selected from lung cancer, colorectal cancer, and prostate cancer.

74. The pharmaceutical composition of claim 73, wherein the lung cancer is non-small cell lung cancer.

75. 72. The pharmaceutical agent of claim 71, wherein the cancer is selected from Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), diffuse large B-cell lymphoma (DLBCL), ovarian cancer, urothelial carcinoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer, squamous non-small cell lung cancer (sqNSCLC), squamous head and neck cancer, non-Hodgkin's lymphoma, pancreatic cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), fallopian tube cancer, and peritoneal cancer.

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