Peptide conjugates of cytotoxins as therapeutics

PEPTIDE CONJUGATES OF CYTOTOXINS AS THERAPEUTIC

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

Cancer is a group of diseases characterized by aberrant control of cell growth. It is estimated that the annual incidence of cancer exceeds 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 will die of cancer each year.

The treatment of cancer in humans by the systemic administration of pharmaceutical agents often works by slowing or stopping the uncontrolled replication that is a characteristic of cancer cells. One class of such agents are topoisomerase I inhibitors. Topoisomerase 1 enzymes function to relax supercoiled DNA and relieve DNA helical constraints and play a role in transcriptional regulation. See Li, M., Genomics Proteomics Bioinformatics 14 (2016), 166-171. Topoisomerase I is essential for development in the mammary system due to its dynamic roles in DNA replication and transcription. However, due to its direct role in transcriptional regulation, topoisomerase I dysfunction can lead to abnormal cellular functions. See Li, M., Genomics Proteomics Bioinformatics 14 (2016), 166-171. Therefore, several human diseases, such as cancer, neurodegenerative diseases, and autoimmune diseases, are linked to the regulation and activity of topoisomerase I.

Topoisomerase I inhibitors have been and continue to be developed as anticancer agents. In particular, topoisomerase I inhibitors are widely used for the treatment of colorectal, gastric and other cancers. See Ogitani, Bioorg. Med. Chem. Lett. 26 (2016), 5069-5072. Although topoisomerase I inhibitors are useful in the treatment of cancer, the compounds

I give also exhibit side effects, including neutropenia and severe diarrhea. Preferential delivery of topoisomerase inhibitors to these diseased tissues could avoid these serious side effects. Therefore, there is a need for a more selective delivery of topoisomerase I inhibitors to diseased tissue.

BRIEF DESCRIPTION OF THE INVENTION

The present description provides, inter alia, a compound of Formula (I):

r⁸-qr⁷ (I) or a pharmaceutically acceptable salt thereof, wherein the constituent variables are defined herein.

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

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

The present disclosure also provides the use of a compound described herein in the manufacture of a medicament for use in therapy. The present description also provides the compounds described herein for use in therapy.

The present disclosure also provides methods for synthesizing the compounds of the disclosure and intermediates useful in these methods.

BRIEF DESCRIPTION OF THE FIGURES

The FIG. 1 shows a plot of the plasma concentration of Compound 11 and exatecan released after a single 5 mg/kg IV dose of Compound 11 in a rat (data are expressed as mean ± SEM).

The FIG. 2 shows a plot of tumor and bone marrow peptide concentration after a single 10 mg/kg IP dose of Compound 11 in a mouse (data are expressed as mean ± SEM).

Three

The FIG. 3 shows a graph of total bone marrow counts from the femurs of tumor-bearing nude mice after dosing of 2.6 and 5.2 pmoles/kg of either Compound 11 (equivalent to 10, 20 mg/kg conjugate) or exatecan. free (equivalent to 1.15 and 2.3 mg/kg exatecan) dosed once daily for four days (data are expressed as mean ± SEM).

The FIG. 4A shows the stomachs of tumor-bearing nude mice excised after dosing of vehicle or 5.2 pmoles/kg of either Compound 11 (equivalent to 20 mg/kg conjugate) or free exatecan (equivalent to 2.3 mg/kg exatecan). dosed once daily for four days.

The FIG. 4B shows the stomachs of nude mice bearing tumor in situ after dosing of 5.2 pmoles/kg of either Compound 11 (equivalent to 20 mg/kg conjugate) or free exatecan (equivalent to 2.3 mg/kg exatecan) dosed once a day for four days.

The FIG. 5A shows a plot of mean tumor volume resulting from equimolar amounts of dosing of either free exatecan or Compound 11 in nude mice bearing HCT116 colorectal flank tumors. Animals were dosed once a day four times a week intraparenterally for three weeks.

The FIG. 5B displays a Kaplan Meier survival curve for equimolar amounts of dosing of either free exatecan or Compound 11 in nude mice bearing HCT116 colorectal flank tumors.

The FIG. 6A shows the single-agent efficacy of Compound 11 in nude mice bearing MKN45 HER2-negative gastric cancer flank tumors. Animals were dosed once a day four times a week intraparenterally for two weeks.

The FIG. 6B displays a Kaplan Meier survival curve for equimolar amounts of dosing of either free exatecan or Compound 11 in nude mice bearing MKN45 HER2-negative gastric cancer flank tumors.

The FIG. 7A shows a plot of the mean tumor volume resulting from dosing of Compound 11 in SCID mice bearing JIMT-1 intermediate HER2 breast cancer flank tumors. Animals were dosed once a day four times a week intraparenterally for three weeks.

The FIG. 7B shows a diagram of the percent change in body weight in

Four SCID mice bearing HER2 intermediate breast cancer flank tumors

JIMT-1 dosed with Compound 11.

The FIG. 8A shows a plot of mean tumor volume in nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11. Animals were dosed once daily four times weekly intraparenterally. for three weeks.

The FIG. 8B shows a plot of the percent change in body weight relative to day 0 in nude mice bearing triple negative MDA-MB-231 breast cancer flank tumors dosed with Compound 11.

The FIG. 9A shows a plot of the mean tumor volume of nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11 and talazoparib. Animals were dosed once daily four times weekly intraparenterally for three weeks with Compound 11 and once daily for 18 days orally with talazoparib.

The FIG. 9B shows a plot of the percent change in body weight relative to day 0 of nude mice bearing triple negative MDA-MB-231 breast cancer flank tumors dosed with Compound 11 and talazoparib.

The FIG. 10 shows a graph of the degradation of Compound 11 and Compound 29 resulting from treatment with 10 mM glutathione for 16 h. As shown in FIG. 10, Compound 29 is released much faster than Compound 11 under similar exposure to glutathione.

DETAILED DESCRIPTION OF THE INVENTION

A compound of Formula (I) is provided herein:

r⁸-qr⁷_w or a pharmaceutically acceptable salt thereof, wherein:

R.⁷ is a peptide;

R.⁸ is a small molecule topoisomerase I targeting portion, which binds topoisomerase I; and

Q is a linker, which is covalently linked to the R moiety⁷ and R⁸.

Also provided herein is a compound of Formula (I):

five r⁸-qr⁷ (I) or a pharmaceutically acceptable salt thereof, wherein:

R.⁷ is a peptide capable of selectively delivering R8Q- across a cell membrane having an acidic or hypoxic mantle having a pH less than about 6.0;

R.⁸ is a small molecule topoisomerase I targeting portion, which binds topoisomerase I; and

Q is a linker, which is covalently linked to the R moiety⁷ and R⁸.

A compound of Formula (I) is provided herein:

r⁸-qr⁷ (I) or a pharmaceutically acceptable salt thereof, wherein:

R.⁷ is a peptide;

R.⁸ is selected from the group consisting of:

<img file="ECSP22010228A_D0001.tif" />

<img file="ECSP22010228A_D0002.tif" />

Six

<img file="ECSP22010228A_D0003.tif" />

<img file="ECSP22010228A_D0004.tif" />

<img file="ECSP22010228A_D0005.tif" />

<img file="ECSP22010228A_D0006.tif" />

<img file="ECSP22010228A_D0007.tif" />

Seven

<img file="ECSP22010228A_D0008.tif" />

Eight

<img file="ECSP22010228A_D0009.tif" />

<img file="ECSP22010228A_D0010.tif" />

<img file="ECSP22010228A_D0011.tif" />

<img file="ECSP22010228A_D0012.tif" />

Nine

Q is a linker, which is covalently linked to the R moiety⁷ and R⁸.

A compound of Formula (I) is provided herein:

r⁸-qr⁷_(he) or a pharmaceutically acceptable salt thereof, wherein:

R.⁷ is a peptide;

R.⁸ is selected from the group consisting of:

<img file="ECSP22010228A_D0013.tif" />

<img file="ECSP22010228A_D0014.tif" />

<img file="ECSP22010228A_D0015.tif" />

Ten

<img file="ECSP22010228A_D0016.tif" />

Eleven

<img file="ECSP22010228A_D0017.tif" />

voice

<img file="ECSP22010228A_D0018.tif" />

Q is a linker, which is covalently linked to the R moiety⁷ and R⁸.

A compound of Formula (I) is provided herein:

R.⁸—Q—R⁷ (I) or a pharmaceutically acceptable salt thereof, wherein:

R.⁷ is a peptide;

R.⁸ is selected from the group consisting of:

<img file="ECSP22010228A_D0019.tif" />

Thirteen

<img file="ECSP22010228A_D0020.tif" />

<img file="ECSP22010228A_D0021.tif" />

<img file="ECSP22010228A_D0022.tif" />

<img file="ECSP22010228A_D0023.tif" />

Fourteen

<img file="ECSP22010228A_D0024.tif" />

Fifteen

<img file="ECSP22010228A_D0025.tif" />

<img file="ECSP22010228A_D0026.tif" />

<img file="ECSP22010228A_D0027.tif" />

<img file="ECSP22010228A_D0028.tif" />

Q is selected from the group consisting of

R1 R2

<img file="ECSP22010228A_D0029.tif" />

<img file="ECSP22010228A_D0030.tif" />

Sixteen

<img file="ECSP22010228A_D0031.tif" />

<img file="ECSP22010228A_D0032.tif" />

seventeen-seventeen-

<img file="ECSP22010228A_D0033.tif" />

<img file="ECSP22010228A_D0034.tif" />

<img file="ECSP22010228A_D0035.tif" />

<img file="ECSP22010228A_D0036.tif" />

Eighteen-

<img file="ECSP22010228A_D0037.tif" />

<img file="ECSP22010228A_D0038.tif" />

<img file="ECSP22010228A_D0039.tif" />

<img file="ECSP22010228A_D0040.tif" />

Nineteen·

<img file="ECSP22010228A_D0041.tif" />

<img file="ECSP22010228A_D0042.tif" />

;

R.¹,R²,R³,R⁴,R⁵,R⁶,R⁹,R¹⁰,R¹¹, and R¹² are each independently selected from H, C1-4 alkyl, C1-4 alkenyl, C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,

NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1, wherein said C1-4 alkyl, C1-4 alkenyl, C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl, and 4-10 membered heterocycloalkyl, are each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R¹ and R² together with the carbon atom to which they are attached form a 4-14 membered C3-14 cycloalkyl group or heterocycloalkyl group, each optionally substituted with 1,2, or 3 substituents independently selected from C1-4 alkyl, halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R¹ and R³ together with the carbon atoms to which they are attached form a 4-14 membered C3-14 cycloalkyl group or heterocycloalkyl group, each optionally substituted with 1, 2, or 3 substituents independently selected from C1-4 alkyl, halo , CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R² and R³ together with the carbon atoms to which they are attached form a 4-14 membered C3-14 cycloalkyl group or heterocycloalkyl group, each optionally substituted with 1, 2, or 3 substituents independently selected from C1-4 alkyl, halo , CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R³ and R⁴ together with the carbon atom to which they are attached form a C3-14 cycloalkyl group or 4-14 membered heterocycloalkyl group, each optionally substituted with

Twenty

1,2, or 3 substituents independently selected from C1-4alkyl, halo, CN,

NO2,OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,

NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R⁵ and R6 together with the carbon atom to which they are attached form a C3-14 cycloalkyl group or 4-14 membered heterocycloalkyl group, each optionally substituted with 1,2, or 3 substituents independently selected from C1-4 alkyl, halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

R.¹³ is H or C1-6 alkyl;

A is H or C1-4 alkyl;

R.^a1,R^b1,R^c1, and R^d1 are each independently selected from H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, OH, CN, NO2, and CO2CH3; wherein said C1-6 alkyl and C2-6 alkenyl are each optionally substituted with OH, CN, NO2, or CO2CH3;

I Aril or \

I Heteroaryl I is C6-10 aryl or 5-10 membered heteroaryl; wherein the 5-10 membered heteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4 ring-forming heteroatoms independently selected from N, O, and S;

Ring G is a 4-14 membered C3-14 cycloalkyl or heterocycloalkyl group, each optionally substituted with 1, 2, or 3 substituents independently selected from C1-4 alkyl, halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

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

[N,O] is NH or O;

[C,N,O] is CR^XR.^AND, NH, or O; and each RX and RY are independently selected from H and C1-4 alkyl.

A compound of Formula (I) is provided herein:

twenty-one8_q_r7_(|) or a pharmaceutically acceptable salt thereof, wherein:

R.⁷ is a peptide;

R.⁸ is selected from the group consisting of:

<img file="ECSP22010228A_D0043.tif" />

<img file="ECSP22010228A_D0044.tif" />

Twenty two

<img file="ECSP22010228A_D0045.tif" />

Twenty three

<img file="ECSP22010228A_D0046.tif" />

Twenty four

<img file="ECSP22010228A_D0047.tif" />

Q is selected from the group consisting of

<img file="ECSP22010228A_D0048.tif" />

<img file="ECSP22010228A_D0049.tif" />

<img file="ECSP22010228A_D0050.tif" />

<img file="ECSP22010228A_D0051.tif" />

^R.4 R.₃

<img file="ECSP22010228A_D0052.tif" />

Twenty-five-

<img file="ECSP22010228A_D0053.tif" />

Twenty six-

<img file="ECSP22010228A_D0054.tif" />

Twenty seven

<img file="ECSP22010228A_D0055.tif" />

<img file="ECSP22010228A_D0056.tif" />

<img file="ECSP22010228A_D0057.tif" />

R.¹,R²,R³,R⁴,R⁵,R⁶,

R.⁹,

R.¹⁰,R¹¹, and R¹² are each independently selected from H, C1-4 alkyl, C1-4 alkenyl, C6-10 aryl, 5-10 membered heteroaryl, halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1,

OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1, wherein said C1-4 alkyl, C1-4 alkenyl, C6-10 aryl, and 5-10 membered heteroaryl are each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R¹ and R² together with the carbon atom to which they are attached form a C3-7 cycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and

twenty-eightNR^c1C(O)NR^c1R.^d1, or R¹ and R³ together with the carbon atom to which they are attached form a C3-7 cycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R² and R³ together with the carbon atom to which they are attached form a C3-7 cycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R³ and R⁴ together with the carbon atom to which they are attached form a C3-7 cycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R⁵ and R⁶ together with the carbon atom to which they are attached form a C3-7 cycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

R.¹³ is H or C1-6 alkyl;

A is H or C1-4 alkyl;

R.^a1,R^b1,R^c1, and R^d1 are each independently selected from H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, OH, CN, NO2, and CO2CH3; wherein said C1-6 alkyl and C2-6 alkenyl are each optionally substituted with OH, CN, NO2, or CO2CH;

<img file="ECSP22010228A_D0058.tif" />

Aryl or Heteroaryl is C6-10 aryl or 5-10 membered heteroaryl; wherein the 5-10 membered heteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4 ring-forming heteroatoms independently selected from

Twenty-nine starting from N, O, and S;

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

[N,O] is NH or O;

[C,N,O] is CR^XR.^AND, NH, or O; and each RX and RY are independently selected from H and C1-4 alkyl.

In some modalities, the left side of Q is attached to R8 and the right side of Q is attached to R⁷.

In some embodiments, a sulfur atom from the disulfide moiety of Q is part of a cysteine ​​residue of R⁷.

As used herein, "peptide" refers to a target moiety comprising a sequence of 10-50 amino acids, integrated from naturally occurring amino acid residues and optionally one or more non-naturally occurring amino acids. In some embodiments, the R peptide⁷ is a peptide of 20 to 40, 20 to 30 amino acids, or 30 to 40 residues. Peptides suitable for use in the compounds of the invention are those that can insert through a cell membrane via a conformational change or a change in secondary structure in response to environmental pH changes. In this way, the peptide can target acidic tissue and selectively translocate cell-impermeable polar molecules across cell membranes in response to low extracellular pH. In some embodiments, the peptide is capable of selectively delivering a conjugated portion (eg, R⁸Q-) across a cell membrane that has an acid or hypoxic mantle that has a pH less than about 6.0. In some embodiments, the peptide is capable of selectively delivering a conjugated portion (eg, R⁸Q-) across a cell membrane that has an acidic or hypoxic mantle that has a pH less than about 6.5. In some embodiments, the peptide is capable of selectively delivering a conjugated portion (eg, R⁸Q-) across a cell membrane that has an acid or hypoxic mantle that has a pH less than about 5.5. In some embodiments, the peptide is capable of selectively delivering a conjugated portion (eg, R⁸Q-) across a cell membrane having an acidic or hypoxic mantle having a pH between about 5.0 and about 6.0.

In certain embodiments, the R peptide⁷ includes a cysteine ​​residue that can form the binding site for a payload moiety (for example, R⁸Q-) that is

Thirty supplied through a cell membrane. In some modalities, R⁷ binds to Q via a cysteine ​​residue of R⁷. In some embodiments, the sulfur atom of the cysteine ​​residue may form part of the disulfide bond of the disulfide bond-containing Q linker.

Suitable peptides, which can change conformationally based on pH and insert through a cell membrane, are described, for example, in US Patents 8,076,451 and 9,289,508 (each of which are incorporated herein). for reference 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.

In some modalities, R⁷ is a peptide comprising at least one of the following sequences:

ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1; Pv1),

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

ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3);

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

AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID No. 5; Pv5);

where R⁷ binds to Q via a cysteine ​​residue of R⁷.

In some modalities, R⁷ is a peptide comprising at least one of the following sequences:

ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1; Pv1),

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

ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3), where R⁷ binds to Q via a cysteine ​​residue of R⁷.

In some modalities, R⁷ is a peptide comprising the sequence ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1; Pv1).

In some modalities, R⁷ is a peptide comprising the sequence

AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO. 2; Pv2).

In some modalities, R⁷ is a peptide comprising the sequence

ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3).

Thirty-one

In some modalities, R⁷ is a peptide comprising the sequence

Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO. 4; Pv4).

In some modalities, R⁷ is a peptide comprising the sequence

AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO. 5; Pv5).

In some modalities, R⁷ is a peptide consisting of the sequence

ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1; Pv1).

In some modalities, R⁷ is a peptide consisting of the sequence

AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO. 2; Pv2).

In some modalities, R⁷ is a peptide consisting of the sequence

ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO. 3; Pv3).

In some modalities, R⁷ is a peptide consisting of the sequence AcAAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO. 4; Pv4).

In some modalities, R⁷ is a peptide consisting of the sequence AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO. 5; Pv5).

In some modalities, R⁷ is a peptide comprising at least one sequence selected from SEQ ID NO: 6 to SEQ ID NO: 311 as shown in Table 1.

In some modalities, R⁷ is a peptide consisting of a sequence selected from SEQ ID NO: 6 to SEQ ID NO: 311 as shown in Table 1.

Table 1. R sequences.⁷ additional

<td>SEQ ID NO.</td><td>Sequence</td>

<td> 6</td><td>AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 7</td><td>AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT</td>

<td> 8</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT</td>

<td> 9</td><td>AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT</td>

<td> 10</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 11</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 12</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG</td>

<td> 13</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT</td>

<td> 14</td><td>AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT</td>

<td> 15</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG</td>

<td> 16</td><td>AKEQNPIYWARYADWLFTTPLLLLDLALLVDADECT</td>

Thirty-two

<td> 17</td><td>ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG</td>

<td> 18</td><td>ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG</td>

<td> 19</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT</td>

<td> 20</td><td>AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGT</td>

<td> 21</td><td>AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGT</td>

<td> 22</td><td>AAEQNPIYWARYADWLFTTALLLLLDLALLVDADEGT</td>

<td> 23</td><td>AAEQNPIYWARYADWLFTTTPLLLLELALLVDADEGT</td>

<td> 24</td><td>AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGT</td>

<td> 25</td><td>AAEQNPIIYWARYADWLFTDLPLLLLDLLALLVDADEGT</td>

<td> 26</td><td>GEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 27</td><td>GGEQNPIYWARYADWLFTTTPLLLDLLALLVDADEGTCG</td>

<td> 28</td><td>GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG</td>

<td> 29</td><td>GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 30</td><td>GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG</td>

<td> 31</td><td>AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT</td>

<td> 32</td><td>DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT</td>

<td> 33</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGCT</td>

<td> 34</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT</td>

<td> 35</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT</td>

<td> 36</td><td>AEQNPIYWARYADFLFTTPLLLLDLALLVDADET</td>

<td> 37</td><td>AEQNPIYFARYADWLFTTPLLLLDLALLVDADEGT</td>

<td> 38</td><td>AEQNPIYFARYADFLFTTPLLLLDLALLWDADET</td>

<td> 39</td><td>AKEDQNPYWARYADWLFTTPLLLLDLALLVDG</td>

<td> 40</td><td>ACEDQNPYWARYADWLFTTPLLLLDLALLVDG</td>

<td> 41</td><td>AEDQNPYWARYADWLFTTPLLLLDLALLVDCG</td>

<td> 42</td><td>AEDQNPYWARYADWLFTTTPLLLLELALLVECG</td>

<td> 43</td><td>AKEDQNPYWRAYADLFTPLTLLDLLALWDG</td>

<td> 44</td><td>ACEDQNPYWRAYADLFTPLTLLDLLALWDG</td>

<td> 45</td><td>ACDDQNPWRAYLDLLFPTDTLLLDLLW</td>

<td> 46</td><td>TEDADVLLALDLLLLPTTFLWD</td>

<td> 47</td><td>AEQNPIYWARYADWLFTTPL</td>

<td> 48</td><td>AEQNPIYWARYADWLFTTPCL</td>

<td> 49</td><td>ACEQNPIYWARYADWLFTTPL</td>

<td> 50</td><td>AEQNPIYFARYADWLFTTPL</td>

<td> 51</td><td>KEDQNPWARYADLLFPTTLAW</td>

<td> 52</td><td>ACEDQNPWARYADLLFPTTLAW</td>

<td> 53</td><td>ACEDQNPWARYADWLFPTTLLLLD</td>

<td> 54</td><td>ACEEQNPWARYAELLFPTTLAW</td>

<td> 55</td><td>ACEEQNPWARYAEWLFPTTLLLLE</td>

thirty thirty

<td> 56</td><td>ACEEQNPWARYLEWLFPTETTLLLEL</td>

<td> 57</td><td>GGEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT</td>

<td> 58</td><td>ACEQNPIY WARYADWLFTTPLLLLDLALLV</td>

<td> 59</td><td>WARYADWLFTTTPLLLLDLALLV DADEGTCG</td>

<td> 60</td><td>WARYADWLFTTTPLLLLDLALLV DADEGCT</td>

<td> 61</td><td>GGEQNPIY WARYADWLFTTPLLLLDLALLV DADEGTCG</td>

<td> 62</td><td>ACEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT</td>

<td> 63</td><td>AKEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT</td>

<td> 64</td><td>AKEQNPIY WARYADWLFTTPLLLLDLALLV DADECT</td>

<td> 65</td><td>AAEQNPIY WARYADWLFTTALLLLDLALLV DADEGT</td>

<td> 66</td><td>ACAEQNPIY WARYADWLFTTGLLLLDLALLV DADEGT</td>

<td> 67</td><td>AEQNPIY WARYADFLFTTALLLLDLALLV DADE_T</td>

<td> 68</td><td>AEQNPIY FARYADWLFTTPLLLLDLALLV DADEGT</td>

<td> 69</td><td>AEQNPIY FARYADFLFTTPLLLLDLALLW DADE_T</td>

<td> 70</td><td>AKEDQNP_Y WARYADWLFTTPLLLLDLALLV DG____</td>

<td> 71</td><td>ACEDQNP_Y WARYADWLFTTPLLLLDLALLV DG____</td>

<td> 72</td><td>AEDQNP_Y WARYADWLFTTPLLLLDLALLV DG____</td>

<td> 73</td><td>AEDQNP_Y WARYADWLFTTPLLLLELALLV ECG___</td>

<td> 74</td><td>AKEDQNP_Y WRAYAD_LFT_PLTLLDLLALW DG____</td>

<td> 75</td><td>ACEDQNP_Y WRAYAD_LFT_PLTLLDLLALW DG____</td>

<td> 76</td><td>AKEDQNDP_Y WARYADWLFTTPLLLLDLALLV G_____</td>

<td> 77</td><td>TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA</td>

<td> 78</td><td>GGEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT</td>

<td> 79</td><td>AEQNPIY WARYADWLFTTPL</td>

<td> 80</td><td>AEQNPIY WARYADWLFTTPCL</td>

<td> 81</td><td>ACEQNPIY WARYADWLFTTPL</td>

<td> 82</td><td>ACEQNPIY FARYADWLFTTPL</td>

<td> 83</td><td>ACDDQNP WRAYLDLLFPTDTLLLDLLW</td>

<td> 84</td><td>ACEEQNP WRAYLELLFPTETLLLELLW</td>

<td> 85</td><td>ACDDQNP WARYLDWLFPTDTLLLDL</td>

<td> 86</td><td>CDNNNP WRAYLDLLFPTDTLLLDW</td>

<td> 87</td><td>ACEEQNP WARYLEWLFPTETTLLLEL</td>

<td> 88</td><td>ACEDQNP WARYADWLFPTTLLLLD</td>

<td> 89</td><td>ACEEQNP WARYAEWLFPTTLLLLE</td>

<td> 90</td><td>ACEDQNP WARYADLLFPTTLAW</td>

<td> 91</td><td>ACEDQNP WARYAELLFPTTLW</td>

<td> 92</td><td>KEDQNP WARYADLLFPTTLW</td>

<td> 93</td><td>DDDEDNP IYWARYAHWLFTTPLLLLHGALLVDADECT</td>

<td> 94</td><td>DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT</td>

Thirty four-

<td> 95</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT</td>

<td> 96</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT</td>

<td> 97</td><td>DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT</td>

<td> 98</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGIG</td>

<td> 99</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET</td>

<td> 100</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT</td>

<td> 101</td><td>GGEQNPIYWARYADWLFTTTPLLLDLLALLVDADEGTCG</td>

<td> 102</td><td>GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG</td>

<td> 103</td><td>GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG</td>

<td> 104</td><td>AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 105</td><td>AAEQNPIYWARYAEWLFTTTPLLLLELALLVDADEGTCG</td>

<td> 106</td><td>GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 107</td><td>GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 108</td><td>GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 109</td><td>AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 110</td><td>ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG</td>

<td> 111</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT</td>

<td> 112</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT</td>

<td> 113</td><td>DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT</td>

<td> 114</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT</td>

<td> 115</td><td>DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT</td>

<td> 116</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT</td>

<td> 117</td><td>AAEQNPIYWARYADWLFTTTPLLLLELALLVDADEGTCG</td>

<td> 118</td><td>AAEQNPIYWARYAEWLFTTTPLLLLELALLVDADEGTCG</td>

<td> 119</td><td>AAEQNPIYWARYADWLFTTTPLLLLELALLVDADEGTKCG</td>

<td> 120</td><td>GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 121</td><td>GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 122</td><td>GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 123</td><td>GGEQNPIYWARYADWLFTTPLLLLDALLVNANQGT</td>

<td> 124</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT</td>

<td> 125</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT</td>

<td> 126</td><td>ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG</td>

<td> 127</td><td>GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 128</td><td>GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 129</td><td>GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 130</td><td>AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 131</td><td>AAEQNPIYWARYADWLFTDLPLLLLDLLALLVDADEGT</td>

<td> 132</td><td>GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG</td>

<td> 133</td><td>GGEQNPIYWARYADWLFTTTPLLLDLLALLVDADEGTCG</td>

Thirty-five

<td> 134</td><td>AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT</td>

<td> 135</td><td>AEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 136</td><td>GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 137</td><td>GGEQNPIYWAQDYAWLFTTPLLLLDLALLDADEGTCG</td>

<td> 138</td><td>GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 139</td><td>AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGTCG</td>

<td> 140</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG .........EGTK(rhodamine)C(phalloidin)G</td>

<td> 141</td><td>AAEQNPIYWARYADWLFTTTPLLLLELALLDADEGTKCG</td>

<td> 142</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 143</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC(phalloidin)G</td>

<td> 144</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 145</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET</td>

<td> 146</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG</td>

<td> 147</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT</td>

<td> 148</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT</td>

<td> 149</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT</td>

<td> 150</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT</td>

<td> 151</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 152</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC(phalloidin)G</td>

<td> 153</td><td>AAEQNPIYWARYADWLFTTTPLLLLELALLVDADEGTKCG</td>

<td> 154</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG</td>

<td> 155</td><td>DDDEDNPIYWARYAHWLFTTPLLLLBGALLVDADECT</td>

<td> 156</td><td>DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT</td>

<td> 157</td><td>DDDEDNPIYWARYAHWLFTTPLLLLBGALLVNADECT</td>

<td> 158</td><td>DDDEDNPIYWARYAHWLFTTPLLLLBGALLVNANECT</td>

<td> 159</td><td>DDDEDNPIYWARYADWLFTTPLLLLIBGALLVDADECT</td>

<td> 160</td><td>DDDEDNPIYWARYADWTFTTPLLLLHGALLVDADECT</td>

<td> 161</td><td>DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT</td>

<td> 162</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT</td>

<td> 163</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT</td>

<td> 164</td><td>DDDEDNPIYWARYHWLFTTPLLLLHGALLVNANECT</td>

<td> 165</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT</td>

<td> 166</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT</td>

<td> 167</td><td>DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT</td>

<td> 168</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT</td>

<td> 169</td><td>DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT</td>

<td> 170</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT</td>

<td> 171</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT</td>

Thirty-six

<td> 172</td><td>DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT</td>

<td> 173</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT</td>

<td> 174</td><td>DDDEDNPIYWARYAHMLFTTPLLLLDGALLVDADECT</td>

<td> 175</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT</td>

<td> 176</td><td>DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT</td>

<td> 177</td><td>DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT</td>

<td> 178</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT</td>

<td> 179</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT</td>

<td> 180</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT</td>

<td> 181</td><td>AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT</td>

<td> 182</td><td>GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG</td>

<td> 183</td><td>GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 184</td><td>GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 185</td><td>GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 186</td><td>AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 187</td><td>GGEQNPIYWARYADWLFTTPLLLLDALLVDADEGTCG</td>

<td> 188</td><td>GGEQNPIYWARYADWLFTTTPLLLDLLALLVDADEGTCG</td>

<td> 189</td><td>GGEQNPIYWARYADWLFTTTPLLLDLLALLVDADEGTCG</td>

<td> 190</td><td>GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG</td>

<td> 191</td><td>GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 192</td><td>GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 193</td><td>GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 194</td><td>GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 195</td><td>GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 196</td><td>GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 197</td><td>AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 198</td><td>GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 199</td><td>GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 200</td><td>GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 201</td><td>AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 202</td><td>AAEQNPIYWARYADWLFTTTPLLLLELALLVDADEGTKCG</td>

<td> 203</td><td>................EGTK(ridamine)C(phalloidin)G</td>

<td> 204</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG</td>

<td> 205</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG</td>

<td> 206</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC(phalloidin)G</td>

<td> 207</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG</td>

<td> 208</td><td>AAEQNPIYWARYADWLFTTTPLLLLELALLVDADEGTKCG</td>

<td> 209</td><td>AAEQNPIYWARYADWLFTDLPLLLLDLLALLVDADEGT</td>

<td> 210</td><td>AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG</td>

Thirty-seven

<td> 211</td><td>GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 212</td><td>GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 213</td><td>GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 214</td><td>AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 215</td><td>AAEQNPIYWARYAEWLFTTTPLLLLELALLVDADEGTCG</td>

<td> 216</td><td>AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGTCG</td>

<td> 217</td><td>AAEQNPIYWARYADWLFTTTPLLLLELALLVDADEGTCG</td>

<td> 218</td><td>AAEQNPIYWARYAEWLFTTTPLLLLELALLVDADEGTCG</td>

<td> 219</td><td>AAEQNPIYWARYADWLFTTTPLLLLELALLVDADEGTKCG</td>

<td> 220</td><td>ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG</td>

<td> 221</td><td>ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG</td>

<td> 222</td><td>AAEQNPIYWARYADWLFTTALLLLLDLALLVDADEGT</td>

<td> 223</td><td>AEQNPIYFARYADLLFPTTLAW</td>

<td> 224</td><td>AEQNPIYWARYADLLFPTTLAF</td>

<td> 225</td><td>AEQNPIYWARYADLLFPTTLAW</td>

<td> 226</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET</td>

<td> 227</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT</td>

<td> 228</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 229</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG</td>

<td> 230</td><td>AKEQNPIYWARYADWLFTTPLLLLDLALLVDADECT</td>

<td> 231</td><td>CCTCTTACCTCAGTTACA</td>

<td> 232</td><td>D-Arg8 D-Arg8-CCTCTTACCTCAGTTACA</td>

<td> 233</td><td>D-Lys4 D-Lys4-CCTCTTACCTCAGTTACA</td>

<td> 234</td><td>SS-CCTCTTACCTCAGTTACA</td>

<td> 235</td><td>SS-CCTCTGACCTCATTTACA</td>

<td> 236</td><td>D-Arg8-Deca D-Arg8-Deca-CCTCTTACCTCAGTTACA</td>

<td> 237</td><td>D-Arg8-Deca-mismatch D-Arg8-Deca-CCTCTGACCTCATTTACA</td>

<td> 238</td><td>SS-CCTCTTACCTCAGTTACA</td>

<td> 239</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 240</td><td>AEDQNPYWARYDWLFTTPLLLLDLALLVDCG</td>

<td> 241</td><td>AEDQNPYWARYADWLFTTTPLLLLELALLVECG</td>

<td> 242</td><td>AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGCT</td>

<td> 243</td><td>ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET</td>

<td> 244</td><td>AE-QN-PI YWARYADWLFTTPLLLLDLALLV DADEGT-COOH</td>

<td> 245</td><td>AEDQN-P-YWARYADWLFTTPLLLLDLALLV D---G--COOH</td>

<td> 246</td><td>AEDQNDP-YWARYADWLFTTPLLLLDLALLV----G--COOH</td>

<td> 247</td><td>AEQNPI YWARYADFLFTTPLLLLDLALLV DADET-COOH</td>

<td> 248</td><td>AEQNPI YFARYADWLFTTPLLLLDLALLV DADET-COOH</td>

<td> 249</td><td>AEQNPI YFARYADFLFTTPLLLLDLALLW DADET-COOH</td>

Thirty-eight-

<td> 250</td><td>AE-QN-PI YWARYADWLFTTPLLLLDLALLV DADEGCT-COOH</td>

<td> 251</td><td>AEDQN-PI YWARYADWLFTTPLLLLDLALLV DC--GT-COOH</td>

<td> 252</td><td>AEDQNDPI YWARYADWLFTTPLLLLELALLV EC--GT-COOH</td>

<td> 253</td><td>Chelated-ACEEQNPWARYLEWLFPTETLLLEL</td>

<td> 254</td><td>AEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT-COOH</td>

<td> 255</td><td>AKEDQNPY WARYADWLFTTPLLLLDLALLV DG-COOH</td>

<td> 256</td><td>AKEDQNDPY WARYADWLFTTPLLLLDLALLV G-COOH</td>

<td> 257</td><td>AEQNPI YWARYADWLFTTPLLLLDLALLV DADEGC-Biotin-T-COO H</td>

<td> 258</td><td>AEDQNP YWARYADWLFTTPLLLLDLALLV DC-Biotin-G-COOH</td>

<td> 259</td><td>AEDQNP YWARYADWLFTTTPLLLLELALLV EC-Biotin-G-COOH</td>

<td> 260</td><td>ACEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT</td>

<td> 261</td><td>ACEDQNPY WARYADWLFTTPLLLLDLALLV DG</td>

<td> 262</td><td>ACEDQNPY WRAYADLFTPLTLLDLLALW DG</td>

<td> 263</td><td>ACDDQNP WRAYLDLLFPTDTLLLDLLW</td>

<td> 264</td><td>WRAYLELLFPTETLLLELLW</td>

<td> 265</td><td>WARYLDWLFPTDTLLLDL</td>

<td> 266</td><td>WRAYLDLLFPTDTLLLDW</td>

<td> 267</td><td>WARYLEWLFPTETTLLLEL</td>

<td> 268</td><td>WAQYLELLFPTETLLEW</td>

<td> 269</td><td>WRAYLELLFPTETLLEW</td>

<td> 270</td><td>WARYADWLFPTTLLLLD</td>

<td> 271</td><td>WARYAEWLFPTTLLLLE</td>

<td> 272</td><td>ACEDQNP WARYADLLFPTTLAW</td>

<td> 273</td><td>ACEEQNP WARYAELLFPTTLAW</td>

<td> 274</td><td>Ac-TEDAD VLLALDLLLLPTTFLWDAYRAW YPNQECA-Am</td>

<td> 275</td><td>CDDDDDNPNY WARYANWLFTTPLLLLNGALLV EAEET</td>

<td> 276</td><td>CDDDDDNPNY WARYAPWLFTTPLLLLPGALLV EAEET</td>

<td> 277</td><td>Ac-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGCT</td>

<td> 278</td><td>Ac-AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG</td>

<td> 279</td><td>ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGT</td>

<td> 280</td><td>Ac-AAEQNPIYWARYADWLFTTTPLLLLELALLVDADEGTKCG</td>

<td> 281</td><td>DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADET</td>

<td> 282</td><td>CDDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADET</td>

<td> 283</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGT</td>

<td> 284</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADEGT</td>

<td> 285</td><td>DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANEGT</td>

<td> 286</td><td>AKEDQNDPYWARYADWLFTTPLLLLDLALLVG</td>

<td> 287</td><td>AEDQNPYWARYADWLFTTTPLLLLELALLVCG</td>

<td> 288</td><td>AKDDQNPWRAYLDLLFPTDTLLLDLLWC</td>

Thirty nine

<td> 289</td><td>ACEEQNPWRAYLELLFPTETLLLELLW</td>

<td> 290</td><td>ACDDQNPWARYLDWLFPTDTLLLDL</td>

<td> 291</td><td>CDNNNPWRAYLDLLFPTDTLLLDW</td>

<td> 292</td><td>CEEQQPWAQYLELLFPTETLLLEW</td>

<td> 293</td><td>EEQQPWRAYLELLFPTETLLLEW</td>

<td> 294</td><td>CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET</td>

<td> 295</td><td>CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEE</td>

<td> 296</td><td>AEQNPIYFARYADLLFPTTLAW</td>

<td> 297</td><td>AEQNPIYWARYADLLFPTTLAF</td>

<td> 298</td><td>AEQNPIYWARYADLLFPTTLAW</td>

<td> 299</td><td>KEDQNPWARYADLLFPTTLW</td>

<td> 300</td><td>ACEEQNPQAEYAEWLFPTTLLLLE</td>

<td> 301</td><td>AAEEQNPWARYLEWLFPTETTLLLEL</td>

<td> 302</td><td>AKEEQNPWARYLEWLFPTETTLLLEL</td>

<td> 303</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTGG</td>

<td> 304</td><td>XXEXNPIYWAXXXXXXLFTXXXLLLXXXALLVXAXXXTXG</td>

<td> 305</td><td>DAAEQNPIYWARYADWLFTTTLPLLLLDLLALLVDADEGTKGG</td>

<td> 306</td><td>GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTGG</td>

<td> 307</td><td>XXEXNPIYWAXXXXXXLFTXXXLLLXXXALLVXAXXXTGG</td>

<td> 308</td><td>DGGEQNDPIYWARYADWLFTTLPLLLDLLALLVDADEGCTXGG</td>

<td> 309</td><td>AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG</td>

<td> 310</td><td>AEDQNPYWARYDWLFTTPLLLLDLALLVDCG</td>

<td> 311</td><td>GLAGLAGLLGLEGLLGLPLGLLEGLWLGLELEGN</td>

Any of the foregoing peptides useful in the present invention can be modified to include a cysteine ​​residue by replacing a non-cysteine ​​residue with cysteine, or by appending a cysteine ​​residue to either the N-terminus or C-terminus.

In some embodiments, the R peptide⁷ it is a conformationally restricted peptide. A conformationally restricted peptide can include, for example, macrocyclic peptides and stapled peptides. A stapled peptide is a peptide restricted by a covalent bond between two amino acid side chains, forming 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; Thesaurus 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

Forty incorporates herein by reference in its entirety.

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

The Q moiety is a linking group, covalently connecting R⁷ and R⁸ which serves as a tether between the peptide and topoisomerase I inhibitor that can be cleaved when the conjugate or portion thereof is inside a cell. In some embodiments, Q is a chain of 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 atoms in the chain, which is optionally substituted with substituents 1-10 R.^what, and wherein one or more carbon atoms of Q may be oxidized to form a carbonyl (C=O), and wherein one or more N and S atoms in the chain may each be optionally oxidized to form an oxide group amine, sulfoxide or sulfonyl; where each R^what is independently selected from OH, CN, -COOH, NH2, halo, haloC1-6alkyl, C1-6alkyl, C1-6alkoxy, haloC1-6alkoxy, C1-6alkylthio, phenyl, 5-6 membered heteroaryl , 4-6 membered heterocycloalkyl, C3-6 cycloalkyl, NH(C1-6 alkyl) and N(Ci-6 alkyl)2, where C1-6 alkyl, phenyl, C3-6 cycloalkyl, 4-6 heterocycloalkyl membered, and 5-6 membered heteroaryl of R^what are each optionally substituted with halo, OH, CN, -COOH, NH2, C1-4 alkyl, C1-4 alkoxy, haloC1-4 alkyl, haloC1-4 alkoxy, phenyl, C3-10 cycloalkyl, 5- or 6-membered heteroaryl or 4-6 membered heterocycloalkyl; and two R groups^what together with the chain atoms to which they are attached they can form a phenyl, 5-6 membered heteroaryl, 4-6 membered heterocycloalkyl, or C3-6 cycloalkyl ring.

In some modalities, R^what is independently selected from OH, CN, -COOH, NH2, halo, haloC1-6alkyl, C1-6alkyl, C1-6alkoxy, haloC1-6alkoxy, NH(C1-6alkyl), and N(Ci-6alkyl). 6)2.

Forty-one

In some embodiments, Q is selected from:

<img file="ECSP22010228A_D0059.tif" />

<img file="ECSP22010228A_D0060.tif" />

<img file="ECSP22010228A_D0061.tif" />

<img file="ECSP22010228A_D0062.tif" />

<img file="ECSP22010228A_D0063.tif" />

Forty-two

<img file="ECSP22010228A_D0064.tif" />

<img file="ECSP22010228A_D0065.tif" />

<img file="ECSP22010228A_D0066.tif" />

<img file="ECSP22010228A_D0067.tif" />

forty three

<img file="ECSP22010228A_D0068.tif" />

<img file="ECSP22010228A_D0069.tif" />

<img file="ECSP22010228A_D0070.tif" />

<img file="ECSP22010228A_D0071.tif" />

forty four

<img file="ECSP22010228A_D0072.tif" />

<img file="ECSP22010228A_D0073.tif" />

<img file="ECSP22010228A_D0074.tif" />

In some embodiments, Q is:

R1 R2

<img file="ECSP22010228A_D0075.tif" />

In some embodiments, Q is:

<img file="ECSP22010228A_D0076.tif" />

In some embodiments, Q is:

Forty-five-

<img file="ECSP22010228A_D0077.tif" />

In some embodiments, Q is:

<img file="ECSP22010228A_D0078.tif" />

In some embodiments, Q is:

<img file="ECSP22010228A_D0079.tif" />

In some modalities:

R.¹,R²,R³, and R⁴ are each independently selected from H and Ci-4 alkyl, halo, CN, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1,

OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1, wherein said C1-4 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R¹ and R² together with the carbon atom to which they are attached form a C3-10 cycloalkyl group or 4-10 membered heterocycloalkyl group, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R¹ and R³ together with the carbon atom to which they are attached form a C3-10 cycloalkyl group or 4-10 membered heterocycloalkyl group, each optionally substituted with 1,2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R¹ and R⁴ Together with the carbon atom to which they are attached, they form a cycloalkyl group.

Forty-six

C3-10 or 4-10 membered heterocycloalkyl group, each optionally substituted with

1,2, or 3 substituents independently selected from halo, CN, NO2, OR^a1,

MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,

NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R² and R³ together with the carbon atom to which they are attached form a C3-10 cycloalkyl group or 4-10 membered heterocycloalkyl group, each optionally substituted with 1,2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R² and R⁴ together with the carbon atom to which they are attached form a C3-10 cycloalkyl group or 4-10 membered heterocycloalkyl group, each optionally substituted with 1,2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R³ and R⁴ together with the carbon atom to which they are attached form a C3-10 cycloalkyl group or 4-10 membered heterocycloalkyl group, each optionally substituted with 1,2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1.

In some modalities:

R.¹,R²,R³, and R⁴ they are each independently selected from H and C1-4 alkyl;

or R¹ and R² together with the carbon atom to which they are attached form a C3-10 cycloalkyl group or 4-10 membered heterocycloalkyl group, each optionally substituted with 1,2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R¹ and R³ together with the carbon atom to which they are attached form a C3-10 cycloalkyl group or 4-10 membered heterocycloalkyl group, each optionally substituted with 1,2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or R³ and R⁴ Together with the carbon atom to which they are attached, they form a cycloalkyl group.

Forty-seven C3-10 or 4-10 membered heterocycloalkyl group, each optionally substituted with

1,2, or 3 substituents independently selected from halo, CN, NO2, OR^a1,

MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,

NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1.

In some modalities, R¹ and R² are each independently selected from H and methyl, and R³,R⁴,R⁵, and R⁶are each hydrogen.

In some modalities, R¹,R²,R³, and R⁴ are each independently selected from H and methyl, and R⁵, and R⁶are each hydrogen.

In some modalities, R¹ and R² they are each independently selected from H and methyl.

In some modalities, R³ and R⁴ they are each independently selected from H and methyl.

In some modalities, R¹ and R² are each H.

In some modalities, R¹ and R² together with the carbon atom to which they are attached form a C3-7 cycloalkyl group optionally substituted with 1,2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1.

In some modalities, R¹ and R² Together with the carbon atom to which they are attached, they form a C3-7 cycloalkyl group.

In some modalities, R¹ and R² Together with the carbon atom to which they are attached, they form a cyclobutyl group.

In some modalities, R³ and R⁴ are each H.

In some modalities, R¹ and R³ together with the carbon atom to which they are attached form a C3-7 cycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1.

In some modalities, R¹ and R³ together with the carbon atom to which they are attached they form a cyclopentyl, cyclohexyl, cycloheptyl, 1,2,3,4-tetrahydronaphthyl, tetrahydrofuranyl, or tetrahydropyranyl.

In some modalities, R¹ and R³ together with the carbon atom to which they are attached

Forty-eight form a C3-7 cycloalkyl group.

In some modalities, R¹ and R³ Together with the carbon atom to which they are attached, they form a cyclohexyl group.

In some modalities, R² and R⁴ are each H.

In some modalities, R⁵ and R6 are each H.

In some modalities, R9, R¹⁰,R¹¹, and R¹² they are each independently selected from H and methyl.

In some embodiments, the compound of the invention is a compound of Formula (II):

<img file="ECSP22010228A_D0080.tif" />

s.

Mr⁷ (II) or a pharmaceutically acceptable salt thereof, wherein:

R.⁷ is a peptide;

R.⁸ it is a topoisomerase I inhibitor;

Ring Z is a monocyclic C5-7 cycloalkyl ring or a monocyclic 5-7 membered heterocycloalkyl ring;

each R^z is independently selected from C1-4 alkyl, halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,

NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

or two R's^z together with the atoms to which they are attached form a fused monocyclic C5-7 cycloalkyl ring, a fused monocyclic 5-7 membered heterocycloalkyl ring, a fused C6-10 aryl ring, or a fused 6-10 membered heteroaryl ring, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from C1-4 alkyl, halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1;

R.^a1,R^b1,R^c1, and R^d1 are each independently selected from H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, each optionally substituted with 1, 2, or 3 substituents independently selected from halo, OH, CN, and NO2 ; and

Forty-nine n is 0, 1, 2, or 3.

In some embodiments of the compounds of Formula (II), R⁷ is a peptide comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ

ID NO:4, or SEQ ID NO:5.

In some embodiments of the compounds of Formula (II), R⁷ it is Pv1, Pv2, Pv3, Pv4, or Pv5.

In some embodiments of the compounds of Formula (II), R⁷ binds to the nucleus via a cysteine ​​residue of R⁷ wherein one of the sulfur atoms of the disulfide moiety in Formula II is derived from the cysteine ​​residue.

In some embodiments of the compounds of Formula (II), R⁸ is camptothecin, opotecan, irinotecan (CPT-11), silatecan (DB-67, AR-67), cositecan (BNP-1350), lurtotecan, gimatecan (ST1481), belotecan (CKD-602), rubitecan, topotecan, deruxtecan, or exatecan.

In some embodiments of the compounds of Formula (II), R⁸it is exatecan.

In some embodiments of the compounds of Formula (II), R⁸It is attached to the nucleus through an N atom.

In some embodiments of the compounds of Formula (II), Ring Z is a monocyclic C5-7 cycloalkyl ring.

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

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

In some embodiments of the compounds of Formula (II), Ring Z is a cycloheptyl ring.

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

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

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

In some embodiments of the compounds of Formula (II), Ring Z is a

Fifty 7-membered heterocycloalkyl ring.

In some embodiments of the compounds of Formula (II), two adjacent RZs together with the atoms to which they are attached form a fused monocyclic C5-7 cycloalkyl ring, a fused monocyclic 5-7 membered heterocycloalkyl ring, a fused C6-aryl ring. fused 10, or a fused 6-10 membered heteroaryl ring, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from C1-4 alkyl, halo, CN, NO2, OR^a1, MR^a1, C(O)R^b1, C(O)NR^c1R.^d1, C(O)OR^a1, OC(O)R^b1, OC(O)NR^c1R.^d1,NR^c1R.^d1,NR^c1C(O)R^b1,NR^c1C(O)OR^a1, and NR^c1C(O)NR^c1R.^d1.

In some embodiments of the compounds of Formula (II), n is 0.

In some embodiments of the compounds of Formula (II), n is 1.

In some embodiments of the compounds of Formula (II), n is 2 .

In some embodiments of the compounds of Formula (II), n is 3.

In some embodiments, the compound of the invention is a compound of Formula (III), Formula (IV), or Formula (V):

<img file="ECSP22010228A_D0081.tif" />

or a pharmaceutically acceptable salt thereof, wherein R⁷, R8, RZ and n are defined as in any of the above embodiments for Formula (II).

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

Fifty-one

<img file="ECSP22010228A_D0082.tif" />

<img file="ECSP22010228A_D0083.tif" />

Fifty; and two

<img file="ECSP22010228A_D0084.tif" />

<img file="ECSP22010228A_D0085.tif" />

,

<img file="ECSP22010228A_D0086.tif" />

<img file="ECSP22010228A_D0087.tif" />

^, , and

s.

pv2

<img file="ECSP22010228A_D0088.tif" />

pv2

Pv2 or a pharmaceutically acceptable salt of any of the above.

Fifty-three

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

<img file="ECSP22010228A_D0089.tif" />

Fifty-four-

<img file="ECSP22010228A_D0090.tif" />

<img file="ECSP22010228A_D0091.tif" />

Fifty five

<img file="ECSP22010228A_D0092.tif" />

<img file="ECSP22010228A_D0093.tif" />

<img file="ECSP22010228A_D0094.tif" />

EITHER

Fifty-six

<img file="ECSP22010228A_D0095.tif" />

Fifty seven-

<img file="ECSP22010228A_D0096.tif" />

In some embodiments, a compound having Formula (IIA) is provided herein:

<img file="ECSP22010228A_D0097.tif" />

s.^yes—^C.and¹ (IIA) or a salt thereof, wherein:

cy¹ is C6-10 aryl or 5-10 membered heteroaryl; wherein the 510-membered heteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4 ring-forming heteroatoms independently selected from N, O, and S; and wherein said C6-10 aryl and 5-10 membered heteroaryl are each optionally substituted with 1, 2, or 3 substituents independently selected from C1-4 alkyl, halo, OH, C1-6 alkoxy, CN, and NO2;

and R⁸, Ring Z, R^z, and n are as defined herein.

In some modalities, Cy¹ it is 5-10 membered heteroaryl. In some modalities, Cy¹ it is pyridinyl. In some modalities, Cy¹ it's phenyl.

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

yes_x 's

<img file="ECSP22010228A_D0098.tif" />

<img file="ECSP22010228A_D0099.tif" />

either

<img file="ECSP22010228A_D0100.tif" />

<img file="ECSP22010228A_D0101.tif" />

or a salt thereof.

Fifty-eight In some embodiments, a composite of

Formula (IIA):

<img file="ECSP22010228A_D0102.tif" />

or a salt thereof, for use in the preparation of a compound of the invention (for example, a compound of Formula (I) or Formula (II)), wherein Cy¹,R⁸, Ring Z, R^z,R^a1,R^b1,R^c1,R^d1, and n are as defined herein.

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

<img file="ECSP22010228A_D0103.tif" />

or a salt thereof, for use in the preparation of a compound of the invention (eg a compound of Formula (I) or Formula (II)).

Molecules of the invention can be labeled, for example, with a probe such as a fluorophore, radioisotope, and the like. In some embodiments, the probe is a fluorescent probe, such as LIQUOR. A fluorescent probe can include any portion that can re-emit light upon excitation of light (eg, a fluorophore).

Amino acids are represented by IUPAC abbreviations, as follows: Alanine (Ala; A), Arginine (Arg; R), Asparagine (Asn; N), Aspartic Acid (Asp; D), Cysteine ​​(Cys; C), Glutamine (Gln; Q), Glutamic Acid (Glu; E), Glycine (Gly; G), Histidine (His; H), Isoleucine (Ile; I), Leucine (Leu; L), Lysine (Lys; K), Methionine (Met; M), Phenylalanine (Phe; F), Proline (Pro; P), Serine (Ser; S), Threonine (Thr; T), Tryptophan (Trp; W), Tyrosine (Tyr; Y), Valine ( Val; V).

The term "Pv1" means ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID

NOT: 1).

Fifty-nine

The term "Pv2" means AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ ID NO:2).

The term "Pv3" means ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO:3).

The term “Pv4” means

AcAAEQNPlYWARYADWLFTTPLLLLDLALLVDADEGTKCG (SEQ ID NO:4).

The term “Pv5” means

AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (SEQ ID NO:5).

In the compounds of the invention, the R peptides⁷ they are attached to the disulfide moiety in the Q linker via an amino acid residue comprising a sulfur atom, such as a cysteine ​​residue. Normally, the sulfur atom of the disulfide moiety in the Q linker that is the point of attachment to the R peptide⁷ is derived from an amino acid residue of the peptide, such as a cysteine ​​residue.

The term "acid and/or hypoxic mantle" refers to the cell environment in the diseased tissue in question that has a pH less than 7.0 and preferably less than 6.5. An acid or hypoxic blanket more preferably has a pH of about 5.5 and most preferably has a pH of about 5.0. Compounds of formula (I) insert through a cell membrane having an acidic and/or hypoxic mantle in a pH-dependent manner to insert R⁸Q in the cell, after which the disulfide linker is cleaved to release R⁸H. Since the compounds of formula (I) are pH dependent, they preferentially insert through a cell membrane only in the presence of an acidic or hypoxic mantle surrounding the cell and not through the cell membrane of cells "normal", which do not have an acid or hypoxic mantle. An example of a cell that has an acid or hypoxic mantle is a cancer cell.

The terms "pH sensitive" or "pH dependent" as used herein to refer to peptide R⁷ or to the mode of insertion of the peptide R⁷ or of the compounds of the invention across a cell membrane, mean that the peptide has a higher affinity for a cell membrane lipid bilayer having an acidic or hypoxic mantle than a membrane lipid bilayer at neutral pH. Therefore, compounds of the invention preferentially insert through the cell membrane to insert R⁸Q inside the cell (and thus supply R⁸H as described above) when the cell membrane lipid bilayer has an acid or hypoxic mantle (a “diseased” cell) but does not insert through

Sixty one cell membrane when the mantle (the environment of the cell membrane lipid bilayer) is not acidic or hypoxic (a “normal” cell). This preferential insertion is believed to be achieved as a result of the R7 peptide forming a helical configuration, which facilitates membrane insertion.

It is further appreciated that certain features of the invention which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment (while the embodiments are intended to be combined as if written in multiple forms). dependents). Conversely, various features of the invention which, for the sake of brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. Therefore, it is contemplated that the features described as embodiments of the compounds of Formula (I) may be combined in any suitable combination.

In various places in this specification, certain characteristics of compounds are described in groups or in ranges. Such description is specifically intended to include any and all individual subcombinations of members of such groups and ranges. For example, the term "C1-6 alkyl" is specifically intended to individually describe (without limitation) methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

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

At various places in the present specification, variables that define divalent linking groups may be described. Each linking substituent is specifically intended to include both the forward and reverse forms of the linking substituent. For example, -NR(CR'R'')n- includes both -NR(CR'R'')n- and -(CR'R'')nNR- and is intended to describe each of the forms individually. When the structure requires a linkage group, the Markush variables listed for that group will be understood as linkage groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists “alkyl” or “aryl”, then “alkyl” or “aryl” is understood to represent an alkylene or arylene linking group. , respectively.

Sixty-one

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

The term "Cn-m" indicates a range including endpoints, where n and m are integers and indicate the number of carbons. Examples include C1-4, C1-6, and the like.

The term "alkyl" used alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight or branched chain. The term "Cn-m alkyl" refers to an alkyl group having nam carbon atoms. An alkyl group formally corresponds to an alkane with a CH bond replaced by the point of attachment of the alkyl group to the rest of the compound. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 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; higher homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and the like.

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 carbon-carbon double bonds. An alkenyl group formally corresponds to an alkene with a CH bond replaced by the point of attachment of the alkenyl group to the rest of the compound. The term "Cn-m alkenyl" refers to an alkenyl group having nam carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Exemplary alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

The term "alkynyl" used alone or in combination with other terms, refers to a straight or branched chain hydrocarbon group corresponding to a

Sixty-four alkyl group having one or more carbon-carbon triple bonds. An alkynyl group formally corresponds to an alkyne with a CH bond replaced by the point of attachment of the alkyl group to the rest of the compound. The term "Cn-m alkynyl" refers to an alkynyl group having nam 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 contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

The term "alkylene", used alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene group formally corresponds to an alkane with two CH bonds replaced by points of attachment of the alkylene group to the rest of the compound. The term "Cn-m alkylene" refers to an alkylene group having nam carbon atoms. Examples of alkylene groups include, but are not limited to, ethane-1,2-diyl, ethane-1,1-diyl, propan-1,3-diyl, propan-1,2-diyl, propan-1,1 -diyl, butan-1,4diyl, butan-1,3-diyl, butan-1,2-diyl, 2-methyl-propan-1,3-diyl and the like.

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

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

The term "cyano" or "nitrile" refers to a group of formula -CΞN, which can also be written as -CN.

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

The term "haloalkyl" as used herein refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by a halogen atom. The term "Cn-m haloalkyl" refers to a Cn-m alkyl group having nam carbon atoms and from at least one to {2(nam)+1} halogen atoms, which may be the same or different. In some embodiments, the halogen atoms are fluoro atoms. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 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.

The term "haloalkoxy", used alone or in combination with other terms, refers to a group of the formula -O-haloalkyl, where the haloalkyl group is as

Sixty-three was defined above. The term "Cn-m haloalkoxy" refers to a haloalkoxy group, which haloalkyl group has nam carbons. Exemplary haloalkoxy groups include trifluoromethoxy and the like. In some embodiments, the haloalkoxy group has 6, 1 to 4, or 1 to 3 carbon atoms.

The term "oxo" refers to an oxygen atom as a divalent substituent, which forms a carbonyl group when attached to carbon, or attached to a heteroatom which forms a sulfoxide or sulfone group, or an N-oxide group. In some embodiments, heterocyclic groups may be optionally substituted with 1 or 2 oxo (=O) substituents.

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

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

The term "aromatic" refers to a carbocycle or heterocycle having one or more polyunsaturated rings that are aromatic in character (ie, having delocalized π (pi) electrons (4n + 2) where n is an integer).

The term "aryl", used alone or in combination with other terms, refers to an aromatic hydrocarbon group, which can be monocyclic or polycyclic (eg, having 2 fused rings). The term "Cn-m aryl" refers to an aryl group having nam carbon atoms in the ring. Aryl groups include, for example, phenyl, naphthyl, and the like. In some embodiments, aryl groups have from 6 to about 10 carbon atoms. In some embodiments, aryl groups have 6 carbon atoms. In some embodiments, aryl groups have 10 carbon atoms. In some embodiments, the aryl group is phenyl.

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 ring members heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, heteroaryl has 5-14 ring atoms including carbon atoms and 1, 2, 3, or 4 ring-membered heteroatoms independently selected from nitrogen, sulfur, and oxygen. In

In some 64 embodiments, heteroaryl has 5-10 ring atoms including carbon atoms and 1, 2, 3, or 4 ring members heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 ring members heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a five or six membered heteroaryl ring. In other embodiments, the heteroaryl is an eight-membered, nine-membered, or ten-membered fused bicyclic heteroaryl ring.

A five-membered heteroaryl ring is a heteroaryl group having five ring atoms wherein one or more (for example, 1, 2, or 3) ring atoms are independently selected from N, O, and S.

A six-membered heteroaryl ring is a heteroaryl group having six ring atoms wherein one or more (for example, 1, 2, or 3) ring atoms are independently selected from N, O, and S.

The term "cycloalkyl", used alone or in combination with other terms, refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic or polycyclic), including cyclized alkyl and alkenyl groups. The term "Cn-m cycloalkyl" refers to a cycloalkyl having nam carbon atoms on the ring members. Cycloalkyl groups can include mono- or polycyclic groups (eg, having 2, 3, or 4 fused rings) and spirocycles. Cycloalkyl groups can have 3, 4, 5, 6, or 7 ring-forming carbons (C3-7). 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 a monocyclic C3-6cycloalkyl group. The ring-forming carbon atoms of a cycloalkyl group can optionally be oxidized to form an oxo or sulfido group. Cycloalkyl groups also include cycloalkylidenes. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Also included in the definition of cycloalkyl are moieties having one or more aromatic rings fused (ie having a bond in common) with the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclohexane and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom, including a ring-forming atom of the fused aromatic ring. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,

Sixty-five cyclopentenyl, cyclohexenyl, cyclohexadienyl and the like. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

The term "heterocycloalkyl", used alone or in combination with other terms, refers to a non-aromatic ring or ring system, which may optionally contain one or more alkenylene groups as part of the ring structure, having at least one member ring heteroatom independently selected from nitrogen, sulfur, oxygen, and phosphorous, and having 4-10 ring members, 4-7 ring members, or 4-6 ring members. Included within the term "heterocycloalkyl" are monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups can include monocyclic or bicyclic (eg, having two fused or bridged rings) or spirocyclic ring systems. In some embodiments, the heterocycloalkyl group is a monocyclic group having 1, 2, or 3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. The ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can optionally be oxidized to form an oxo or sulfide group or other oxidized bond (for example, C(O), S(O), C(S), or S(O) 2, N-oxide, etc.) or a nitrogen atom can be quaternized. The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties having one or more aromatic rings fused to (ie having a bond in common with) the heterocycloalkyl ring, for example, benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom, including a ring-forming atom of the fused aromatic ring. Examples of heterocycloalkyl groups include 2-pyrrolidinyl; morpholinul; azetidinyl; and piperazinyl.

In certain places, the definitions or embodiments refer to specific rings (eg, an azetidine ring, a pyridine ring, etc.). Unless otherwise stated, these rings can be attached to any member of the ring as long as the valency of the atom is not exceeded. For example, an azetidine ring can be attached at any position on the ring, while an azetidin-3-yl ring is attached at the 3-position.

The compounds described herein can be asymmetric (eg, having one or more stereocenters). All stereoisomers, such as

Sixty-six enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated in racemic or optically active forms. Methods on how to prepare optically active forms from optically inactive starting materials, such as by resolution of racemic mixtures or by stereoselective synthesis, are known in the art. Many geometric isomers of olefins, C=N double bonds, and the like may also be present in the compounds described herein, and all of these stable isomers are contemplated by the present invention. The 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 separate isomeric forms.

Resolution of racemic mixtures of compounds can be accomplished by any of a number of methods known in the art. One method includes fractional recrystallization using a chiral resolving acid which is an optically active, salt-forming organic acid. Resolving agents suitable for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various camphorsulfonic acids. optically active such as β-camphorsulfonic acid. Other suitable resolving agents for fractional crystallization methods include stereoisomerically pure forms of α-methylbenzylamine (for example, S and R forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1, 2-diaminocyclohexane and the like.

Resolution of racemic mixtures can also be performed by elution on a column packed with an optically active resolving agent (eg, dinitrobenzoylphenylglycine). One skilled in the art can determine the proper elution solvent composition.

In some embodiments, the compounds of the invention have the (R) configuration. In other embodiments, the compounds have the (S) configuration. In compounds with more than one chiral center, each of the chiral centers in the compound can independently be (R) or (S), unless otherwise indicated.

Compounds of the invention also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond along with the concomitant migration of a proton. Tautomeric forms include

Sixty-seven prototropic tautomers that are isomeric protonation states that have the same empirical formula and total charge. Exemplary prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamineimine pairs, and ring forms where a proton can occupy two or more positions of a heterocyclic system, e.g., 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2Hisoindole and 1H- and 2H-pyrazole. The tautomeric forms may be in equilibrium or sterically locked in an appropriate substitutional form.

The compounds of the invention may also include all isotopes of atoms that occur in the intermediate or final compounds. Isotopes include those atoms that have 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 invention may be replaced or substituted with isotopes of the atoms in natural or unnatural abundance. In some embodiments, the compound includes at least one deuterium atom. For example, one or more hydrogen atoms in a compound of the present disclosure may be replaced or substituted with deuterium. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 deuterium atoms. Synthetic methods for including isotopes in 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 Labeling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in various studies such as NMR spectroscopy, metabolism experiments and/or assays.

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

The term "compound," as used herein, is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. The term also refers to the compounds of the invention, regardless of how they are prepared, for example synthetically, through a

Sixty-eight biological process (eg, metabolism or enzymatic conversion), or a combination thereof.

All of the compounds, and pharmaceutically acceptable salts thereof, may be present together with other substances such as water and solvents (eg, hydrates and solvates) or they may be isolated. When in the solid state, the compounds described herein and salts thereof can occur in various forms and can, for example, take the form of solvates, including hydrates. Compounds can be in any solid state form, such as a polymorph or solvate, so unless clearly stated otherwise, reference in the specification to compounds and salts thereof should be understood to encompass any form of solid state of the compound.

In some embodiments, the compounds of the invention, or salts thereof, are substantially isolated. By "substantially isolated" it is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. The partial separation can include, for example, a composition enriched in the compounds of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds of the invention, or salts thereof.

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

The terms "room temperature" and "room temperature" as used herein are understood in the art and generally refer to a temperature, for example, a reaction temperature, that is approximately the temperature of the room in which which the reaction is carried out. for example, a temperature from about 20°C to about 30°C.

The present invention also includes pharmaceutically acceptable salts of the compounds described herein. The term "pharmaceutically acceptable salts"

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

Synthesis

The compounds of the invention, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of a number of possible synthetic routes, such as those in the following reaction Schemes.

Reactions to prepare compounds of the invention can be carried out in suitable solvents that are readily selectable by one skilled in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reagents), intermediates or products at the temperatures at which the reactions are carried out, for example, temperatures that can range from the freezing temperature of the solvent to the temperature of boiling of the solvent. A given reaction can be carried out in one solvent or in a mixture of more than one solvent. Depending on the particular reaction step, the person skilled in the art can select suitable solvents for a particular reaction step.

Seventy The preparation of 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 skilled in the art. The chemistry of protecting groups is described, 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., Protecting Groups in Carbohydrate Chemistry, J. Chem. Educ., 1997, 74(11), 1297; and Wuts et al., Protective Groups in Organic Synthesis, 4th Ed., (Wiley, 2006).

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (for example,¹H o¹³C), infrared spectroscopy, spectrophotometry (eg UV-visible), mass spectrometry or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

The following reaction Schemes provide general guidance in relation to the preparation of the compounds of the invention. One skilled in the art will understand that the preparations shown in the Reaction Schemes can be modified or optimized using general knowledge of organic chemistry to prepare various compounds of the invention.

Compounds of Formula (I) can be prepared, for example, using a process as illustrated in the following reaction schemes.

Reaction Scheme 1: Synthesis of Compounds Linked to Carbonate and Carbamate

Cluster

outgoing 1

EITHER

<img file="ECSP22010228A_D0104.tif" />

Outgoing Group 2

R1 R2

II

<img file="ECSP22010228A_D0105.tif" />

base

<img file="ECSP22010228A_D0106.tif" />

R1 R2

II

HS

<img file="ECSP22010228A_D0107.tif" />

<img file="ECSP22010228A_D0108.tif" />

R1 R2

Seventy-one

Intermediate II, which is flanked by orthogonal leaving groups, can be reacted with a compound R⁸nucleophilic H to produce intermediate III. Intermediate III can then be reacted with a thiol-containing peptide (HS-R⁷) that participates in a disulfide exchange reaction to produce the final compound. Suitable leaving groups are described below.

Reaction Scheme 2: Synthesis 1 of Conjugates Linked to Thio Propionate

<img file="ECSP22010228A_D0109.tif" />

IV.

<img file="ECSP22010228A_D0110.tif" />

<img file="ECSP22010228A_D0111.tif" />

V

<img file="ECSP22010228A_D0112.tif" />

Disulfide propionate IV with previously installed Leaving Groups 1 and 2 are

0 can selectively react with R⁸nucleophilic -H to produce V. This compound can then be reacted with R⁷-SH to provide the desired conjugate.

Reaction Scheme 3: Synthesis 2 of Conjugates Linked to Thio Propionate

<img file="ECSP22010228A_D0113.tif" />

<img file="ECSP22010228A_D0114.tif" />

<img file="ECSP22010228A_D0115.tif" />

<img file="ECSP22010228A_D0116.tif" />

VI VII VIII

<img file="ECSP22010228A_D0117.tif" />

The thionoester VI can be reacted with R⁸-H nucleophilic to produce propionate thiol VII. This compound can participate in a disulfide exchange reaction to provide Intermediate VIII. This compound can be treated with R⁷-SH to provide the desired conjugate.

Reaction Scheme 4: Synthesis 1 of Para Benzyl Linked Conjugates

seventy-one give

<img file="ECSP22010228A_D0118.tif" />

<img file="ECSP22010228A_D0119.tif" />

eleventh

<img file="ECSP22010228A_D0120.tif" />

<img file="ECSP22010228A_D0121.tif" />

<img file="ECSP22010228A_D0122.tif" />

<img file="ECSP22010228A_D0123.tif" />

<img file="ECSP22010228A_D0124.tif" />

The alcohol group of para-aminobenzyl alcohol IX can be selectively protected to produce Intermediate X. This intermediate can then be reacted at the aniline position with Intermediate II to provide aryl 5-carbamate XI. The protecting group can be removed to produce free alcohol XII, which can be treated with an activating agent to provide Intermediate XIII, which contains orthogonal leaving groups. Reaction of Intermediate XIII with R8-H can provide Intermediate XIV, followed by treatment with R⁷-SH

Seventy-trey can produce the desired parabenzyl-linked conjugate.

Reaction Scheme 5: Synthesis of Para Benzyl Linked Conjugates

<img file="ECSP22010228A_D0125.tif" />

<img file="ECSP22010228A_D0126.tif" />

The 4-mercaptobenzyl alcohol XV can be reacted in a disulfide exchange reaction to produce the 4-mercaptobenzyl alcohol disulfide XVI containing Leaving Group 2. The remaining benzyl alcohol can be treated with an appropriate carbonyl compound to provide the activated compound XVII. This intermediate can be further selectively reacted with R⁸-Nucleophilic H to provide Intermediate XVIII, which can be treated with R⁷io SH to produce the desired conjugate.

Reaction Scheme 6: Synthesis of Conjugates Linked to Ortho Benzyl

<img file="ECSP22010228A_D0127.tif" />

XIX

<img file="ECSP22010228A_D0128.tif" />

XX

Seventy-four-

<img file="ECSP22010228A_D0129.tif" />

<img file="ECSP22010228A_D0130.tif" />

xxii

2-Mercapto benzyl alcohol XXIII can be reacted as previously described to produce the desired conjugate.

Reaction Scheme 7: Cleavage of Peptide Conjugates

<img file="ECSP22010228A_D0131.tif" />

glutathione

37°C, Buffer

<img file="ECSP22010228A_D0132.tif" />

<img file="ECSP22010228A_D0133.tif" />

Cleavage of the final compound to release R⁸ -H can be achieved by treating the compound with excess glutathione (GSH) in buffer with incubation at 37°C. Reversed phase HPLC analysis over a desired time course is used to follow the course of cleavage.

R peptides⁷ they 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 may also be employed. The Merrifield technique is well known and is a common method for the preparation of peptides. Useful techniques for solid phase peptide synthesis are described in several books, such as the text "Principles of Peptide Synthesis" by Bodanszky, Springer Verlag 1984. This method of synthesis involves the stepwise addition of protected amino acids to a growing peptide chain that was covalently attached to a solid resin particle. Using this procedure, the reagents and

Seventy-five by-products are 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 in the chain to a solid polymer by a covalent bond, followed by the addition of the next protected amino acids, one at a time, in a stepwise fashion until the desired sequence is assembled. . Finally, the protected peptide is removed from the solid resin support and the protecting groups are cleaved.

R peptides⁷ they can also be produced by fermentation, for example, by modification of E. coli. Protein production in E. coli can be controlled to produce recombinant polypeptides having an R-peptide sequence⁷ described herein. The production of recombinant polypeptides in E. coli is described in the following references: Zhao, Q., Xu, W., Xing, L. et al. Recombinant production of medium- to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag. Microb Cell Fact 15, 136 (2016); of Marco, Recombinant polypeptide production in E. coli: towards a rational approach to improve the yields of functional proteins; Microbial Cell Factories 2013, 12:101; and Kleiner-Grote GM, Risse, JM, Friehs, K; Secretion of recombinant proteins from E. coli; Eng. Life Sci. 2018, 18, 532-550, each of which is incorporated by reference in its entirety.

The amino acids can be attached to any suitable polymer. The polymer must be insoluble in the solvents used, must have a stable physical form that allows easy filtration, and must contain a functional group to which the first protected amino acid can be firmly attached by a covalent bond. Various polymers are suitable for this purpose, such as cellulose, polyvinyl alcohol, polymethylmethacrylate, and polystyrene.

Methods of Use

Provided herein is the use of the 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 the compounds of formula (I) in the treatment of diseases involving acidic or hypoxic diseased tissue, such as cancer or neurodegenerative disease. Hypoxia and acidosis are physiologic markers of many disease processes, including cancer. In cancer, hypoxia is a mechanism responsible for the development of an acidic environment within solid tumors. As a result, hydrogen ions must be removed from the cell (for example, by a proton pump) to maintain a normal pH within the cell. As a consequence of

Seventy-six this export of hydrogen ions, cancer cells often have an increased pH gradient across the lipid bilayer of the cell membrane and a lower pH in the extracellular milieu compared to normal cells. One approach to improving the efficacy and therapeutic index of cytotoxic agents is to take advantage of this physiological characteristic to provide selective delivery of the compound to hypoxic cells over healthy tissue.

In the methods of treatment of the invention, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered as a single agent or in combination with other forms of therapy, such as ionizing radiation or agents. cytotoxic in the case of cancer. In combination therapy, the compound of formula (I) can be administered before, at the same time, or after the other therapeutic modality, as will be appreciated by those skilled in the art. Any method of treatment (single agent or combination with other forms of therapy) can be administered as a course of treatment involving multiple doses or treatments over a period of time.

Examples of cancers that can be treated using the compounds of the present disclosure include, but are not limited to, colorectal cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous malignant melanoma. or intraocular cancer, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, endometrial carcinoma, endometrial cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the gland parathyroid gland, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemias 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 carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis , spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, and combinations of such cancers.

In some embodiments, cancers treatable with compounds of the present

Seventy-seven description include bladder cancer, bone cancer, glioma, breast cancer (eg, triple negative breast cancer), cervical cancer, colon cancer, colorectal cancer, endometrial cancer, epithelial cancer, colon cancer, esophagus, Ewing's sarcoma, pancreatic cancer, gallbladder cancer, gastric cancer, gastrointestinal tumors, head and neck cancer (upper aerodigestive cancer), intestinal cancers, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liver cancer (for example, hepatocellular carcinoma), lung cancer (for example, non-small cell lung cancer, adenocarcinoma), melanoma, prostate cancer, rectal cancer, clear cell renal cell carcinoma, cancer skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer.

In some embodiments, cancers treatable with compounds of the present disclosure include melanoma (eg, metastatic malignant melanoma), kidney cancer (eg, clear cell carcinoma), prostate cancer (eg, hormone refractory adenocarcinoma of the prostate ), breast cancer, triple negative breast cancer, colon cancer, and lung cancer (for example, non-small cell lung cancer and small cell lung cancer). Furthermore, the disclosure includes refractory or recurrent malignancies the growth of which can be inhibited using the compounds of the disclosure.

In some embodiments, cancers that can be treated using the compounds of the present disclosure include, but are not limited to, solid tumors (eg, prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer). , uterine cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancers, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.) , hematologic cancers (for example, lymphoma, leukemia such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin lymphoma (including relapsed or refractory and recurrent follicular NHL), Hodgkin lymphoma or multiple myeloma) and combinations of such cancers.

Compounds of the invention (eg, a compound of formula (I)) comprising a topoisomerase I targeting moiety derived from a topoisomerase I inhibitor (eg, exatecan) may exhibit certain therapeutic advantages over the topoisomerase I inhibitor. per se. For example, administration of a compound of formula (I) may show reduced toxicity (for example,

78 gastric or bone marrow toxicity) compared with administration of the corresponding topoisomerase I inhibitor (eg exatecan). In some embodiments, bone marrow toxicity is measured by total bone marrow count of samples from the subject (eg, total bone marrow count in mouse femora). In some embodiments, bone marrow toxicity is measured by PARylation in bone marrow tissue. In some embodiments, bone marrow toxicity is measured by total nucleated bone marrow cells. In some embodiments, gastric toxicity is assessed using photographs of the subject's (eg, mouse) stomachs taken both in situ and ex vivo.

In certain embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof may be used in combination with a chemotherapeutic agent, targeted anticancer therapy, immunotherapy, or radiotherapy. The agents can be combined with the present compounds in a single dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms. In some modalities, the chemotherapeutic agent, targeted cancer therapy, immunotherapy, or radiation therapy is less toxic to the patient, for example, by showing reduced gastric or bone marrow toxicity, when administered in conjunction with a compound of formula (I) or a pharmaceutically acceptable salt thereof, compared to when administered in combination with the corresponding topoisomerase inhibitor (for example, R⁸-H).

Suitable chemotherapeutic or other anticancer agents include, for example, alkylating agents (including, without limitation, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes) such as uracil mustard, chlormethin, cyclophosphamide (Cytoxan^TM), ifosfamide, melphalan, chlorambucil, pipobromane, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

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

Seventy-nine

Suitable chemotherapeutic or other anticancer agents include, for example, antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogues, purine analogues, and adenosine deaminase inhibitors) such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

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

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

Also suitable are cytotoxic agents such as, for example, epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitors; antihormonal therapeutic agents; leucovorin; tegafur; and hematopoietic growth factors.

Other anticancer agents 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.).

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

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

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.

Other agents suitable for use in combination with the compounds in the

Eighty present inventions include chemotherapy combinations such as platinum-based doublets used in lung cancer and other solid tumors (cisplatin or carboplatin plus gemcitabine; cisplatin or carboplatin plus docetaxel; cisplatin or carboplatin plus paclitaxel; cisplatin or carboplatin plus pemetrexed) or gemcitabine plus particles bound to paclitaxel (Abraxane®).

The compounds of this invention may be effective in combination with antihormonal agents for the treatment of breast cancer and other tumors. Suitable examples are antiestrogen agents, including, but not limited to, tamoxifen and toremifene, aromatase inhibitors, including, but not limited to, letrozole, anastrozole, and exemestane, adrenocorticosteroids (for example, prednisone), progestogens (for example, acetate megastrol) and estrogen receptor antagonists (for example, fulvestrant). Suitable antihormonal agents used for the treatment of prostate and other cancers may also be combined with 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 (for example, degarelix), androgen (eg enzalutamide) and agents that inhibit androgen production (eg abiraterone).

The compounds of the present invention can 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 and against cancer-associated 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. Inhibitors of c-Met can be used in combination with the compounds of the invention. These include onartumzumab, tivantnib, and INC-280. Agents against Abl (or Bcr-Abl) include imatinib, dasatinib, nilotinib, and ponatinib and those against Alk (or EML4-ALK) include crizotinib.

Angiogenesis inhibitors may be effective in some tumors in combination with compounds of the invention. These include antibodies against VEGF or VEGFR or inhibitors of VEGFR kinases. Antibodies or other therapeutic proteins

Eighty-one against VEGF include bevacizumab and aflibercept. Inhibitors of VEGFR kinases and other anti-angiogenesis inhibitors include, but are not limited to, sunitinib, sorafenib, axitinib, cediranib, pazopanib, regorafenib, brivanib, and vandetanib.

Activation of intracellular signaling pathways is common in cancer, and agents targeting components of these pathways have been combined with receptor-targeting agents to improve efficacy and reduce resistance. Examples of agents that can be combined with compounds of the present invention include inhibitors of the PI3K-AKT-mTOR pathway, inhibitors of the Raf-MAPK pathway, inhibitors of the JAKSTAT pathway, and inhibitors of chaperone proteins and progression. of the cell cycle.

Agents against PI3 kinase include, but are not limited to topilalisib, idelalisib, buparlisib. mTOR inhibitors such as rapamycin, sirolimus, temsirolimus and everolimus can be combined with compounds of the 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 JAKs (eg, ruxolitinib, baricitinib, tofacitinib), Hsp90 (eg, tanespimycin), cyclin-dependent kinases (eg, palbociclib), HDACs (eg, panobinostat), PARP (eg, olaparib) and proteasomes (eg, bortezomib, carfilzomib) can also be combined with compounds of the present invention. A further example of a PARP inhibitor that can be combined with a compound of the invention is talazoparib.

Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, its 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, eg, 1996 edition, Medical Economics Company, Montvale, NJ), the disclosure of which is incorporated herein by reference as if it were fully described.

The phrase "therapeutically effective amount" of a compound (therapeutic agent, active ingredient, drug, etc.) refers to an amount of the compound to be administered to a subject in need of therapy or treatment that alleviates a symptom, improves a condition, or slows the onset of disease conditions, according to clinically acceptable standards for the disorder or condition being treated. For example, a therapeutically effective amount can be an amount that has been shown to have a therapeutic effect.

Eighty-two desired in an in vitro test, an in vivo animal test, or a clinical trial. The therapeutically effective amount may vary based on the particular dosage form, method of administration, treatment protocol, specific disease or condition to be treated, benefit/risk ratio, etc., among many other factors.

Said therapeutically effective amount can be obtained from a clinical trial, an animal model or an in vitro cell culture assay. It is known in the art that the effective amount suitable for human use can be calculated from the effective amount determined from an animal model or an 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 assays) = "mg/kg body weight/day" (effective amount for a mouse). Furthermore, the effective amount for a human can be calculated from the effective amount for a mouse based on the fact that the metabolism rate of mice is 6 times faster than that of humans.

As an example of treatment using a compound of formula (I) in combination with a cytotoxic agent, a therapeutically effective amount of a compound of formula (I) may be administered to a patient suffering from cancer as part of a treatment regimen that also involves a therapeutically effective amount of ionizing radiation or a cytotoxic agent. In the context of this treatment regimen, the term "therapeutically effective" amount is to be understood to mean effective in the combination therapy. Experts in the field of cancer treatment will understand how to adjust dosages to achieve the optimal therapeutic result.

Similarly, those skilled in the medical art can readily determine appropriate doses of the compounds of the invention for the treatment of non-cancerous diseases or conditions (such as cardiovascular disease).

The term "treat" as used herein includes the administration of a compound or composition that reduces the frequency, delays the onset, or reduces the progression of symptoms of a disease involving acidic or hypoxic diseased tissue, such as cancer, stroke, myocardial infarction, or long-term neurodegenerative disease, in a subject relative to a subject not receiving the compound or composition. This may include reversing, reducing, or stopping the symptoms, clinical signs, or underlying pathology of a condition in a way to

Eighty-three to improve or stabilize a subject's condition (eg, regression of tumor growth, for cancer or decrease or ameliorate myocardial ischemia reperfusion injury in myocardial infarction, stroke, or similar cardiovascular disease). The terms "inhibit" or "reduce" are used for cancer in reference to methods of inhibiting or reducing tumor growth (for example, decreasing the size of a tumor) in a population compared to an untreated control population. .

All publications (including patents) mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications, which might be used in connection with the description described herein. The publications discussed throughout the text are provided solely for description prior to the filing date of the present application.

Several types of ranges are described here. When describing or claiming a range of any kind, the intent is to individually describe or claim each possible number that such a range could reasonably encompass, including the endpoints of the range, as well as any subranges and combinations of subranges encompassed. in this. When a range of therapeutically effective amounts of an active ingredient is described or claimed, for example, the intent is to individually describe or claim every possible number that such a range might encompass, consistent with the description herein. For example, by a description that the therapeutically effective amount of a compound may range from about 1 mg/kg to about 50 mg/kg (of subject's body weight).

Formulation, Dosage Forms and Administration

To prepare the pharmaceutical compositions of the present invention, a compound of Formula (I) or a pharmaceutically acceptable salt thereof is combined as the active ingredient in intimate admixture with a pharmaceutical carrier in accordance with conventional pharmaceutical compounding techniques, the carrier it can take a wide variety of forms depending on the form of preparation desired for administration, eg, oral or parenteral. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of such oral liquid preparations. as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, diluents, granulating agents, lubricants,

Eighty-four binders, disintegrating agents, and the like in the case of solid oral preparations, such as, for example, powders, capsules, and tablets. Due to their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets can be sugar coated or enteric coated by standard techniques. For parenterals, the carrier will normally comprise sterile water, although other ingredients may be included, for example, to aid solubility or for preservation purposes. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, and the like may be employed. One skilled in the pharmaceutical and medical arts will readily be able to determine a suitable dosage of the pharmaceutical compositions of the invention for the particular disease or condition to be treated.

EXAMPLES

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

• Brine: a saturated solution of NaCl in water • DCM: dichloromethane • TFA: trifluoroacetic acid • DIPEA: diisopropylethylamine • DMA: dimethylacetamide • DME: dimethoxyethane • DMF: dimethylformamide • DMSO: methyl sulfoxide • DTT: dithiothreitol • MSD: detector spectrometry of masses • Et2O: ethyl ether • EtOAc: ethyl acetate

EtOH: ethyl alcohol

Eighty-five • HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'tetramethyluronium hexafluorophosphate • HOBt: 1-hydroxybenzotriazole • RP: reverse phase • HPLC: high performance liquid chromatography • IPA: isopropanol • LAH: lithium aluminum hydride • N-BuLi: n-butyl lithium • LC-MS: liquid chromatography-mass spectrometry • LDA: lithium diisoproylethylamide • Me: methyl • MeOH: methanol • MTBE: methyl t -butyl ether • NMP: N-methylpyrrolidine • Ph: phenyl • PNPC: para-nitrophenylchloroformate • RT or rt: room temperature • SFC: supercritical fluid chromatography • TBAI: tetrabutylammonium iodide • TBME: tert-butylmethyl ether • tBu: tertiary butyl • THF: tetrahydrofuran • TEA: triethylamine • TMEDA: tetramethylethylenediamine • GSH: Glutathione • GS: Sulfur bound glutathione • LiOH: lithium hydroxide

Eighty-six • DPPA: diphenylphosphoryl azide • Sn(Bu)2(laurate)2: dibutyltin dilaurate • PBS: phosphate buffered saline • ACN: acetonitrile • AcOH: acetic acid • EEDQ: N-ethoxycarbonyl-2- ethoxy-1,2-dihydroquinoline • DMAP: 4-dimethylaminopyridine • EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

The source of the starting materials used in the Examples is described below in the following tables.

Table 2. Starting materials for R⁸

<td>R code⁸</td><td>structure of R⁸h</td><td>Synthesis or Acquired Reference</td>

<td>R.⁸H-1</td><td>zP L II 1 nY and 0 /H ' OH 0</td><td>medchem express HY-16560</td>

<td>R.⁸H-2</td><td>HO. TO GOES THE A^No.z 'v⁰/'Ή</td><td>medkoo 406280</td>

<td>R.⁸H-3</td><td>nh₂THE A^No.V γ 0 /Ή 'OH 0</td><td>asta tech F11420</td>

<td>R.⁸H-4</td><td>0^ +„O“ No. Λύυ^noTO and 0 /'Ή 'OH 0</td><td>Asta Tech 42333</td>

Eighty-seven-

<td>R.⁸H-5</td><td>_ΛΝΗ₂and L II Ί NZ K~~~\ V⁰/'Ή ' OH 0</td><td>medchem express HY-13631A</td>

<td>R.⁸H-6</td><td>ooh_λνη rt r V^to AL II I nZ⁰/Ή ' OH 0</td><td>medchem express HY-13631D</td>

<td>R.⁸H-7</td><td>1 HO. Á rYrt^ zP L II Ί \ 0 /Ή 'OH 0</td><td>Asta Tech 21428</td>

<td>R.⁸H-8</td><td> /^{Yes /}Kick-/ ''''^no'v⁰/Ή 'OH 0</td><td>medchem express HY-14812</td>

<td>R.⁸H-9</td><td>\L II NZ /'jO ' OH</td><td>US 20030105109 A1</td>

<td>R.⁸H-10</td><td>7? No. rtrV^ /7° L II 1 'v⁰rVA 'OH 0</td><td>WO 9902530 A1</td>

Eighty-eight-

<td>R.⁸H-11</td><td>ex ^X⁰^N=TO^No. 0 X HO or</td><td>Medchem Express: Cat. No.: HY-16562</td>

<td>R.⁸H-12</td><td>T°Y° O. I / X γΥΥο £ f AY /A and/ EITHER O U- CO you</td><td>Widdison et al., ACS Medical Chemistry Letters 2019 10 (10), 1386-1392</td>

Table 3. Starting Materials for Linkers

<td>Linker Code</td><td>Linker Structure</td><td>Synthesis or Acquired Reference</td>

<td>L-1</td><td>zx A^hHO^z</td><td>synthesized WO2013055987A1</td>

<td>L2</td><td>zx SH HO^z</td><td>synthesized WO2013055987A1</td>

<td>L3</td><td>zx A^hho^z γς</td><td>synthesized ACS Med. Chem. Lett. 2016, 7, 988-993</td>

<td>L4</td><td>I ΗΟ^Χ</td><td>R,R*</td>

<td>L5</td><td>NOW ^^shHO^z</td><td>H.H*</td>

Eighty-nine

<td>L6</td><td>1 ΗΟ^Ζ</td><td>R,S*</td>

<td>L7</td><td>JL SH ΗΟ^Ζ</td><td>MR*</td>

<td>L8</td><td>HO .SH</td><td>R,R*</td>

<td>L9</td><td>HO^ ^SH</td><td>H.H*</td>

<td>XV-1</td><td>^h°nn</td><td>Combiblocks OR-5865</td>

*Absolute configuration randomly assigned

The HPLC methods used are described below:

HPLC methods

A: Sunfire C18 150x4.6mm; H2O/Acetonitrile with TFA modifier (0.05%); 5 Flow Rate: 1ml/min; Wavelength = 217 nM.

B: Ace Equivalence 250x4.6mm; H2O/Acetonitrile with TFA modifier (0.05%); Flow Rate: 1ml/min; Wavelength = 217 nM.

C: Sunfire C18 150x30mm; H2O/Acetonitrile with TFA modifier (0.05%); Flow Rate: 30ml/min; Wavelength = 217 nM.

Mass Spectrometry Methods

Maldi-TOF (Matrix Assisted Laser Desorption/Ionization - Time of Flight) mass spectrometry was measured on an Applied Biosystems Voyager System 6268. The sample was prepared as an α-cyano hydroxy cinnamic acid matrix on an AB Science plate ( Part # V700666).

ESI (Electrospray Ionization) mass spectrometry was measured already

NoventaSea on Agilent 1100 Series LC-MS with a 1946 MSD or high-resolution MS

Waters Xevo Qtof, both provided a mass/charge of species (m/z=3).

Synthesis of cis-S-(3-hydroxybutan-2-yl) ethanethioate (L-4 and L-5)

<img file="ECSP22010228A_D0134.tif" />

h₂OR, RT, 4 p.m.

<img file="ECSP22010228A_D0135.tif" />

racemic cis

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

Synthesis of cis -3-Mercaptobutan-2-ol

<img file="ECSP22010228A_D0136.tif" />

Racemic Cis

LAH, THF, 0°C, RT, 3h

<img file="ECSP22010228A_D0137.tif" />

Racemic Cis

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

Synthesis of trans -S-(3-hydroxybutan-2-yl) ethanethioate (L-6 and L-7)

<img file="ECSP22010228A_D0138.tif" />

either

Ah h₂o, rt, h

<img file="ECSP22010228A_D0139.tif" />

racemic trans

To a stirred solution of cis-2,3-dimethyloxirane (1.0 g, 13.9 mmol) in water (15 mL) was added thioacetic acid (1.1 mL, 15.6 mmol) at room temperature and stirred.

<img file="ECSP22010228A_D0140.tif" />

<img file="ECSP22010228A_D0141.tif" />

Ninety-one for 16 h. The reaction mixture was quenched with sodium bicarbonate solution (10ml) and extracted with ethyl acetate (20ml). The organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to give trans-S(3-hydroxybutan-2-yl)ethanethioate as a yellow oil (0.7 g crude).

Synthesis of trans -3-mercaptobutan-2-ol

LAH, THF, 0°C, RT, 3h

S. /

Π ------* HO

EITHER

Trans Racemic Trans Racemic

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

Synthesis of trans-S-(2-hydroxycyclohexyl) ethanethioate (L-8 and L-9)

<img file="ECSP22010228A_D0142.tif" />

Trans; racemic

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

Synthesis of trans -2-mercaptocyclohexan-1-ol

<img file="ECSP22010228A_D0143.tif" />

. SH

Trans Racemic

Trans Racemic

ninety and give

To a stirred solution of trans-S-(2-hydroxycyclohexyl)ethanethioate (3.8 g, 21.8 mmol) in THF (20.0 mL) was added 1 M LiAH4 in THF (21.8 mL, 21.8 mmol) at 0°C. The reaction mixture was allowed to gradually warm to room temperature and stirred for 1 h. The progress of the reaction was monitored by TLC (20% EtOAc/Hex). After completion of the reaction, the reaction mixture was cooled to room temperature and quenched with 1.0N HCl (30 mL). The reaction mixture was extracted into CH2Cl2 (30.0 mL). The organic layer was washed with brine solution (30.0 mL), concentrated, and the crude trans-2-sulfanylcyclohexanol was taken for the next step. (2.88g, raw).

Synthesis of Intermediate I from L

<img file="ECSP22010228A_D0144.tif" />

L

Yo

<td>Int.</td><td>R.¹,R²</td><td>R.³,R⁴</td><td>X</td><td>Method/Ref.</td><td>MH+</td>

<td>I-1</td><td>H, I</td><td>H H</td><td>h</td><td>WO2013055987A1</td><td> 202.1</td>

<td>I-2</td><td>I,H</td><td>H H</td><td>h</td><td>WO2013055987A1</td><td> 202.1</td>

<td>I-3</td><td>-CH2CH2CH2-</td><td>H H</td><td>NO2</td><td>ACS Med. Chem. Lett. 2016,7,988-993</td><td> 272.9</td>

<td>I-4</td><td>H, I</td><td>I,H</td><td>h</td><td></td><td> 216.1</td>

<td>I-5</td><td>I,H</td><td>H, I</td><td>h</td><td></td><td> 216.1</td>

<td>I-6</td><td>I,H</td><td>I,H</td><td>h</td><td></td><td> 215.9</td>

<td>I-7</td><td>H, I</td><td>H, I</td><td>h</td><td></td><td> 216.2</td>

<td>I-8</td><td colspan="2">CH2,HCH2,H ch₂ch₂ch₂ch₂</td><td>h</td><td></td><td></td>

<td>I-9</td><td colspan="2">H, CH2 H, ^₂CH₂CH₂CH₂CH₂</td><td>h</td><td></td><td></td>

Synthesis of Intermediate I-1: (2R)-2-(2-pyridyldisulfanyl)propan-1-ol

<img file="ECSP22010228A_D0145.tif" />

L-1

<img file="ECSP22010228A_D0146.tif" />

EtOH, RT, 16h

<img file="ECSP22010228A_D0147.tif" />

I-1

Ninety three

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

Intermediate I-2 was prepared from L-2 in an analogous manner.

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

<img file="ECSP22010228A_D0148.tif" />

<img file="ECSP22010228A_D0149.tif" />

^L-3 I-3

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

Synthesis of I-4 and I-5: 3-(pyridin-2-yldisulfanyl)butan-2-ol Isomer 1 and Isomer 2

<img file="ECSP22010228A_D0150.tif" />

cis Racemic

<img file="ECSP22010228A_D0151.tif" />

<img file="ECSP22010228A_D0152.tif" />

MeOH

<img file="ECSP22010228A_D0153.tif" />

I-5: Isomer 1

I-4: Isomer 2

chiral separation

A stirred solution of 2,2-dipyridyldisulfide (520 mg, 2.35 mmol) in MeOH (15 mL) was purged with nitrogen gas for 5 min. The nitrogen gas purged solution of cis -3-mercaptobutan-2-ol (500 mg) in CH2Cl2 (10 mL) was added thereto at 0°C. The reaction mixture was allowed to gradually warm to room temperature and stirred for 16h. The reaction mixture was concentrated under reduced pressure to

Ninety-four provided crude material that was purified by column chromatography using 30-40% EA/hexane. The racemic product was separated with Chiral Preparative HPLC (CHIRALPAK IG; 100mm X 4.6mm X 3mic; Mobile phase: nHexane:Ethanol 80:20 with 0.1% DEA; Flow rate: 1.0 mL/min) to separate the respective enantiomers. Solvents were removed to obtain (2S,3S)-3-(2-pyridyldisulfanyl)butan-2-ol* (140 mg, Isomer-1) MS m/z 216.1 [M+H]+ y (2R,3R) -3-(2-pyridyldisulfanyl)butan-2-ol (140 mg, Isomer-2). MS m/z 216.1 [M+H]⁺.

Synthesis of I-6 and I-7: 3-(pyridin-2-yldisulfanyl)butan-2-ol Isomer 1 and Isomer 2

<img file="ECSP22010228A_D0154.tif" />

^HEp^{chiral prayer}^{trans rac}and^monkey I-6: Isomer 1 I-7: Isomer 2

A stirred solution of 2,2-dipyridyldisulfide (520 mg, 2.35 mmol) in MeOH (15 mL) was purged with nitrogen gas for 5 min. The nitrogen gas purged solution of cis-3-mercaptobutan-2-ol (500mg) in CH2Q2 (10ml) was added thereto at 0°C. The reaction mixture was allowed to gradually warm to room temperature and stirred for 16h. The reaction mixture was concentrated under reduced pressure to provide crude material which was purified by column chromatography using 30-40% EA/hexane. The racemic product was separated with Chiral Preparative HPLC (Column: CHIRALPAK IG (100mm X 4.6mm X 3mic) Mobile phase: nHexane:Ethanol with 0.1% DEA (80:20) Flow rate: 1.0 mL/min) to separate enantiomers respective. The solvents were removed to obtain (2R,3S)-3-(2-pyridyldisulfanyl)butan-2-ol* (0.6 g, Isomer-I) MS m/z 215.9 [M+H]⁺y (2S,3R)-3-(2-pyridyldisulfanyl)butan-2-ol* (0.6 g, Isomer-II) MS m/z 216.2 [M+H]⁺ as oily compounds.

Synthesis of Intermediate I-6: Trans-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol

<img file="ECSP22010228A_D0155.tif" />

^{trans Ra}EC^myco trans Racemic

To a solution of 1,2-di(pyridin-2-yl)disulfan (2.41 g, 10.9 mmol) in MeOH (degassed with N2) (30 mL) was added trans-2-sulfanylcyclohexanol (2.88 g, 21.0 mmol) ( degassed with N2) dropwise and stirred for 16 h at room temperature under N2 atmosphere. The reaction mixture was concentrated to dryness.

Ninety-five under empty. The resulting crude was purified by column chromatography using 30% EtOAc/hexanes to provide trans-2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol as a yellow liquid.

<img file="ECSP22010228A_D0156.tif" />

<img file="ECSP22010228A_D0157.tif" />

I-8: Isomer 1 I-9: Isomer 2

Trans Racemic

Quinal separation was performed by chiralpak IG (100 mm X 4.6 mm X 3 mie) using n-hexane:IPA with 0.1% diethylamine (80:20) to give (1R,2R)-2-(2-pyridyldisulfanyl)cyclohexanol* lsomer- 1 (350 mg) and (1S,2S)-2-(2-pyridyldisulfanyl)cyclohexanol* lsomere-2 (400 mg).

Intermediary XV from the XXI

<img file="ECSP22010228A_D0158.tif" />

<img file="ECSP22010228A_D0159.tif" />

fifteenth

16th

<td>Int.</td><td>R.⁵,R⁶</td><td>R.⁹,R¹⁰,R¹¹,R¹²</td><td>MH⁺</td>

<td>XV-1</td><td>Η,H</td><td>Η, Η, Η, H</td><td> 250.1</td>

Synthesis of Intermediate XV-1: r4-(2-pyhdildisulfanyl)phenyl]methanol

<img file="ECSP22010228A_D0160.tif" />

EtOH, AcOH, RT 12h

<img file="ECSP22010228A_D0161.tif" />

XV-1

XVI-1

A stirred solution of 1,2-di(pyridin-2-µl)disulfane (2.68 g, 12.1 mmol) in mixed solvent of AcOH : ethanol (5 mL, 1:10) was degassed under N2. This was followed by the addition of 4-mercaptophenyl)methanol (0.74 g, 5.2 mmol) in mixed AcOH/ethanol solvent (5 mL) dropwise over 20 min and stirred for 12 h under

Ninety-seven atmospheres of N2 at room temperature. The reaction was concentrated under reduced pressure to provide the crude product which was purified by column chromatography (S1O2, 60-70% EtOAc/hexanes) to provide [4-(2-pyrindild¡sulfan¡l)phenyl]methanol as a colorless liquid ( 800 mg, 61% yield).

Intermediate II of Carbonate Leaving Group from Intermediate I or

<img file="ECSP22010228A_D0162.tif" />

II II

<td>Int.</td><td>R.¹,R²</td><td>R.³,R⁴</td><td>X</td><td>MH⁺</td>

<td> 11-1</td><td>H, I</td><td>Η,H</td><td>h</td><td> 367.1</td>

<td>II-2</td><td>I,H</td><td>Η,H</td><td>h</td><td> 367.1</td>

<td>II-3</td><td>-CH2CH2CH2-</td><td>Η,H</td><td>NO2</td><td> 438.0</td>

<td>II-4</td><td>H, I</td><td>I,H</td><td>h</td><td> 381.0</td>

<td>II-5</td><td>I,H</td><td>H, I</td><td>h</td><td> 381.0</td>

<td>II-6</td><td>I,H</td><td>I,H</td><td>h</td><td> 381.0</td>

<td>II-7</td><td>H, I</td><td>H, I</td><td>h</td><td> 381.0</td>

<td>II-8</td><td colspan="2">&lt;pH₂, H Cj H₂,H ch₂ch₂ch₂ch₂</td><td>h</td><td> 407.0</td>

<td>II-9</td><td colspan="2">H,pH₂ Η, &lt; γΗ₂CH₂CH₂CH₂CH₂</td><td>h</td><td> 406.7</td>

Synthesis of 11-1: (4-nitrophenyl) i(2R)-2-(2-pyridyldisulfanyl)propyl1 carbonate

<img file="ECSP22010228A_D0163.tif" />

THF, RT

PNPC, Pyridine, DMAP,

<img file="ECSP22010228A_D0164.tif" />

1-1

11-1

To (2R)-2-(2-pyrid¡ld¡sulfanyl)propan-1-ol (0.39 g, 1.94 mmol) in THF under N2 was added pyridine (0.16 mL, 1.94 mmol) and the (4-nitrophenyl)carbonochloridate. (0.59g, 2.91mmol). The mixture was stirred for 16h under N2. The mixture was diluted with EtOAc and quenched with 20 mL sat. NH4Cl. The mixture was washed with water and brine and the organic layer was concentrated. The crude mixture was purified by column chromatography.

Ninety-seven (SiO2, 0-50% EtOAc/Hexanes) to provide 0.59 g, 83% of (4-nitrophenyl)[(2R)-2(2-pyridyldisulfanyl)propyl]carbonate. MS m/z found 367.1 [M+H]+.

Intermediates II-2 and II-3 were synthesized analogously to II-1.

Synthesis of II-4: 4-nitrophenyl((2R,3R)-3-(pyridin-2-yldisulfanyl)butan-2-yl)carbonate cAci

<img file="ECSP22010228A_D0165.tif" />

Pyridine, Cat. DMAP, DMC, RT, 48 h

To a stirred solution of (2R,3R)-3-(pyridin-2-yldisulfanyl)butan-2-ol (140mg, 0.651mmol) in CH2O2 (2.0mL) was added pyridine (0.11mL, 1.43mmol), 4- nitrophenyl carbonochloridate (150 mg, 0.781 mmol) and catalytic amount of 4-dimethylaminopyridine at room temperature. The reaction vessel was sealed and stirred at RT for 48h. The reaction mixture was diluted with CH2O2 (10 ml) and then washed with water (10 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to provide the crude which was purified by column chromatography using 30-40% ethyl acetate/hexane. The fractions were concentrated to obtain the crude which was further purified on a C18 reverse phase column. The pure fractions were concentrated to obtain 4-nitrophenyl((2R,3R)-3-(pyridin-2-yldisulfaneyl)butan-2-yl)carbonate (70 mg, 28%) as an oily compound. MS m/z 381.0 [M+H]⁺.

Synthesis of II-5: 4-nitrophenyl((2S,3S)-3-(pyridin-2-yldisulfanyl)butan-2-yl)carbonate

<img file="ECSP22010228A_D0166.tif" />

To a stirred solution of (2S,3S)-3-(pyridin-2-yldisulfanyl)butan-2-ol (80mg, 0.372mmol) in CH2Cl2 (1.0mL) was added pyridine (0.066mL, 0.818mmol), 4-nitrophenylcarbonochloridate ( 89 mg, 0.446 mmol) and catalytic amount of 4-dimethylaminopyridine at room temperature. The reaction vessel was sealed and stirred at RT for 48h. The reaction mixture was diluted with CH2Cl2 (5 mL) and then

Ninety-eight washed with water (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to provide crude title product which was purified by column chromatography using 30-40% ethyl acetate/Hexane. The fractions were concentrated to obtain the crude which was further purified on a C18 reverse phase column. The pure fractions were concentrated to obtain 4-nitrophenyl ((2S,3S)-3-(pyridin-2-yldisulfanyl)butan-2-yl)carbonate (140 mg, 58%) as an oily compound. MS m/z 381.0 [M+H]+.

Synthesis of II-6: 4-nitrophenyl((2R,3S)-3-(pyridin-2-yldisulfanyl)butan-2-yl)carbonate

<img file="ECSP22010228A_D0167.tif" />

To a stirred solution of (2R,3S)-3-(pyridin-2-yldisulfanyl)butan-2-ol (0.4 g, 1.86 mmol) in CH2CL2 (10 mL) was added pyridine (0.36 mL, 4.09 mmol), 4 -nitrophenyl carbonochloridate (0.44 g, 2.32 mmol) and catalytic amount of 4-dimethylaminopyridine at 0°C. The reaction vessel was sealed and stirred at room temperature for 48h. The reaction mixture was diluted with CH2Cl2 (20 ml) and washed with water (20 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to provide the crude which was purified by flash column chromatography on silica gel using 30-40% ethyl acetate/hexane. The compound was eluted as a mixture in 30% EtOAc:Hexane. The fractions were concentrated to obtain the crude which was further purified on a C18 reverse phase column. The pure fractions were evaporated to obtain 4-nitrophenyl ((2R,3S)-3-(pyridin-2-yldisulfanyl)butan-2-yl)carbonate (0.17 g, 24.2%) as an oily compound. MS m/z 381.0 [M+H]⁺.

Synthesis of II-7: 4-nitrophenyl((2S,3R)-3-(pyridin-2-yldisulfanyl)butan-2-yl)carbonate cAci

<img file="ECSP22010228A_D0168.tif" />

Pyridine, Cat. DMAP, DMC, RT, 48 h

To a stirred solution of (2S,3R)-3-(pyridin-2-yldisulfanyl)butan-2-ol (0.4 g, 1.86

Ninety-ninemmol) in CH2CI2 (10 mL) was added pyridine (0.36 mL, 4.09 mmol), 4-nitrophenyl carbonochloridate (0.44 g, 2.32 mmol) and catalytic amount of 4-dimethylaminopyridine at 0°C. The reaction vessel was sealed and stirred at room temperature for 48h. The reaction mixture was diluted with CH2O2 (20 ml) and washed with water (20 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to provide the crude which was purified by flash column chromatography on silica gel using 30-40% ethyl acetate/hexane. The compound was eluted as a mixture in 30% EtOAc:Hexane. The fractions were concentrated to obtain the crude which was further purified on a C18 reverse phase column. The pure fractions were concentrated to obtain 4-nitrophenyl ((2S,3R)-3-(pyridin-2-yldisulfanyl)butan-2-yl)carbonate (0.18 g, 26%) as an oily compound. MS m/z 381.0 [M+H]⁺.

Synthesis of II-8: (4-nitrophenyl) [(1R,2R)-2-(2-pyridyldisulfanyl)cyclohexyl1 carbonate

<img file="ECSP22010228A_D0169.tif" />

1-8

THF, RT, 48h

PNP, K2CO3, DMAP

<img file="ECSP22010228A_D0170.tif" />

II-8

To a solution of (1R,2R)-2-(2-pyridyldisulfanyl)cyclohexanol* (130.0 mg, 0.5 mmol) in THF (3.0 mL) was added potassium carbonate (0.20 g, 1.5 mmol), catalytic amount of DMAP, and 4-nitrophenyl chloroformate (0.21 g, 0.10 mmol) at room temperature. The reaction vessel was sealed and stirred at RT for 48h. The progress of the reaction was monitored by TLC (20% EtOAc/Hex). After completion of the reaction, the reaction mixture was quenched with water (20.0 mL) and extracted with EtOAc (20.0 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to provide the crude product which was purified by column chromatography using 20-30% EtOAc/hexanes to provide 4-nitrophenyl. (4-nitrophenyl)[(1R,2R)-2-(2-pyridyldisulfanyl)cyclohexyl-1-carbonate* as an off-white solid (89 mg, 40% yield). MS m/z 407.0 [M+H]⁺.

Synthesis of II-9: (4-nitrophenyl) [(1S,2S)-2-(2-pyridyldisulfanyl)cyclohexyl1 carbonate

<img file="ECSP22010228A_D0171.tif" />

I-8

PNP, K2CO3, DMAP

THF, RT, 48h

<img file="ECSP22010228A_D0172.tif" />

To a solution of (1S,2S)-2-(2-pyridyldisulfanyl)cyclohexanol* (0.42 g, 1.7 mmol) in

THF (10.0 mL) was added potassium carbonate (0.70g, 5.1 mmol), catalytic amount of

100

100

CentoDMAP and 4-nitrophenyl chloroformate (0.69 g, 3.4 mmol) at room temperature. The reaction vessel was sealed and stirred at RT for 48h. The progress of the reaction was monitored by TLC (20% EtOAc/Hex). After completion of the reaction, the reaction mixture was quenched with water (20.0 mL) and extracted with EtOAc (20.0 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to provide the crude product which was purified by column chromatography using 20-30% EtOAc/hexanes to provide 4-nitrophenyl (4 -nitrophenyl)[(1R,2R)-2-(2-pyridyldisulfanyl)cyclohexyl]carbonate* as an off-white solid (250 mg, 35% yield). MS m/z 406.7 [M+H]⁺.

Intermediate XV of Carbonate Leaving Group from XIV

<img file="ECSP22010228A_D0173.tif" />

<img file="ECSP22010228A_D0174.tif" />

<td>Intermediary</td><td>R.⁵,R⁶</td><td>R.⁹,R¹⁰,R¹¹,R¹²</td><td>X</td><td>MH⁺</td>

<td>XV-1</td><td>H H</td><td>H, H, H, H</td><td>h</td><td> 415.0</td>

Synthesis of XV-1: (4-nitrophenyl)[4-(2-pyridyldisulfanyl)phenyl]methyl carbonate

<img file="ECSP22010228A_D0175.tif" />

Pyridine, DMAP

DCM, RT, 2h

<img file="ECSP22010228A_D0176.tif" />

XIV-1 XV-1

To a stirred solution of (4-(pyridin-2-yldisulfanyl)phenyl)methanol (0.40 g, 1.60 mmol) in CH2Cl2 (10 mL) were added 4-nitrophenyl chloroformate (0.65 g, 3.2 mmol), pyridine (0.25 mL, 3.20 mmol), catalytic amount of DMAP (0.005 g) at 0°C. The mixture was allowed to stir for 2h at room temperature. The reaction mixture was quenched with 1.5N HCl solution. The organic layer was separated and washed with brine, dried over anhydrous Na2SO4, and concentrated. The crude product was purified by column chromatography (SiO2, 20-30% EtOAc/hexanes) to provide (4-nitrophenyl)[4-(2-pyridyldisulfanyl)phenyl]methyl carbonate as a colorless liquid (600 mg, 91% performance); MS m/z 415.0 [M+H]^-.

Intermediates III Linked to Carbonate and Carbamate

101

101

One hundred one-

<img file="ECSP22010228A_D0177.tif" />

<img file="ECSP22010228A_D0178.tif" />

x

II III

<td>Intermediary</td><td>R.⁸h</td><td>R.¹,R²</td><td>R.³,R⁴</td><td>X</td><td>MH-</td>

<td>III-1</td><td>R.⁸H-5</td><td>H, I</td><td>H H</td><td>h</td><td> 663.0</td>

<td>III-2</td><td>R.⁸H-5</td><td>I,H</td><td>H H</td><td>h</td><td> 663.0</td>

<td>III-3</td><td>R.⁸H-5</td><td>-CH2CH2CH2-</td><td>H H</td><td>NO2</td><td> 734.0</td>

<td>III-4</td><td>R.⁸H-5</td><td>H, I</td><td>I,H</td><td>h</td><td> 677.0</td>

<td>III-5</td><td>R.⁸H-5</td><td>I,H</td><td>H, I</td><td>h</td><td> 677.1</td>

<td>III-6</td><td>R.⁸H-5</td><td>I,H</td><td>I,H</td><td>h</td><td> 677.0</td>

<td>III-7</td><td>R.⁸H-5</td><td>H, I</td><td>H, I</td><td>h</td><td> 677.1</td>

<td>III-8</td><td>R.⁸H-5</td><td colspan="2">CH2, HC^h2,H ch₂ch₂ch₂ch₂</td><td>h</td><td> 703.0</td>

<td>III-9</td><td>R.⁸H-5</td><td colspan="2">H, CH2 H, CH2 ch₂ch₂ch₂ch₂</td><td>h</td><td> 703.1</td>

Synthesis of III-1: [(2S)-2-(2-pyr¡dyldisulfanyl)prop¡l1 N-[(10S,23S)-10-ethyl-18-fluoro-10 hydroxy-19-methyl-5,9 -dioxo-8-oxa-4,15diazahexacyclori4.7.1.02,14.04,13.06,11.020,241tetracosa-1,6(11),12,14,16(24),17,19heptaen-23-illcarbamate

<img file="ECSP22010228A_D0179.tif" />

HOBt, Pyridine 4 A Sieve

DMF, RT

<img file="ECSP22010228A_D0180.tif" />

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To a mixture of 1-hydroxybenzotriazole hydrate (8.64 mg, 0.0564 mmol), finely ground 4 A molecular sieve (50 mg) of (10S,23S)-23amino-10-ethyl-18-fluoro-10-methanesulfonic acid hydroxy-19-methyl-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose-1,6(11),12,14,16(24),17,19heptaene-5, 9-Dione (25.0 mg, 0.0470 mmol) and Pyridine (0.0190 mL, 0.235 mmol) in 2 mL of anhydrous DMF was added (4-nitrophenyl)[(2S)-2-(2-pyridyldisulfanyl)propyl]carbonate (19.0 mg , 0.0517 mmol). After stirring for 16h at room temperature the mixture was filtered and the solution was concentrated. The residue was purified by column chromatography (0-5% MeOH/DCM) to yield [(2S)-2-(2-pyridyldisulfanyl)propyl] N-[(10S,23S)-10-ethyl-18-fluoro10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24) ,17,19heptaen-23-yl]carbamate (29.0 mg, yield: 93.0%). MS m/z 663.0 [M+H]⁺.

Intermediates III-2 through III-9 were prepared from II-2 through II-9 in a manner analogous to III-1.

XVI Intermediates Linked to Carbonate and Carbamate

<img file="ECSP22010228A_D0181.tif" />

R8-H, Base, HOBT

<img file="ECSP22010228A_D0182.tif" />

<td>Int.</td><td>R.⁸h</td><td>R.⁵,R⁶</td><td>R.⁹,R¹⁰,R¹¹,R¹²</td><td>X</td><td>MH⁺</td>

<td>XVI-1</td><td>R.⁸H-5</td><td>H H</td><td>H, H, H, H</td><td>h</td><td> 711.0</td>

The Intermediate XVI-1 was prepared from the XV-1 in a manner analogous to the III-1.

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

<img file="ECSP22010228A_D0183.tif" />

Step 1: Synthesis of racemic trans-(4-hydroxytetrahydrofuran-3-yl) ethanethioate

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one hundred and three

<img file="ECSP22010228A_D0184.tif" />

To a stirred solution of 3,6-dioxabicyclo[3.1.0]hexane (5.0 g, 0.051 mol) in water (40.0 mL) was added thioacetic acid (4.98 mL, 0.069 mol) and the resulting reaction mixture was stirred at room temperature. environment for 16 h. The progress of the reaction was monitored by TLC (20% EtOAc/Hexane). After the completion of the reaction, the reaction mixture was diluted with diethyl ether and washed with 10% sodium bicarbonate solution. The organic layer was separated and washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide the crude product, which was purified by column chromatography using 20% ​​EtOAc : n-Hexane to obtain the title product as a brown liquid (4.0 g, 42% yield).¹HNMR (400 MHz, CDCh): δ 4.35 - 4.28 (m, 2H), 4.02 - 3.98 (m, 1H), 3.81 - 3.73 (m, 2H), 3.69 - 3.62 (m, 1H), 2.73 (s, 3H ).

Step 2: Synthesis of racemic Trans-4-mercaptotetrahydrofuran-3-ol

<img file="ECSP22010228A_D0185.tif" />

To a stirred solution of racemic trans-(4-hydroxytetrahydrofuran-3-yl)ethanethioate (4.0 g, 24.7 mmol) in dry THF (20.0 mL) under a nitrogen atmosphere was added LAH (1 M in THF) (27.1 mL, 27.1 mmol) dropwise at 0°C. The reaction mixture was allowed to gradually warm to room temperature and stirred for 2 h. The progress of the reaction was monitored by TLC (20% EtOAc : n-Hexane). After completion of the reaction, the reaction mixture was cooled to room temperature and quenched with 1.0N HCl (50 mL). The reaction mixture was extracted into DCM (3 x 20 ml), the organic layer was washed with brine solution (20 ml), dried over anhydrous sodium sulfate, filtered, partly distilled and taken as such for the next step. (2.9g, raw).

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

<img file="ECSP22010228A_D0186.tif" />

Isomer 1 trans (1RS,2RS) Isomer 2 trans (1SR, 2SR)

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one hundred four

To a solution of 2-(pyridin-2-yldisulfanyl)pyridine (0.9 g, 21.7 mmol) in MeOH (degassed with N2) (10 mL) was added 4-sulfaniloxolan-3-ol (2.9 g, 24.1 mmol) (degassed with with N2) dropwise and stirred at room temperature under a nitrogen atmosphere for 16 h. The reaction mixture was concentrated to dryness under vacuum. The resulting crude was purified by flash column chromatography using 30% EtOAc : n-Hexane to provide the title compound 4-(pyridin-2-yldisulfanyl)oxolan-3-ol (racemic) as a yellow oil. Isomers were separated by Chiral preparative HPLC.

Chiral preparative HPLC conditions:

Column: Chiralpak IA (250 mm X 20 mm X 5 mic)

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

Flow rate : 19 ml/min

Separate fractions of the resolved isomers were collected from chiral preparative HPLC and evaporated under reduced pressure to give the title compounds as Isomer 1 (600mg) and Isomer 2 (620mg).

Isomer 1: (trans-(4RS,3RS)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol): LCMS m/z calcd for C9H11NO2S2, 229; found 230 [M+H]+. 1H-NMR (400 MHz, CDCl3): δ 8.53 - 8.52 (m, 1H), 7.67 - 7.63 (m, 1H), 7.56 (d, J = 8.0Hz, 1H), 7.23 - 7.19 (m, 1H), 4.45 - 4.48 (m, 1H), 4.25 (t, J = 8.8Hz,1H), 4.12 (t, J = 6.8Hz,1H), 3.74 -3.67 (m, 2H), 3.48 - 3.41 (m, 1H) .

Isomer 2: (trans-(4SR,3SR)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol): LCMS m/z calcd for C9H11NO2S2, 229; found 230 [M+H]⁺. 1H-NMR (400 MHz, CDCl3): δ 8.54 - 8.53 (m, 1H), 7.68 - 7.64 (m, 1H), 7.56 (d, J = 8.0Hz, 1H), 7.23 - 7.20 (m, 1H), 4.49 - 4.45 (m, 1H), 4.25 (t, J = 7.6Hz,1H), 4.12 - 4.10 (m,1H), 3.74 - 3.67 (m, 2H), 3.47 - 3.44 (m, 1H).

The absolute stereochemistry of the isomers was assigned arbitrarily.

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

<img file="ECSP22010228A_D0187.tif" />

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Slow C- dnco To a stirred solution of trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol (0.61 g, 2.69 mmol) in DMF (10 mL) under a nitrogen atmosphere DIPEA (1.45 mL, 8.08 mmol) and bis(4-nitrophenyl)carbonate (1.64 g, 5.38 mmol) at room temperature. The reaction vessel was sealed and stirred at room temperature for 12h. The progress of the reaction was monitored by TLC (20% EtOAc : n-Hexane). After completion of the reaction, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide the crude product which was purified by flash column chromatography using 20-30% EtOAc : n Hexane. to provide 4-nitrophenyl(trans-(3RS,4RS)-4-(pyridin-2yldisulfanyl)tetrahydrofuran-3-yl)carbonate as an off-white solid (790 mg, 77% yield).¹HNMR (400 MHz, CDCl3): δ 8.50 (d, J = 4.4 Hz, 1H), 8.27 (d, J = 8.8 Hz, 2H), 7.67 - 7.59 (m, 2H), 7.36 (d, J = 8.8 Hz , 2H), 7.15 (t, J = 5.2 Hz, 1H), 5.44 - 5.43 (m, 1H), 4.40 - 4.25 (m, 2H), 4.03 (d, J = 11.2 Hz, 1H), 3.92 - 3.86 ( m, 1H), 3.85-3.79 (m, 1H); LCMS m/z calculated for C16H14N2O6S2, 394; found 395 [M+H]⁺.

Synthesis of 4-nitrophenyl (trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran3-yl)carbonate

<img file="ECSP22010228A_D0188.tif" />

To a stirred solution of trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-ol (550 mg, 2.46 mmol) in DMF (10.0 mL) under nitrogen was added DIPEA (1.32 mL, 7.38 mmol) and bis(4-nitrophenyl)carbonate (1.5 g, 4.92 mmol) at room temperature. The reaction vessel was sealed and stirred at room temperature for 12h. The progress of the reaction was monitored by TLC (20% EtOAc : n-Hexane). After completion of the reaction, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide the crude product which was purified by flash column chromatography using 20-30% EtOAc:n- Hexane to provide 4-nitrophenyl (trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydrofuran-3-yl)carbonate as an off-white solid (0.6 g, 70% yield).¹HNMR (400 MHz, CDCl3): δ 8.85 (d, J = 4.4 Hz, 1H), 8.26 (d, J = 8.8 Hz, 2H), 7.68 - 7.59 (m, 2H), 7.35 (d, J = 8.8 Hz , 2H), 7.14 (t, J = 5.2 Hz, 1H), 5.44 - 5.43 (m, 1H), 4.40 - 4.25 (m, 2H), 4.03 (d, J =

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A hundred and six

11.2 Hz, 1H), 3.92 - 3.86 (m, 1H), 3.85 - 3.79 (m, 1H); LC-MS m/z calculated for

C16H14N2O6S2, 394; found 395 [M+H]+.

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

<img file="ECSP22010228A_D0189.tif" />

Step 1: Synthesis of racemic trans-(5-hydroxycyclopentan-1-yl) ethanethioate

<img file="ECSP22010228A_D0190.tif" />

<img file="ECSP22010228A_D0191.tif" />

To a stirred solution of 6-oxabicyclo[3.1.0]hexane (3.0 g, mmol) in water (30 mL) was added thioacetic acid (3 mL, 39.2 mmol) at room temperature and stirred for 16 h. The reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 x 10 mL). The organic layer was separated and washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give racemic trans-(5-hydroxycyclopentan-1-yl)ethanethioate as an oily compound (2.6 g, raw). LC-MS m/z calculated for C7H12O2S, 160.2; found 143.3 [M+H - 17]⁺.

Step 2: Synthesis of racemic Trans-2-mercaptocyclopentan-1-ol

ooh

<img file="ECSP22010228A_D0192.tif" />

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

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Step 3: Synthesis of trans-(1RS, 2RS)-2-(pyridin-2-yldisulfanill)cyclopentan-1-ol and trans-(1SR, 2SR)-2-(pyridin-2-yldisulfanill)cyclopentan-1-ol

<img file="ECSP22010228A_D0193.tif" />

<img file="ECSP22010228A_D0194.tif" />

Isomer 1 trans (1RS,2RS)

2-trans isomer (1SR, 2SR)

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

Chiral preparative HPLC conditions:

Column: Chiralpak IA (250 mm X 20 mm X 5 mic)

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

Flow rate : 19 ml/min

The separated fractions of the separated isomers were collected from chiral preparative HPLC and evaporated under reduced pressure to give the title compounds as Isomer 1 (300mg) and Isomer 2 (300mg) as a colorless oil.

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

Isomer 2 (trans-(1SR, 2SR)-2-(pyridin-2-yldisulfanill)cyclopentan-1-ol): LC-MS m/z calcd for C10H13NOS2, 227.34; found 228.1 [M+H]⁺. ¹HNMR (400 MHz, CDCl3): δ 8.51 - 8.50 (m, 1H), 7.61 - 7.57 (m, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.17 - 7.14 (m,

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One hundred and eight1H), 4.03 - 3.97 (m, 1H), 3.0 - 2.87 (m,1H), 2.11 - 2.02 (m, 3H), 1.75 - 1.65 (m, 4H).

The absolute stereochemistry of the isomers was assigned arbitrarily.

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

<img file="ECSP22010228A_D0195.tif" />

To a stirred solution of trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol (0.3g, 1.34mmol) in DMF (10ml) under a nitrogen atmosphere was added DIPEA (0.65ml , 3.96 mmol) and Bis(4-nitrophenyl)carbonate (0.8 g, 2.64 mmol) at room temperature. The reaction vessel was sealed and stirred at room temperature for 16h. The reaction mixture was quenched with water (20ml) and extracted with EtOAc (3 x 10ml). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was purified by flash column chromatography on silica gel. The compound was eluted in 10% EtOAc : n-Hexane as a mixture. Fractions were evaporated to obtain the crude compound which was purified on reverse phase column chromatography. Fractions containing the product were evaporated under reduced pressure to obtain 4-nitrophenyl(trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cyclopentyl)carbonate as a colorless oil (305 mg, 59%).¹HNMR (400 MHz, CDCl3): δ 8.46 (d, J = 4.1Hz, 1H), 8.25 (d, J = 6.8 Hz, 2H), 7.66 - 7.62 (m, 2H), 7.34 (d, J = 6.4 Hz , 2H), 7.10 - 7.08 (m, 1H), 5.29 - 5.10 (m, 1H), 3.52 - 3.45 (m, 1H), 2.32 - 2.28 (m, 2H), 1.9 -1.76 (m, 4H). LC-MS m/z calculated for C17H16N2O5S2, 392.44; found 393.0 [M+H]⁺.

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

<img file="ECSP22010228A_D0196.tif" />

To a stirred solution of (1SR,2SR)-2-(pyridin-2-yldisulfanyl)cyclopentan-1-ol (0.26 g, 1.14 mmol) in DMF (10.0 mL) under a nitrogen atmosphere was added DIPEA (0.57 mL, 3.43 mmol) and bis(4-nitrophenyl)carbonate (0.7 g, 2.29 mmol) at room temperature. He

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One hundred and nine reaction vessel was sealed and stirred at room temperature for 16 h. The reaction mixture was quenched with water (20.0 mL) and extracted with EtOAc (3 x 10 mL). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was purified by flash column chromatography on silica gel. The compound was eluted in 10% EtOAc : n-Hexane as a mixture. Fractions were evaporated to obtain the crude compound which was purified on reverse phase column chromatography. Fractions containing the product were evaporated under reduced pressure to obtain 4-nitrophenyl(trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cyclopentyl)carbonate (330 mg, 73.5%) as a colorless oil.¹HNMR (400 MHz, CDCb): δ 8.46 (d, J = 4Hz, 1H), 8.25 (d, J = 6.8 Hz, 2H), 7.66 - 7.62 (m, 2H), 7.34 (d, J = 6.4 Hz, 2H), 7.10 - 7.08 (m, 1H), 5.29 - 5.10 (m, 1H), 3.52 - 3.45 (m, 1H), 2.32 - 2.28 (m, 2H), 1.9 -1.76 (m, 4H). LC-MS m/z calculated for C17H16N2O5S2, 392.44; found 393.0 [M+H]+.

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

<img file="ECSP22010228A_D0197.tif" />

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

<img file="ECSP22010228A_D0198.tif" />

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

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Hundred- d-ie^¹HNMR (400 MHz, CDCI3): δ 7.14 (t, J = 3.2 Hz, 2H), 7.05 (t, J = 3.2 Hz, 2H),

3.48 (s, 2H), 3.32 (d, J = 17.6 Hz, 2H), 3.19 (d, J = 17.6 Hz, 2H).

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

<img file="ECSP22010228A_D0199.tif" />

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

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

Step 3: Synthesis of racemic Trans-3-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol

<img file="ECSP22010228A_D0200.tif" />

To a stirred solution of [(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)sulfanyl](phenyl)methanone (115 mg, 404 pmol) in methanol (3.00 mL) was added K2CO3 (113 mg , 2 eq., 809 pmol) and the reaction mixture was stirred for 0.5 h at RT. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was concentrated (to remove methanol) and then acidified with 1N HCl solution until pH reached 2-3, to obtain trans -3-sulfanyl-1,2 Racemic ,3,4-tetrahydronaphthalen-2-ol (70.0 mg, 388 pmol) which was further taken as such for the next step.

Step 4: Synthesis of trans-(2RS, 3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4111 tetrahydronaphthalen-2-ol and trans-(2SR, tetrahydronaphthalen-2-ol

<img file="ECSP22010228A_D0201.tif" />

<img file="ECSP22010228A_D0202.tif" />

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Hundred eleven

3SR)-3-(pyridin-2-yldisulfanyl)-1,2,3,4Isomer 1 trans (2RS,3RS)

2-trans isomer (2SR, 3SR)

To a stirred solution of racemic trans-3-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol (350 mg, 1.94 mmol) in methanol (2.50 mL) under a nitrogen atmosphere was added 2-(pyridin-2 -yldisulfanyl)pyridine (428 mg, 1 eq., 1.94 mmol) and stirred at RT for 16 h. The progress of the reaction was monitored by TLC and LC-MS. After the completion of the reaction, the reaction mass was concentrated and then diluted with DCM, washed with water followed by brine, and dried over sodium sulfate. The obtained crude product was purified by flash column chromatography on silica gel. The desired product was eluted in 20% EtOAc : Hexane. The product was repurified by reverse phase column chromatography (10-20% of 0.1% Formic acid in water/Acetonitrile). Fractions containing the desired product were collected and evaporated under vacuum to give 3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (350 mg, 1.21 mmol) as a yellow solid. Isomers were separated by Chiral preparative HPLC.

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

Preparatory conditions:

Column: CHIRALPAK IA (250 mm X 420 mm X 5 mic)

Mobile phase : n-Hexane : Ethanol with 0.1% DEA (50:50)

Flow rate : 19 ml/min

The isomers were separated and the respective fractions collected from the chiral preparative HPLC were combined and evaporated to give the respective isomers. Isomer 1 was collected first and assigned as trans-(2RS, 3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol. Isomer 2 was collected second and assigned as trans-(2SR, 3SR)-3-(pyridin-2112

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One hundred and twelve yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol. The absolute stereochemistry of the isomers was assigned arbitrarily.

Step 5: Synthesis of 4-nitrophenyl (trans-(2RS, 3RS)-3-(pyridin-2-yldisulfanyl)1,2,3,4-tetrahydronaphthalen-2-yl)carbonate

To a stirred solution of trans-(2RS, 3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (150 mg, 518 pmol) in Dimethylformamide (3.00 mL, 38.7 mmol) bis(4-nitrophenyl)carbonate (315 mg, 2 eq., 1.04 mmol) was added followed by N,NDiisopropylethylamine (271 pL, 3 eq., 1.55 mmol). The reaction mixture was stirred at RT for 12h. After completion of the reaction, the reaction mass was quenched with water, extracted with DCM (3 x 5), the combined organic phase dried over sodium sulfate, filtered and evaporated under reduced pressure to give the product raw. The crude product was purified by flash column chromatography on silica gel (0-40% EtOAc : n-Hexane) and also further purified by reverse phase column chromatography (10-50% of 0.1% formic acid in water). : ACN), to yield (trans-(2RS, 3RS)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl)carbonate (133 mg, 293 pmol) as an off-white solid .

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

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

<img file="ECSP22010228A_D0203.tif" />

To a stirred solution of trans-(2SR,3SR)-3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (130 mg, 449 pmol) in dimethylformamide (2.60 mL, 33.6 mmol) bis(4-nitrophenyl)carbonate (273 mg, 2 eq., 898 pmol) was added followed by diisopropylethylamine (13.0 mL, 3 eq., 74.6 mmol). The reaction mixture was stirred at RT for 12h. After completion of the reaction (reaction progress was monitored by TLC), the reaction mass was quenched with water, extracted with DCM (3 x 5), the combined organic phase was dried over sodium sulfate, filtered and evaporated under reduced pressure to give the crude product which was purified by flash column chromatography (0-40% EtOAc : n -Hexane). The product was further purified by

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One hundred and thirteen reverse phase column chromatography (10-50% of 0.1% formic acid in water: ACN) to yield 4-nitrophenyl(trans-(2SR,3SR)-3-(pyridin-2-yldisulfanyl)-1, 2,3,4-Tetrahydronaphthalen-2-yl)carbonate (30.0 mg, 66.0 pmol) as an off-white solid.

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

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

<img file="ECSP22010228A_D0204.tif" />

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

<img file="ECSP22010228A_D0205.tif" />

To a stirred solution of 3,6-dihydro-2H-pyran (2.0 g, 23.8 mmol) in dichloromethane (20.0 mL) at 0°C was added 3-chlorobenzene-1-carboperoxoic acid (4.92 g, 1.2 eq., 28.5 mmol) slowly in_portions and stirred under a nitrogen atmosphere for 16 h at room temperature. The progress of the reaction was monitored by TLC. After the completion of the reaction, the reaction mass was quenched with saturated sodium bicarbonate solution, and the organic layer was separated and washed with water followed by brine solution, dried over anhydrous sodium sulfate, filtered, and evaporated to give the title compound 3,7-dioxabicyclo[4.1.0]heptane (1.00 g, 9.99 mmol) as a colorless oil.¹H NMR (400 MHz, CDCla) : 4.03 - 3.94 (m, 2H), 3.55 3.49 (m, 1H), 3.46 - 3.41 (m, 1H), 3.35 (m, 1H), 3.18 (m, 1H), 2.00 (m, 2H).

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

<img file="ECSP22010228A_D0206.tif" />

To a stirred solution of 3,7-dioxabicyclo[4.1.0]heptane (1.00 g, 9.99 mmol) in ethoxyethane (40 mL) at room temperature was added S-benzenecarbothioic acid (5.88 mL, 5 eq., 49.9 mmol) followed by of silanedione (3.00 g, 5 eq., 49.9 mmol) and the

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One hundred and fourteen reaction mixture was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC, after completion of starting material the reaction mass was quenched with saturated sodium bicarbonate solution and then extracted with ethyl acetate (2 x 10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and then evaporated under reduced pressure to give the crude product which was purified by flash column chromatography (0-30% EtOAc : n-Hexane). The compound eluted at 20% EtOAc : n-Hexane. The pure fractions were collected and evaporated to yield racemic [trans-(3-hydroxytetrahydropyran4-yl)sulfanyl](phenyl)methanone (2.0 g, 8.39 mmol).

LC-MS m/z calculated for C12H14O3S; 238.3, found 239.1 [M+H]+;¹HNMR (400 MHz, CDCI3): δ 7.43 (d, J = 7.6 Hz, 2H), 7.09 (t, J = 17.2 Hz, 1H), 6.93 (t, J = 8.0 Hz, 2H), 3.55 (dd, J = 4.0 Hz, 4.0 Hz, 1H), 3.37 (d, J = 11.2 Hz, 1H), 3.28 - 3.24 (m, 1H), 3.22 - 3.18 (m, 1H), 3.01 (t, J = 10.8 Hz, 1H ), 2.83 (t, J = 12.4 Hz, 1H), 1.36 1.27 (m, 1H), 1.27 (s, 2H).

Step 3: Synthesis of racemic Trans-4-sulfaniloxan-3-ol

<img file="ECSP22010228A_D0207.tif" />

To a stirred solution of racemic [ trans-3-hydroxytetrahydropyran-4yl)sulfanyl](phenyl)methanone (2.50 g, 10.5 mmol) in dichloromethane (25 mL) at room temperature was added hydrazine hydrate (5.15 mL, 10 eq., 105 mmol) slowly and the reaction mixture was stirred for 1 h. The progress of the reaction was monitored by TLC, after completion of the reaction, the reaction mass was quenched with 1N HCl so that the pH was adjusted to 2-3. The two layers were separated and the organic layer was dried over sodium sulfate, filtered and partially evaporated and the crude racemic trans4-sulfanyltetrahydropyran-3-ol was taken for the next step.

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

<img file="ECSP22010228A_D0208.tif" />

Isomer 1 trans (2RS,3RS) Isomer 2 trans (2SR, 3SR)

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Hundred fifteen

To a stirred solution of 2-(pyridin-2-yldisulfanyl)pyridine (1.85 g, 0.8 eq., 8.41 mmol) in Methanol (40 mL) was added racemic trans-4-sulfanyltetrahydropyran-3-ol (1.41 g, 10.5 mmol ) in DCM at 0 °C and then the reaction mixture was stirred at room temperature for 12 h. After the completion of the reaction, the entire reaction mass was evaporated under reduced pressure to give the crude which was purified by flash column chromatography. The product was eluted in 20% EtOAc : n Hexane, the pure fractions collected and evaporated to give the title product 4-(pyridin-2-yldisulfanyl)oxan-3-ol (racemic). Isomers were separated by Chiral preparative HPLC.

Chiral preparative HPLC conditions:

Column : CHIRALPAK IA (250 mm X 20 mm X 5 mic)

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

Flow rate : 19 ml/min

The isomers were separated and the respective fractions were collected from chiral preparative HPLC. Fractions were combined and evaporated to provide the respective isomers.

(Isomer 1- 350 mg, Isomer 2 - 350 mg) LC-MS m/z calculated for

C10H13NO2S2; 243.34, found 244 [M+H]+.

Isomer 1 (trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-ol):

¹H-NMR (400 MHz, DMSO): δ 8.53 (s, 1H), 7.60 (t, J = 6.40 Hz, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.23 (t, J = 20.8 Hz , 1H), 4.28 - 4.06 (m, 1H), 3.94 (d, J = 12 Hz, 1H), 3.54 - 3.40 (m, 3H), 3.33 - 3.21 (m, 1H), 3.07 - 2.74 (m, 1H ), 2.04-1.94 (m, 2H).

Isomer 2 (trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3-ol)

1H-NMR (400 MHz, DMSO): δ 8.52 (d, J = 2.8 Hz, 1H), 7.61 (t, J = 6.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.18 (t , J = 5.2 Hz, 1H), 4.12 - 4.09 (m, 1H), 3.94 (d, J = 12 Hz, 1H), 3.53 - 3.47 (m, 1H), 3.47 - 3.37 (m, 1H), 3.25 ( t, J = 10.4 Hz, 1H), 2.80 - 2.73 (m, 1H), 1.96 -1.42 (m, 1H), 1.20 (d, J = 6.0 Hz, 2H).

The absolute stereochemistry of the isomers was assigned arbitrarily.

Step 5: Synthesis of 4-nitrophenyl (trans-(3RS,4RS)-4-(pyridin-2yldisulfanyl)tetrahydropyran-3-yl)carbonate

To a stirred solution of (trans -(3RS,4RS)-4-(pyridin-2116

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One hundred and sixteen yldisulfanyl)tetrahydropyran-3-ol) (300 mg, 1.23 mmol) in DMF (8 mL) was added bis(4-nitrophenyl)carbonate (750 mg, 2 eq., 2.47 mmol) then followed by diisopropylethylramine (644 pL , 3 eq., 3.70 mmol) at room temperature for 12 h. After completion of the reaction, the reaction mass was partitioned between water and DCM. The organic layer was separated and washed with brine solution and dried over sodium sulfate, filtered and evaporated under reduced pressure to give the crude which was purified by flash column chromatography. The desired compound was eluted at 25% EtOAc : n-Hexane as a mixture. The mixture was purified by reverse phase column chromatography (10-60% of 0.1% formic acid in water/ACN). Fractions containing the desired product were combined and evaporated to give 4-nitrophenyl(trans-(3RS,4RS)-4-(pyridin-2-yldisulfanyl)tetrahydropyran-3yl)carbonate (270 mg, 0.66 mmol). LC-MS m/z calculated for C17H16N2O6S2; 408.4, found 409.1 [M+H]⁺; ¹H-NMR (400 MHz, CDCI3): δ 8.46 (d, 1H), 8.28 (d, J = 8.8 Hz, 2H), 7.64 - 7.52 (m, 2H), 7.41 (d, J = 8.8 Hz, 2H) , 7.09 (s, 1H), 4.87 (d, J = 2.8 Hz, 1H), 4.25 - 4.18 (m, 1H), 3.91 (d, J = 11.6 Hz, 1H), 3.52 - 3.42 (m, 1H), 3.20 (d, J = 2.8 Hz, 1H), 2.21 (d, J = 12.4 Hz, 1H), 1.98 (d, J = 7.6 Hz, 1H), 1.25 (s, 1H).

Synthesis of 4-nitrophenyl (trans-(3SR,4SR)-4-(pyridin-2-yldisulfanyl)tetrahydropyran3-yl)carbonate

<img file="ECSP22010228A_D0209.tif" />

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

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One hundred seventeen

C17H16N2O6S2; 408.4, found 409.1 [M+H]⁺; ¹H-NMR (400 MHz, DMSO): δ 8.46 (d,

1H), 8.28 (d, J = 8.0 Hz, 2H), 7.66 - 7.58 (m, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 4.87 (d, J = 3.6 Hz, 1H), 4.25 - 4.22 (m, 1H), 3.91 (d, J = 11.6 Hz, 1H), 3.52 - 3.42 (m, 2H), 3.20 (d, J = 3.6 Hz, 1H), 2.21 (d , J = 12.0 Hz, 1H), 1.98 - 1.95 (m, 1H).

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

<img file="ECSP22010228A_D0210.tif" />

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

cz^b

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

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

<img file="ECSP22010228A_D0211.tif" />

To a stirred solution of 8-oxabicyclo[5.1.0]octane (3.00 g, 26.7 mmol) in toluene (60 mL) at room temperature under a nitrogen atmosphere was added S-benzenecarbothioic acid (4.72 mL, 1.5 eq., 40.1 mmol ), followed by 2-methylpropan-2-aminium chloride (293 mg, 0.1 eq., 2.67 mmol). The reaction mixture was stirred at 50 °C for 16 h (reaction progress was monitored by TLC). Behind the

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One hundred and eighteen completion of the reaction, the reaction mixture was quenched with saturated sodium bicarbonate solution and then extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to provide the crude product, which was purified by flash column chromatography. The desired product was eluted in 20% EtOAc : n-Hexane, the pure fractions collected and evaporated to give the title compound [ trans -(2-hydroxycycloheptyl)sulfanyl](phenyl)methanone racemic (3.0 g, 12.0 mmol). LC-MS m/z calculated for CuH18O2S;250.4,¹H-NMR (400 MHz, CDCh) : δ 7.96 (d, J = 8.0 Hz, 2H), 7.57 (t, J = 6.8 Hz, 1H), 7.4 (t, J = 7.6 Hz, 2H), 3.88 - 3.85 (m, 1H), 3.81 - 3.77 (m, 1H), 2.09 - 2.05 (m, 1H), 2.04 - 1.62 (m, 8H), 1.55 - 1.53 (m, 2H).

Step 3: Synthesis of racemic Trans-4-sulfanylcycloheptan-3-ol aOH

SH

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

Step 4: Synthesis of trans-(1RS, 2RS)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol and trans-(1SR, 2SR)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol^HQW\/HO'\__/

1 trans isomer (1RS, 2RS) 2 trans isomer (1SR, 2SR)

To a stirred solution of 2-(pyridin-2-yldisulfanyl)pyridine (1.73 g, 0.7 eq., 7.85 mmol) in methanol (25 mL) under a nitrogen atmosphere at 0°C was added trans-4119

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One hundred and nineteen racemic sulfanylcycloheptan-3-ol (1.64 g, 11.2 mmol) in DCM and the reaction mixture was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC and LCMS and the reaction mass was evaporated under reduced pressure. The crude product was purified by flash column chromatography and the desired product eluted with 20% EtOAc : n-Hexane. As the product was collected as a mixture, it was repurified by reverse phase column chromatography (10-50% of 0.1% formic acid in water : acetonitrile) to give trans-2-(pyridin-2-yldisulfanyl)cycloheptan-1- Racemic ol (1.5 g, 52%) (Racemic mixture). Isomers were separated by Chiral preparative HPLC.

(Isomer-1: 550 mg, Isomer-2: 550 mg).

Chiral preparative HPLC conditions:

Column : CHIRALPAK IA (250 mm X 20 mm X 5 mic)

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

Flow rate : 19 ml/min

The isomers were separated and the respective fractions were collected from chiral preparative HPLC. Fractions were evaporated separately to provide the respective isomers.

Isomer 1 (trans-(1RS, 2RS)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol):

LC-MS m/z calculated for C12H17NOS2; 255.4, found 256.2 [M+H]+;¹HNMR (400 MHz, CDCh) : δ 8.49 (s, 1H), 7.56 (d, J = 6.8 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 6.17 (s , 1H), 3.51 (m, 1H), 2.75 - 2.73 (m, 1H), 2.08 - 1.95 (m, 2H), 1.82 - 1.67 (m, 4H), 1.57 - 1.25 (m, 4H).

Isomer 2 (trans-(1SR, 2SR)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol):

LC-MS m/z calculated for C12H17NOS2; 255.4, found 256.2 [M+H]⁺; ¹H-NMR (400 MHz, CDCl3): δ 8.50 (d, J = 4.40 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 8.40 Hz, 1H), 7.13 (t , J = 6.4 Hz, 1H), 6.18 (s, 1H), 3.53 - 3.49 (m, 1H), 2.77 - 2.72 (m, 1H), 2.11 2.08 (m, 1H), 2.00 - 1.96 (m, 1H) , 1.84 - 1.67 (m, 4H), 1.59 - 1.45 (m, 4H).

The absolute stereochemistry of the isomers was assigned arbitrarily.

Step 5: Synthesis of 4-nitrophenyl (trans-(1RS,2RS)-2-(pyridin-2-yldisulfanyl)cycloheptyl)carbonate

To a stirred solution of trans -(1RS, 2RS)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol

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One hundred percent (500 mg, 1.96 mmol) in DMF (10 mL) under a nitrogen atmosphere was added bis(4-nitrophenyl)carbonate (1.49 g, 2.5 eq., 4.89 mmol) followed by diisopropylethylamine (1.02 mL, 3 eq. , 5.87 mmol) at room temperature. The reaction mixture was stirred for 12h. After completion of the reaction, the reaction mixture was partitioned between water and DCM. The two layers were separated and the organic layer was washed with brine solution, dried over sodium sulfate, filtered and evaporated under reduced pressure to give the crude product which was purified by flash column chromatography. The desired product was eluted in 23% EtOAc : n-Hexane as a mixture. The mixture was repurified by reverse phase column chromatography (10-60% of 0.1% formic acid in water/ACN) to give the title product 4-nitrophenyl (trans-(1RS,2RS)-2-( pyridin-2-yldisulfanyl)cycloheptyl)carbonate (450 mg, 1.07 mmol)). LC-MS m/z calculated for C19H20N2O5S2; 420.5, found 421.3 [M+H]⁺; ¹H-NMR (400 MHz, CDCb): δ 8.45 (s, 1H), 8.27 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 7.6 Hz, 1H), 7.62 (t, J = 7.6 Hz , 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.09 (m, 1H), 5.04 - 5.03 (m, 1H), 3.22 (m, 1H), 2.15 - 2.00 (m, 3H), 1.87 - 1.79 (m, 2H), 1.72 1.63 (m, 4H), 1.54 - 1.49 (m, 2H).

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

<img file="ECSP22010228A_D0212.tif" />

To a stirred solution of trans-(1SR,2SR)-2-(pyridin-2-yldisulfanyl)cycloheptan-1-ol (580 mg, 2.27 mmol) in DMF (10 mL) under a nitrogen atmosphere was added bis(4- nitrophenyl)carbonate (1.73 g, 2.5 eq., 5.68 mmol) followed by di-isopropylethylamine (1.38 mL, 3.5 eq., 7.95 mmol). The reaction mixture was stirred at room temperature for 12h. After the completion of the reaction, monitored by TLC, the reaction mixture was partitioned between water and DCM. The two layers were separated and the combined organic layer was washed with brine solution, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude product was purified by flash column chromatography. The desired product was eluted in 23-25% EtOAc : n -Hexane as a mixture. The product was further purified by reverse phase column chromatography (10-60% of 0.1% formic acid in water/ACN) to give the title compound 4-nitrophenyl (trans(1SR,2SR)-2-(pyridine- 2-yldisulfanyl)cycloheptyl)carbonate (450 mg, 1.07 mmol)). LC-MS m/z calculated for C19H20N2O5S2; 420.5, found 421.3 [M+H]⁺; 1H-NMR (400MHz,

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One hundred and twenty-one CDCI3): δ 8.46 (s, 1H), 8.27 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 7.6 Hz,

1H), 7.40 (d, J = 8.4 Hz, 1H), 7.12 (m, 1H), 5.04 - 5.03 (m, 1H), 3.23 (m, 1H), 2.12 - 2.00 (m, 2H), 1.87 - 1.79 (m, 3H),1.63 - 1.49 (m, 6H).

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

<img file="ECSP22010228A_D0213.tif" />

<img file="ECSP22010228A_D0214.tif" />

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

<img file="ECSP22010228A_D0215.tif" />

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

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

<img file="ECSP22010228A_D0216.tif" />

To a stirred solution of 1aH,2H,3H,7bH-naphtho[1,2-b]oxirene (2.25 g, 15.4 mmol) in ethoxyethane (20 mL) was added silanedione (4.50 g, 74.9 mmol) and S-benzenecarbothioic acid. drip (9.06 mL, 5 eq., 77.0 mmol). The mixture was allowed to stir at room temperature for 16h. The progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with

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One hundred and twenty-two saturated sodium carbonate solution (25 ml) and extracted with ethyl acetate (2 x 20 ml). The combined organic layer was washed with water, brine and dried over sodium sulfate and concentrated under reduced pressure to obtain a crude which was purified by column chromatography to give [ trans -(2-hydroxy-1,2,3, Racemic 4-tetrahydronaphthalen-1yl)sulfanyl](phenyl)methanone as a yellow liquid (1.57 mg, 35.87%)

Step 3: Synthesis of racemic Trans-1-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol

<img file="ECSP22010228A_D0217.tif" />

SH

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

Step 4: Synthesis of trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)-1,2,3,4tetrahydronaphthalen-2-ol and trans-(1SR,2SR)-1 -(pyridin-2- yldisulfanyl)-1,2,3,4-tetrahydronaphthalen2-ol

<img file="ECSP22010228A_D0218.tif" />

<img file="ECSP22010228A_D0219.tif" />

1 trans isomer (1RS, 2RS) 2 trans isomer (1SR, 2SR)

To a stirred solution of 2-(pyridin-2-yldisulfanyl)pyridine (867 mg, 0.8 eq., 3.94 mmol) in methanol (5 mL) at 0°C. To this, racemic trans-1-sulfanyl-1,2,3,4-tetrahydronaphthalen-2-ol in DCM taken from the previous step was added dropwise. The reaction was allowed to stir at RT for 16h. The reaction was monitored by LCMS and TLC. After the completion of the reaction, the reaction mass was concentrated under reduced pressure to give a crude which was purified by column chromatography to give 1-(pyridine-2123

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One-twenty-three yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol racemic as a yellow oil which was further purified by reverse phase column chromatography to give a colorless oil (380 mg, 26.69%). The obtained racemic product was separated by chiral chromatography to give Isomer-1: 130 mg; Isomer-2: 190 mg.

Preparatory conditions:

Column: CHIRALPAK IA (250 mm X 420 mm X 5 mic)

Mobile phase : n-Hexane : Ethanol with 0.1% DEA (50:50)

Flow rate : 19 ml/min

Isomer-1( trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol):

LC-MS m/z calculated for C15H15NOS2; 289.4, found 290.1 ​​[M+H]+; 1HNMR (400 MHz, CDCh): δ 8.07(d, J = 7.2 Hz, 2H), 7.61 - 7.57(m, 1H), 7.48 - 7.44 (m, 2H), 7.36 - 7.34 (m, 1H), 7.18- 7.13 (m, 3H), 4.98 (d, J=4.4 Hz, 1H), 4.24 (m, 1H), 3.072.99 (m, 1H), 2.91-2.80 (m, 1H).

Isomer-2( trans-( 1SR, 2SR)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol)

LC-MS m/z calculated for C15H15NOS2; 289.4, found 290.1 ​​[M+H]⁺; 1HNMR (400 MHz, CDCh): δ 8.55 (d, J=4.0 Hz, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.58 (t, J=7.6 Hz, 1H), 7.37(d, J =8.4 Hz, 1H), 7.26-7.17 (m, 3H), 7.08 (d, J=7.2 Hz, 1H), 4.15 (d, J=8.0 Hz, 1H), 3.97-3.93 (m, 1H), 2.89 (d, J=4.8 Hz, 2H), 2.32-2.28 (m, 1H), 1.97-1.87 (m, 2H).

The absolute stereochemistry of the isomers was assigned arbitrarily.

Step 5. Synthesis of 4-nitrophenyl (trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)1,2,3,4-tetrahydronaphthalen-2-yl)carbonate

To a stirred solution of trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (170 mg, 587 pmol) in N,N-dimethylformamide (2.50 mL ) bis(4-nitrophenyl)carbonate (447 mg, 2.5 eq., 1.47 mmol) was added followed by diisopropylethylamine (307 pL, 3 eq., 1.76 mmol) dropwise at RT. The reaction mixture was stirred at RT for 12 h in a sealed tube. The reaction was monitored by TLC and LCMS. After the completion of the reaction, the reaction mass was partitioned between water (5 ml) and DCM (5 ml). The organic layer was dried over sodium sulfate and evaporated under reduced pressure to give the crude, which was purified by flash column chromatography (0-40% EA in hexane) and also repurified by reverse phase column chromatography. (10-70% of 0.1% formic acid in water/ACN) to produce 4-nitrophenyl (trans-(1RS,2RS)-1-(pyridin-2-yldisulfanyl)

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124

One-twenty-four 1,2,3,4-tetrahydronaphthalen-2-yl)carbonate (70.0 mg, 154 pmol) as a colorless gummy solid (70 mg, 26.22%). LC-MS m/z calculated for C22H18N2O4S2; 454.5 found 455.3 [M+H]⁺; 1H-NMR (400 MHz, CDCl3): δ 8.73(d, J=20.4 Hz, 1H), 8.22(d, J=8.4 Hz, 2H), 7.67(s, 2H), 7.50(m, 1H), 7.32 (d, J=8.4 Hz, 2H), 7.25-7.16(m, 4H), 5.51(s, 1H), 4.52(s, 1H), 3.01-2.85(m, 2H), 2.63(m, 1H), 2.26-2.22(m, 1H).

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

<img file="ECSP22010228A_D0220.tif" />

To a stirred solution of trans-(1SR,2SR)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (120 mg, 415 pmol) in N,N-dimethylformamide (1.50 mL ) bis(4-nitrophenyl)carbonate (315 mg, 2.5 eq., 1.04 mmol) was added followed by diisopropylethylamine (217 pL, 3 eq., 1.24 mmol) dropwise at RT. The reaction mixture was stirred at RT for 12 h in a sealed tube. The progress of the reaction was monitored by TLC and LCMS. After the completion of the reaction, the reaction mass was partitioned between water (5 ml) and DCM (5 ml), The organic layer was dried over sodium sulfate and evaporated under reduced pressure to give the crude which was purified by flash column chromatography (040% EA in hexane) and also repurified by reverse phase column chromatography (10- 70% of 0.1% formic acid in water/ ACN) to produce 4-nitrophenyl (trans(1SR,2SR)-1-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-yl)carbonate (65.0 mg, 143 pmol)) as a colorless gummy solid (65mg, 34.49%).

LC-MS m/z calculated for C22H18N2O4S2; 454.5 found 455.3 [M+H]⁺; 1HNMR (400 MHz, CDCl3): δ 8.55(m, 1H), 8.22(d, J=7.6 Hz, 2H), 7.69(s, 2H), 7.51(m, 1H), 7.32(d, J=7.6 Hz , 2H), 7.25-7.16(m, 4H), 5.51(s, 1H), 4.52(s, 1H), 3.01-2.86(m, 2H), 2.62(m, 1H), 2.26(m, 1H).

Synthesis of 4-nitrophenyl (trans-4-(pyridin-2-yldisulfanyl)cyclohexyl) carbonate

EITHER₂N./χ and YJ² V|1 O II SN

Step 1: Synthesis of trans-4-mercaptocyclohexan-1-ol

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One hundred and twenty five SH

To a stirred solution of 7-oxabicyclo[2.2.1]heptane (1.00 g, 10.2 mmol) in ethanol (10 mL) was added 4-methylbenzene-1-sulfonic acid (2.63 g, 1.5 eq., 15.3 mmol), thiourea (1.16 g, 1.5 eq., 15.3 mmol) and the reaction mass was heated at 80 °C for 24 h. Then, the reaction mass was cooled to room temperature and 50% aqueous sodium hydroxide solution (1.30 g, 3.2 eq., 32.6 mmol) was added to the reaction mass and heated at 100 °C for 2 h. After completion of the reaction, the reaction mass was cooled to room temperature, concentrated under reduced pressure, and acidified with 10% H2SO4 solution. The reaction mass was then extracted with DCM and recovered for the next step as such.

Step 2: Synthesis of trans-4-(pyridin-2-yldisulfanyl)cyclohexan-1-ol

<img file="ECSP22010228A_D0221.tif" />

To a stirred solution of 2-(pyridin-2-yldisulfanyl)pyridine (1.60 g, 0.8 eq., 7.26 mmol) in methanol (10.0 mL) at 0°C the organic layer of (step 1) 4-sulfanylcyclohexan1-ol was added (1.20g, 9.08mmol). After completion of the addition, the reaction mass was allowed to stir at room temperature for 16 h. After completion of the reaction, the reaction mass was concentrated and the crude product was purified by column chromatography (using 0-40% EtOAc : n-Hexane) to give the desired product. The product was repurified by reverse phase column chromatography using 0.1% formic acid and ACN. Fractions containing the desired product were collected and concentrated under reduced pressure to provide the title product as a yellow oil (1.60 g, 73% yield). LC-MS m/z calculated for C11H15NOS2, 241; found 242 [M+H] +.

Step 3: Synthesis of 4-nitrophenyl (trans-4-(pyridin-2-yldisulfanyl)cyclohexyl) carbonate

To a stirred solution of trans-4-(pyridin-2-yldisulfanyl)cyclohexan-1-ol (400 mg, 1.66 mmol) in N,N-dimethylformamide (3 mL) under a nitrogen atmosphere was added bis(4-nitrophenyl)carbonate ( 907 mg, 1.8 eq., 2.98 mmol), ethylbis(propan-2-yl)amine (892 pL, 3 eq., 4.97 mmol) and stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was

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One hundred twenty-six was quenched with water (15ml) and extracted with DCM (3 x 10ml). The two layers were separated and the combined organic layer was washed with water followed by brine solution, dried over sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was purified by flash column chromatography (0-30% EtOAc : n-Hexane). The product was repurified by reverse phase column chromatography using 0.1% formic acid and ACN. Fractions containing the desired product were collected and concentrated under reduced pressure to provide 4-nitrophenyl (trans-4-(pyridin-2-yldisulfanyl)cyclohexyl)carbonate as a yellow oil (0.3 g, 73% yield). LCMS m/z calculated for C18H18N2O5S2, 407; found 407 [M+H] +;¹HNMR (400 MHz, CDCl3): δ 8.49 - 8.42 (m, 1H), 8.26 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.0 Hz, 1H), 7.65 - 7.60 (m, 1H) , 7.35 (d, J = 8.4 Hz, 2H), 7.12 - 7.05 (m, 1H), 4.75 - 4.65 (m, 1H), 2.98 - 2.87 (m, 1H), 2.28 - 2.18 (m, 4H), 1.68 - 1.50 (m, 4H).

Synthesis of (2R)-3-methyl-2-(pyridin-2-yldisulfanyl)butyl 4-nitrophenyl carbonate

<img file="ECSP22010228A_D0222.tif" />

Step 1. Synthesis of cesium benzoylsulfanide or

_cs+

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

Step 2. Synthesis of (2R)-2-(benzoylsulfanyl)-3-methylbutanoic acid⁰ AA

To a stirred solution of (2S)-2-bromo-3-methylbutanoic acid (2.00 g, 11.0 mmol)

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127

One hundred twenty-seven' in N,N-dimethylformamide (14.0 mL) was added cesium benzoylsulfanide (2.98 g, 11.0 mmol). The reaction mixture was stirred at RT for 20h. The progress of the reaction was monitored by TLC, after the completion of the reaction, the reaction mixture was diluted with diethyl ether (3 x 15 ml) and washed with water (3 x 15 ml). The ethereal layer was separated, dried over sodium sulfate, and concentrated under reduced pressure. The obtained residue was recrystallized from n-hexanes to give (2R)-2-(benzoylsulfanyl)-3-methylbutanoic acid (2.50 g, 10.5 mmol) as an oily compound.¹HNMR (400 MHz, DMSO-d6): δ 12.93 (s, 1H), 7.92 (d, J = 7.2 Hz, 2H), 7.69 (t, J = 7.6 Hz, 1H), 7.55 (t, J = 7.2 Hz , 2H), 4.14 (d, J = 6.8 Hz, 1H), 2.30 - 2.22 (m, 1H), 1.01 - 0.89 (m, 6H).

Step 3. Synthesis of (2R)-3-methyl-2-sulfanylbutan-1-ol

<img file="ECSP22010228A_D0223.tif" />

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

Step 4. Synthesis of (2R)-3-methyl-2-(pyridin-2-yldisulfanyl) butan-1-ol

<img file="ECSP22010228A_D0224.tif" />

To a stirred solution of (2R)-3-methyl-2-sulfanylbutan-1-ol (1.20 g, 9.98 mmol) in MeOH (5 mL) was added 2-(pyridin-2-yldisulfanyl)pyridine (1.76 g, 0.8 eq., 7.99 mmol) under a nitrogen atmosphere and stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC and LC-MS. After the completion of the reaction, the reaction mass was concentrated, and then extracted with DCM. The two layers were separated and the combined organic layer was washed with water followed by brine and dried over sodium sulphate, filtered and evaporated. The crude product was purified by flash column chromatography on silica gel (using 12 g column), which was eluted in 50% EtOAc : n128

128

Hundred twenty eight

Hexanes and also repurified by reverse phase column chromatography (10-20% of 0.1% formic acid in water/Acetonitrile). Fractions containing the product were collected and evaporated under vacuum to obtain the title product. The product was repurified by Preparative HPLC.

Preparative HPLC Conditions:

Column : X-BridgeC-18 (250mm X 4.6mm X 5mic)

Mobile phase (A) : 0.1% Ammonia in water

Mobile phase (B) : Acetonitrile

Flow rate : 19 ml/min

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

The collected fractions from the Prep HPLC were combined and evaporated to give the title product 3-(pyridin-2-yldisulfanyl)-1,2,3,4-tetrahydronaphthalen-2-ol (350 mg, 1.21 mmol) as a yellow solid. .¹HNMR (400 MHz, CDCl3): δ 8.49 (d, J = 4 Hz,1H), 7.55 - 7.54 (m, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.13 (t, J = 6.4Hz , 1H), 3.82 (dd, J = 12.4 Hz, 1H), 3.66 - 3.60 (m, 1H), 2.75 - 2.70 (m, 1H), 2.01 - 1.92 (m, 1H), 1.10 - 1.01 (m, 7H) ).

Step 5. Synthesis of (2R)-3-methyl-2-(pyridin-2-yldisulfanyl)butyl 4-nitrophenyl carbonate

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

From step 2, the same procedure was followed to synthesize (2S)-3-methyl-2129

129

One-twenty-nine (pyridin-2-yldisulfanyl)butyl 4-nitrophenyl carbonate using (2R)-2-bromo-3-methylbutanoic acid.

Synthesis of the compound of Example 2 from Intermediate III-2

<img file="ECSP22010228A_D0225.tif" />

ch₃cn/h₂either

RT n-Methyl Morpholine

<img file="ECSP22010228A_D0226.tif" />

In a vial with Pv2 (25.0 mg, 0.061 mmol; as a free-flowing solid), [(2S)-2-(2-pyridyldisulfanyl)propyl]N-[(10S,23S)-10-ethyl-18-fluoro -10-hydroxy-19methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11),12,14,16(24) ,17,19-heptaen-23-yl]carbamate (6.03 mg, 0.091 mmol), 1 mL CH3CN and 0.5 mL water were added. To this was added N-Methyl morpholine (22.7 mg, 0.224 mmol). The mixture was stirred overnight at RT. LC-MS indicated a complete reaction.

The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-85% CH3CN/H2O+0.05% TFA, 15 min) to give the desired product (13.0 mg, yield: 47.0%).

The compounds of Examples 1 and 3-9 (see Table 4 below) were synthesized in an analogous manner as the compound of Example 2, from the

Intermediates III-1 and III-3 through III-9, respectively.

Synthesis of the compound of Example 10 from Intermediate XVI-1

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One hundred thirty-

<img file="ECSP22010228A_D0227.tif" />

<img file="ECSP22010228A_D0228.tif" />

pv2

DMF and PBS were degassed using a stream of N2 for 30 min. Pv2 (25.0 mg, 0.061 mmol; as a free-flowing solid), [4-(2-pyridyldisulfanyl)phenyl]methyl N-[(10S,23S)-10-ethyl-18-fluoro- 10-hydroxy-195 methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-,6(11),12,14,16(24 ),17,19-heptaen-23-yl]carbamate (6.5 mg, 0.09 mmol), 1.5 mL DMF and 0.5 mL PBS. To this was added CH3CO2H (0.0347 mL, 0.606 mmol). The mixture was stirred at RT overnight. LC-MS indicated a complete reaction. The reaction mixture was purified by reverse phase HPLC (PrepSlope_4min, 30-100% CH3CN/H2O+0.05%TFA, 18 min) to give the desired product (3.0 mg, yield: 10.7%).

Compounds of the invention and analytical data are presented below.

Table 4. Exemplary Compounds

<td>Example</td><td>Structure</td><td>MS A: MaldiTOF (M+) B: ESI (m/z=3)</td><td>Column % ACN/H2O Run Time Retention Time</td>

<td> 1</td><td></td><td>B:1521.3</td><td>TO</td>

<td></td><td>⁰W'-n^TOeither-^S'S-^P’²</td><td></td><td>2-95% 11 min</td>

<td></td><td>rt^{H Ξ}</td><td></td><td>7.4 min</td>

<td></td><td>XN</td><td></td><td></td>

<td></td><td> )=/ °</td><td></td><td></td>

<td></td><td>^H0 rt O</td><td></td><td></td>

131

131

One hundred thirty one-

<td> 2</td><td>TO AAA /A /\,S, , Pv2 tt N 0 and SA^hAND^No.γγο ho \ / me 0</td><td>B:1521.7</td><td>At 2-95% 11 min 7.4 min</td>

<td> 3</td><td>^FYto A &lt; Y- A -, -A /\ -s, ,Pv2 YAN οχ S nJL^h either AND^No.\ γγ₀ho \ / I 0</td><td>B: 1530.6</td><td>At 2-95% 11 min 7.5 min</td>

<td> 4</td><td>Fx X /\ YY''A^^S'S'^pv2TO^HaAyo TODAY / I either</td><td>B: 1526.5</td><td>At 2-95% 11 min 7.5 min</td>

<td> 5</td><td>^FAyi j । TO^hΛV \=/⁰hoA / I or</td><td>B:1526.1</td><td>At 2-95% 11 min 7.5 min</td>

<td> 6</td><td>^{0 =}W'«Y^yes's-^mvl^hhoA / I 0</td><td>B: 1525.8</td><td>At 2-95% 11 min 7.4 min</td>

<td> 7</td><td>F- /L /\ γΠ ui kxk A·', YY -S. ,Pv2 YYNO '^s A&gt;^{h ξ}TODAY / Me 0</td><td>B:1526.4</td><td>At 2-95% 11 min 7.5 min</td>

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one hundred thirty-one

<td> 8</td><td>OH N&lt;. A S.., Pv2 \ /^yesAA Oh⁰^H0 γο 0</td><td>B: 1534.8</td><td>At 2-95% 11 min 7.6 min</td>

<td> 9</td><td>^FyX^to YY AJA 1 J W n TO.^h S ,Pv2 \ /^Soh Me too TODAY / Me 0</td><td>B:1534.6</td><td>At 2-95% 11 min 7.7 min</td>

<td> 10</td><td>&lt;N CL ω ω P 0 &lt;a ZI O oaxja? -(TO HA \ ?</td><td>B:1537.6</td><td>At 2-95% 11 min 7.5 min</td>

Example 11: Synthesis of Compound 11

<img file="ECSP22010228A_D0229.tif" />

Step 1. Synthesis of 2-(pyridin-2-yldisulfanyl)cyclohexan-1-ol

<img file="ECSP22010228A_D0230.tif" />

To a solution of 1,2-di(pyridin-2-yl)disulfan (15.2 g, 68.9 mmol) in MeOH (degassed with N2) (30 mL) was added (1-mercaptocyclobutyl)methanol (11.4 g, 86.2 mmol) (degassed with N2) dropwise and stirred for 16 h at room temperature under a N2 atmosphere. The reaction mixture was concentrated to dryness under vacuum. The resulting crude material was purified by chromatography

133

133

One hundred thirty three column using 30% EtOAC/hexanes to give the title compound as a yellow liquid.¹HNMR (400 MHz, CDCh): δ 8.54-8.53 (m, 1H), 7.60-7.56 (m, 1H),

7.40-7.38 (m, 1H), 7.17-7.14 (m, 1H), 3.38-3.34 (m, 1H), 2.62-2.57 (m, 1H), 2.11-2.02 (m, 1H), 1.75-1.74 (m , 2H), 1.61-1.60 (m, 1H), 1.42-1.24 (m, 4H).

The title compound was subjected to chiral preparative HPLC conditions (Chiralpak IG : 250 mm x 20 mm x 5 mic; n-Hexane : IPA with 0.1% Diethylamine (80:20); 19 mL/min; 25°C (Temperature (1R,2R)-2-(pyridin-2-yldisulfanyl)cyclohexan1-ol (4.5 g, 18.6 mmol) eluted first (retention time: 3.9 min), followed by (1S,2S)-2- (pyridin-2-yldisulfanyl)cyclohexan-1-ol (retention time: 11.3 minutes). Absolute stereochemistry was confirmed by comparison of the product from Step 2 with chiral material having a reported absolute stereochemistry (see Monaco, MR; J. Am. Chem. Soc. 2014, 136, 49, 16982-16985).

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

<img file="ECSP22010228A_D0231.tif" />

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

Step 3. Synthesis of [(1S,2S)-2-(2-pyridyldisulfanyl)cyclohexyl]N-[(10S,23S)-10-ethyl-18fluoro-10-hydroxy-19-methyl-5,9-dioxo- 8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11), 12,14,16(24),17,19134

One hundred and thirty four

134 heptaen-23-yl]carbamate.

<img file="ECSP22010228A_D0232.tif" />

A (10S,23S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19methyl-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24] methanesulfonic acid tetracose1,6(11),12,14,16(24),17,19-heptaene-5,9-dione (250 mg, 0.470 mmol) in 10 mL dry DMF was added (1R,2R)-2- (pyridin-2-yldisulfanyl)cyclohexan-1-ol (from step 2; 191 mg, 0.470 mmol), N,N-diisopropylethylamine (122 mg, 0.941 mmol), and DMAP (115 mg, 0.941 mmol). The mixture was stirred at room temperature overnight. LC-MS indicated that the desired coupling product had been formed. The reaction mixture was then diluted with EtOAc, washed with saturated aqueous NH4Cl, H2O, and brine. The mixture was dried over sodium sulfate, filtered, and concentrated. The crude residue was purified by column chromatography using 0-5% MeOH/dichloromethane to give 240 mg of the desired product in 72.6% yield (240 mg).

Step 4. Coupling with Pv1 (Compound 11)

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

The reaction mixture was directly purified by reverse phase HPLC (2085% acetonitrile/water, 0.5% acetic acid on a Sunfire Prep C18 column (10 pm, 50x150 mm), retention time: 7022 min) to yield 213 mg of product. desired in 68% yield (213 mg). ESI (M+3H/3)³⁺: 1291.6

Example 12: Synthesis of Compound 12

<img file="ECSP22010228A_D0233.tif" />

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One hundred thirty-five Step 1. Synthesis of [(1R,2R)-1-methyl-2-(2-pyridyldisulfanyl)propyl] N-[(10S,23S)10-ethyl-18-fluoro-10-hydroxy- 19-methyl-5,9-dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11), 12,14,16(24),17,19- heptaen-23-yl]carbamate

To a mixture of 1-hydroxybenzotriazole hydrate (8.64 mg, 0.0564 mmol), finely ground 4 Á molecular sieve (50 mg), and (10S,23S)-23amino-10-ethyl-18-fluoro-10 methanesulfonic acid -hydroxy-19-methyl-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose-1,6(11),12,14,16(24),17,19heptaene-5 ,9-Dione (25.0 mg, 0.0470 mmol) and pyridine (0.0190 mL, 0.235 mmol) in 2 mL of anhydrous DMF was added [(1R,2R)-1-methyl-2-(2-pyridyldisulfanyl)propyl] (4 -nitrophenyl) carbonate (19.7 mg, 0.470 mmol) (see Synthesis of II-4: 4-nitrophenyl((2R,3R)-3-(pyridin-2yldisulfanyl)butan-2-yl)carbonate). After stirring for 16h at room temperature the mixture was filtered and the solution was concentrated. The residue was then purified by column chromatography (0-5% MeOH/DCM) to give the title compound (35.0 mg, 0.0517 mmol, yield: 110%).

Step 2. Coupling with Peptide Pv1 (Compound 12)

In a vial was placed the peptide Pv1 (50.0 mg, 14.7e-5 mol), [(1R,2R)-1-methyl-2(2-pyridyldisulfanyl)propyl]N-[(10S,23S)-10-ethyl -18-fluoro-10-hydroxy-19-methyl-5,9-dioxo8-oxa-4,15-diazahexacyclo[14.7.1.02,14 ]tetracose1,6(11),12, 14,16(24),17,19-heptaen-23-yl]carbamate (0.013 g, 1.92e-5 mol), 2 mL ACN and 1 mL water. To this was added N-methylmorpholine (0.060 ml, 0.000545 mol). The mixture was stirred overnight at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-85% ACN/H2O+0.05% TFA, 13 min; retention time: 6.95 min) to yield Compound 12 (0.0350 g , 9.10e-6 mol, yield: 61.8%). ESI (M+3H/3)³⁺: 1281.9

Example 13: Synthesis of Compound 13

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Compound 13 was made analogous to Compound 11, replacing ((1S,2S)-2-(pyridin-2-yldisulfanyl)cyclohexyl)carbonate with ((1R,2R)-2(pyridin-2-yldisulfanyl )cyclohexyl)carbonate in Step 2. Sunfire Prep C18 column (10 pm, 50x150mm) (20-85% acetonitrile/water, 0.5% acetic acid); retention time: 6,609 minutes. ESI (M+3H/3)³⁺: 1290.3

Example 14: Synthesis of Compound 14

Step 1. Synthesis of (4-Nitrophenyl) [trans-(1RS,2RS)-2-(2pyridyldisulfanyl)cyclopentyl]carbonate

The title compound was synthesized according to the analogous synthetic methods described in the synthesis of Compound 11, using the first stereoisomer that eluted from the chiral chromatographic separation of racemic trans-2-(2-pyridyldisulfanyl)cyclopentyl assigned as trans -(1 RS ,2 RS )-2-(2pyridyldisulfanyl)cyclopentan-1-ol.

Step 2. Synthesis of [trans-(1RS,2RS)-2-(2-pyridyldisulfanyl)cyclopentyl]N[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9 -dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11), 12,14,16(24),17,19-heptaen-23-yl]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol), DMAP (23.0 mg, 0.188 mmol), and (4-nitrophenyl)[trans-(1RS,2RS)-2- (2-pyridyldisulfanyl)cyclopentyl]carbonate (40.6 mg, 0.103 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 pL, 0.188 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-5% MeOH/DCM) to give the title compound (33.0 mg, 0.0479 mmol, yield: 50.9%).

Step 3. Coupling with Peptide Pv1 (Compound 14)

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In a vial was placed the peptide Pv1 (50.0 mg, 1.47e-5 mol), [ trans -(1 RS ,2 RS )2-(2-pyridyldisulfanyl)cyclopentyl]N-[(10S,23S)-10-ethyl -18-fluoro-10-hydroxy-19-methyl-5,9dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12,14, 16(24),17,19-heptaen-23-yl]carbamate (0.0124 g, 1.80e-5 mol), 2 mL ACN and 1 mL water. To this was added N-methylmorpholine (0.060 ml, 0.000545 mol). The mixture was stirred overnight at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-90% ACN/H2O+0.05% TFA, 16 min; retention time: 6.761 min) to yield Compound 14 (0.0360 g , 9.34e-6 mol, yield: 63.3%). ESI (M+3H/3)³⁺: 1286.3.

Example 15: Synthesis of Compound 15

<img file="ECSP22010228A_D0234.tif" />

Step 1. Synthesis of (4-nitrophenyl) [trans-(1SR,2SR)-2-(2pyridyldisulfanyl)cyclopentyl]carbonate

The title compound was synthesized from the second stereoisomer eluting from the chiral chromatographic separation of racemic trans-2-(2-pyridyldisulfanyl)cyclopentyl, assigned as trans -(1 SR ,2 SR )-2-(2-pyridyldisulfanyl) cyclopentan-1-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

Step 2. Synthesis of [trans-(1SR,2SR)-2-(2-pyridyldisulfanyl)cyclopentyl] N[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9 -dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11), 12,14,16(24),17,19-heptaen-23-yl]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol), DMAP (23.0 mg, 0.188 mmol), and (4-nitrophenyl)[trans-(1SR,2SR)-2- (2-pyridyldisulfanyl)cyclopentyl]carbonate (38.2 mg, 0.0974 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 pL, 0.188 mmol). After stirring for 16h at room temperature the mixture was diluted with EtOAc (50ml), washed

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One hundred thirty-eight with 30 mL of saturated NH4Cl, 30 mL of water, and 20 mL of brine. The organic layer was concentrated and the residue was purified by column chromatography (0-5% MeOH/DCM) to give the title compound (29.0 mg, 0.0421 mmol, yield: 44.8%).

Step 3. Coupling with Peptide Pv1 (Compound 15)

In a vial was placed the peptide Pv1 (50.0 mg, 1.47e-5 mol), trans -[(1SR,2SR)2-(2-pyridyldisulfanyl)cyclopentyl]N-[(10S,23S)-10-ethyl-18 -fluoro-10-hydroxy-19-methyl-5,9dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12,14,16( 24),17,19-heptaen-23-yl]carbamate (0.0124 g, 1.80e-5 mol), 2 mL ACN and 1 mL water. To this was added N-methylmorpholine (0.060 ml, 0.000545 mol). The mixture was stirred overnight at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-90% ACN/H2O+0.05% TFA, 16 min; retention time: 6.883 min) to yield Compound 15 (0.0280 g , 7.26e-6 mol, yield: 49.3%). ESI (M+3H/3)³⁺: 1285.9.

Example 16: Synthesis of Compound 16

<img file="ECSP22010228A_D0235.tif" />

Step 1. Synthesis of (4-nitrophenyl) [trans-(3RS,4RS)-4-(2pyridyldisulfanyl)tetrahydrofuran-3-yl] carbonate

The title compound was synthesized from the first stereoisomer eluting from the chiral chromatographic separation of racemic trans-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-ol, assigned as trans-(3RS,4RS)-4-( 2-pyridyldisulfanyl)tetrahydrofuran-3-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

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

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To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol),

DMAP (23.0 mg, 0.188 mmol), and (4-nitrophenyl) [ trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]carbonate (38.2 mg, 0.0969 mmol) in 2 mL of anhydrous DMF were added. added N,N-diisopropylethylamine (35 pL, 0.188 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-5% MeOH/DCM) to give the title compound (40.0 mg, 0.0579 mmol, yield: 61.6%).

Step 3. Coupling with Peptide Pv1 (Compound 16)

In a vial was placed the peptide Pv1 (50.0 mg, 1.47e-5 mol), [ trans-(3RS,4RS)-4-(2pyridyldisulfanyl)tetrahydrofuran-3-yl] N-[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12,14 ,16(24),17,19-heptaen-23-yl]carbamate (0.0124 g, 1.80e-5 mol), 2 mL ACN and 1 mL water. To this was added N-Methylmorpholine (0.060 ml, 0.000545 mol). The mixture was stirred overnight at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-80% ACN/H2O+0.05% TFA, 15 min; retention time: 6.633 min) to yield Compound 16 (0.0290 g , 7.52e-6 mol, yield: 51.0%). ESI (M+3H/3)³⁺: 1286.4.

Example 17: Synthesis of Compound 17

Step 1. Synthesis of (4-nitrophenyl) [trans-(3SR,4SR)-4-(2pyridyldisulfanyl)tetrahydrofuran-3-yl] carbonate

The title compound was synthesized from the second stereoisomer that eluted from the chiral chromatographic separation of racemic trans-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-ol, assigned as trans-(3SR,4SR)-4-( 2-pyridyldisulfanyl)tetrahydrofuran-3-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

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Step 2. Synthesis of [trans-(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]

N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24 ] tetracose1,6(11), 12,14,16(24),17,19-heptaen-23-yl]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol), DMAP (23.0 mg, 0.188 mmol), and (4-nitrophenyl) [ trans- (3SR,4SR)-4- (2pyridyldisulfanyl)tetrahydrofuran-3-yl]carbonate (38.2 mg, 0.0969 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 pL, 0.188 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-5% MeOH/DCM) to yield [ trans -(3SR,4SR)-4-(2-pyridyldisulfanyl)tetrahydrofuran-3-yl]N-[ (10S,23S)-10-ethyl18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose-1, 6(11),12,14,16(24), 17,19heptaen-23-yl]carbamate (31.0 mg, 0.0449 mmol, yield: 47.7%).

Step 3. Coupling with Peptide Pv1 (Compound 17)

In a vial was placed the peptide Pv1 (50.0 mg, 1.47e-5 mol), [ trans-(3SR,4SR)-4-(2pyridyldisulfanyl)tetrahydrofuran-3-yl] N-[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12,14 ,16(24),17,19-heptaen-23-yl]carbamate (0.0124 g, 1.80e-5 mol), 2 mL ACN and 1 mL water. To this was added N-methylmorpholine (0.060 ml, 0.000545 mol). The mixture was stirred overnight at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-85% ACN/H2O+0.05% TFA, 13 min; retention time: 6.670 min) to yield Compound 17 (0.0170 g , 4.41e-6 mol, yield: 29.9%). ESI (M+3H/3)³⁺: 1286.7.

Example 18: Synthesis of Compound 18

<img file="ECSP22010228A_D0236.tif" />

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Step 1. Synthesis of (4-nitrophenyl) [trans-(2RS,3RS)-3-(2-pyridyldisulfanyl)tetralin2-yl] carbonate

The title compound was synthesized from the first stereoisomer eluting from the chiral chromatographic separation of racemic trans-3-(2pyridyldisulfanyl)tetralin-2-ol, assigned as trans -(2RS,3RS)-3-( 2-pyridyldisulfanyl)tetralin-2-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

Step 2. Synthesis of [trans-(2RS,3RS)-3-(2-pyridyldisulfanyl)tetralin-2-yl] N[(10S, 23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl -5,9-dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11), 12,14,16(24),17,19-heptaen-23 -il]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(2RS,3RS)-3- (2pyridyldisulfanyl)tetralin-2-yl]carbonate (32.1 mg, 0.0705 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 pL, 0.941 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to give the title compound (26.0 mg, 0.0346 mmol, yield: 73.6%).

Step 3. Coupling with Peptide Pv1 (Compound 18)

In a vial was placed the peptide Pv1 (25.0 mg, 7.37e-6 mol), [ trans-(2RS,3RS)3-(2-pyridyldisulfanyl)tetralin-2-yl] N-[(10S,23S)-10 -ethyl-18-fluoro-10-hydroxy-19-methyl-5,9dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12, 14,16(24),17,19-heptaen-23-yl]carbamate (0.00719 g, 9.58e-6 mol), 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol). The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN/H2O+0.05% TFA, 20 min; retention time: 6.851 min) to yield Compound 18 (0.0080 g , 2.04e-6 mol, yield: 27.7%). ESI (M+3H/3)³⁺: 1307.4.

Example 19: Synthesis of Compound 19

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<img file="ECSP22010228A_D0237.tif" />

Step 1. Synthesis of (4-nitrophenyl) [trans-(2SR,3SR)-3-(2-pyridyldisulfanyl)tetralin2-yl] carbonate

The title compound was synthesized from the second stereoisomer eluting from the chiral chromatographic separation of racemic trans-3-(2pyridyldisulfanyl)tetralin-2-ol, assigned as trans -(2SR,3SR)-3-( 2-pyridyldisulfanyl)tetralin-2-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

Step 2. Synthesis of [trans-(2SR,3SR)-3-(2-pyridyldisulfanyl)tetralin-2-yl] N[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl -5,9-dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11),12,14,16(24),17,19-heptaen-23 -il]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl) [ trans-(2SR,3SR)-3- (2pyridyldisulfanyl)tetralin-2-yl]carbonate (32.1 mg, 0.0705 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 pL, 0.941 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to yield [trans(2SR,3SR)-3-(2-pyridyldisulfanyl)tetralin-2-yl]N-[( 10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6 (11),12,14,16(24),17,19-heptaen-23-yl]carbamate (10.0 mg, 0.0133 mmol, yield: 28.3%).

Step 3. Coupling with Peptide Pv1 (Compound 19)

In a vial was placed the peptide Pv1 (25.0 mg, 7.37e-6 mol), [ trans-(2SR,3SR)3-(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10 -ethyl-18-fluoro-10-hydroxy-19-methyl-5,9dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12, 14,16(24),17,19-heptaen-23-yl]carbamate (0.010 g, 1.33e-5 mol), 1 mL of

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One-forty-three ACN and 0.5 ml of water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol).

The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN/H2O+0.05% TFA, 20 min; retention time: 6.855) to yield Compound 19 (0.0060 g, 1.33e-5 mol, yield: 20.8%). ESI (M+3H/3)³⁺: 1307.6.

Example 20: Synthesis of Compound 20

<img file="ECSP22010228A_D0238.tif" />

Step 1. Synthesis of (4-nitrophenyl) [trans-(3RS,4RS)-4-(2pyridyldisulfanyl)tetrahydropyran-3-yl] carbonate

The title compound was synthesized from the first stereoisomer eluting from the chiral chromatographic separation of racemic trans-4-(2-pyridyldisulfanyl)tetrahydropyran-3-ol, assigned as trans-(2RS,3RS)-4-( 2-pyridyldisulfanyl)tetrahydropyran-3-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

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

To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl) [ trans-(3RS,4RS)-4- (2pyridyldisulfanyl)tetrahydropyran-3-yl]carbonate (23.1 mg, 0.0564 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 pL, 0.941 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to yield [trans-(3RS,4RS)-4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl]N-[ (10S,23S)-10-ethyl-18144

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One-forty-fourfluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose-1,6(11), 12,14,16(24),17,19heptaen-23-yl]carbamate (30.0 mg, 0.0426 mmol, yield: 90.5%).

Step 3. Coupling with Peptide Pv1 (Compound 20)

In a vial was placed the peptide Pv1 (25.0 mg, 7.37e-6 mol), [ trans-(3RS,4RS)4-(2-pyridyldisulfanyl)tetrahydropyran-3-yl] N-[(10S,23S)-10 -ethyl-18-fluoro-10-hydroxy-19methyl-5,9-dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12,14, 16(24),17,19-heptaen-23-yl]carbamate (0.00779 g, 1.11e-5 mol), , 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol). The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 30-85% ACN/H2O+0.05% TFA, 13 min; retention time: 6.380) to yield Compound 20 (0.0060 g, 1.55e-6 mol, yield: 21.0%). ESI (M+3H/3)³⁺: 1292.3.

Example 21: Synthesis of Compound 21

<img file="ECSP22010228A_D0239.tif" />

Step 1. Synthesis of (4-nitrophenyl) [trans-(3SR,4SR)-4-(2pyridyldisulfanyl)tetrahydropyran-3-yl]carbonate

The title compound was synthesized from the second stereoisomer eluting from the chiral chromatographic separation of racemic trans-4-(2pyridyldisulfanyl)tetrahydropyran-3-ol, assigned as trans-(2SR,3SR)-4-( 2-pyridyldisulfanyl)tetrahydropyran-3-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

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

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To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol),

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

Step 3. Coupling with Peptide Pv1 (Compound 21)

In a vial was placed the peptide Pv1 (25.0 mg, 7.37e-6), [ trans-(3SR,4SR)-4-(2pyridyldisulfanyl)tetrahydropyran-3-yl] N-[(10S,23S)-10-ethyl -18-fluoro-10-hydroxy-19-methyl5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12,14, 16(24),17,19-heptaen-23-yl]carbamate (0.00779 g, 1.11e-5 mol), 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol). The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-70% ACN/H2O+0.05% TFA, 17 min; retention time: 6.765 min) to yield Compound 21 (0.021 g , 5.42e-6 mol, yield: 73.6%). ESI (M+3H/3)³⁺: 1291.1.

Example 22: Synthesis of Compound 22

Step 1. Synthesis of (4-nitrophenyl) [trans-(1RS,2RS)-2-(2pyridyldisulfanyl)cycloheptyl]carbonate

The title compound was synthesized from the first stereoisomer that eluted from the chiral chromatographic separation of trans-2-(2146

146

One hundred forty-six racemic pyridyldisulfanyl)cycloheptan-1-ol, assigned as trans-(1RS,2RS)-2-(2pyridyldisulfanyl)cycloheptan-1-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

Step 2. Synthesis of [trans-(1RS,2RS)-2-(2-pyridyldisulfanyl)cycloheptyl]N[(10S, 23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9 -dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11), 12,14,16(24),17,19-heptaen-23-yl]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(1RS,2RS)-2- (2-pyridyldisulfanyl)cycloheptyl]carbonate (23.7 mg, 0.0564 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 pL, 0.941 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to give the title compound (29.0 mg, 0.0405 mmol, yield: 86.0%).

Step 3. Coupling with Peptide Pv1 (Compound 22)

In a vial was placed the peptide Pv1 (25.0 mg, 7.37e-6), [trans-(1RS,2RS)-2-(2pyridyldisulfanyl)cycloheptyl]N-[(10S,23S)-10-ethyl-18-fluoro -10-hydroxy-19-methyl-5,9-dioxo8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11),12,14,16(24) ,17,19-heptaen-23-yl]carbamate (0.00792 g, 1.11e-5 mol), 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol). The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-70% ACN/H2O+0.05% TFA, 17 min; retention time: 6.868 min) to yield Compound 22 (0.020 g , 5.15e-6 mol, yield: 69.9%). ESI (M+3H/3)³⁺: 1296.3.

Example 23: Synthesis of Compound 23

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one hundred and forty seven

Step 1. Synthesis of (4-nitrophenyl) [trans-(1SR,2SR)-2-(2pyridyldisulfanyl)cycloheptyl]carbonate

The title compound was synthesized from the second stereoisomer that eluted from the chiral chromatographic separation of racemic trans-2-(2-pyridyldisulfanyl)cycloheptan-1-ol, assigned as trans-(1SR,2SR)-2-( 2-pyridyldisulfanyl)cycloheptan-1-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

Step 2. Synthesis of [trans-(1SR,2SR)-2-(2-pyridyldisulfanyl)cycloheptyl]N[(10S, 23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9 -dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11), 12,14,16(24),17,19-heptaen-23-yl]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(1SR,2SR)-2- (2-pyridyldisulfanyl)cycloheptyl]carbonate (23.7 mg, 0.0564 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 pL, 0.941 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to give the title compound (31.0 mg, 0.0432 mmol, yield: 91.9%).

Step 3. Coupling with Peptide Pv1 (Compound 23)

In a vial was placed the peptide Pv1 (25.0 mg, 7.37e-6), [trans-(1SR,2SR)-2-(2pyridyldisulfanyl)cycloheptyl]N-[(10S,23S)-10-ethyl-18-fluoro -10-hydroxy-19-methyl-5,9-dioxo8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11),12,14,16(24) ,17,19-heptaen-23-yl]carbamate (0.00792 g, 1.11e-5 mol), 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol). The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-88% ACN/H2O+0.05% TFA, 17 min; retention time 7.178 min) to yield Compound 23 (0.020 g, 5.15e-6 mol, yield: 69.9%). ESI (M+3H/3)³⁺: 1296.0.

Example 24: Synthesis of Compound 24

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One hundred forty-eight

<img file="ECSP22010228A_D0240.tif" />

Step 1. Synthesis of (4-nitrophenyl) [trans-1-(1RS,2RS)-1-(2pyridyldisulfanyl)tetralin-2-yl] carbonate

The title compound was synthesized from the first stereoisomer eluting from the chiral chromatographic separation of racemic trans-1-(2-pyridyldisulfanyl)tetralin-2-ol, assigned as trans -(1RS,2RS)-1-( 2-pyridyldisulfanyl)tetralin-2-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

Step 2. Synthesis of [trans-(1RS,2RS)-1-(2-pyridyldisulfanyl)tetralin-2-yl]N[(10S, 23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl -5,9-dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11), 12,14,16(24),17,19-heptaen-23 -il]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-1-(1RS,2RS)- 2-pyridyldisulfanyl)tetralin-2-yl]carbonate (32.1 mg, 0.0705 mmol) in 2 mL anhydrous DMF was added N,N-diisopropylethylamine (18 pL, 0.941 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to give the title compound (20.0 mg, 0.0266 mmol, yield: 56.6%).

Step 3. Coupling with Peptide Pv1 (Example 24)

The peptide Pv1 (25.0 mg, 7.37e-6), [ trans-1 - (1RS,2RS)-1(2-pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)- was placed in a vial. 10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12 ,14,16(24),17,19-heptaen-23-yl]carbamate (0.0083 g, 1.11e-5 mol), 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol). The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters

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One hundred forty-nine

SunfirePrep C18, PrepSlope_4 min, 20-95% ACN/H2O+0.05% TFA, 20 min; retention time 6.968) to produce Compound 24 (0.012 g, 3.06e-6 mol, yield:

41.6%). ESI (M+3H/3)³⁺: 1307.2

Example 25: Synthesis of Compound 25

<img file="ECSP22010228A_D0241.tif" />

Step 1. Synthesis of (4-nitrophenyl) [trans-(1SR,2SR)-1-(2-pyridyldisulfanyl)tetralin2-yl] carbonate

The title compound was synthesized from the second stereoisomer that eluted from the chiral chromatographic separation of racemic trans-1-(2-pyridyldisulfanyl)tetralin-2-ol, assigned as trans -(1SR,2SR)-1-( 2-pyridyldisulfanyl)tetralin-2-ol, using the analogous synthesis methods described in the synthesis of Compound 11.

Step 2. Synthesis of [trans-(1SR,2SR)-1-(2-pyridyldisulfanyl)tetralin-2-yl]N[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl -5,9-dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11), 12,14,16(24),17,19-heptaen-23 -il]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (25 mg, 0.0470 mmol), DMAP (11.5 mg, 0.0941 mmol), and (4-nitrophenyl)[trans-(1SR,2SR)-1- (2pyridyldisulfanyl)tetralin-2-yl]carbonate (32.1 mg, 0.0705 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (18 pL, 0.941 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to give the title compound (22.0 mg, 0.0293 mmol, yield: 62.3%).

Step 3. Coupling with Peptide Pv1 (Compound 25)

In a vial was placed the peptide Pv1 (25.0 mg, 7.37e-6), [trans-(1SR,2SR)-1-(2pyridyldisulfanyl)tetralin-2-yl]N-[(10S,23S)-10-ethyl -18-fluoro-10-hydroxy-19-methyl-5.9150

150 One hundred and fifty dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11),12,14,16(24),17,19-heptaen-23 -yl]carbamate (0.0083 g, 1.11e-5 mol), 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol). The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN/H2O+0.05% TFA, 20 min; retention time: 6.944) to yield Compound 25 (0.013 g, 3.32e-6 mol, yield: 45.0%). ESI (M+3H/3)³⁺: 1307.0

Example 26: Synthesis of Compound 26

<img file="ECSP22010228A_D0242.tif" />

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

To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol) and (4-nitrophenyl)[4-(2-pyridyldisulfanyl)cyclohexyl]carbonate (Synthesized from trans-4mercaptocyclohexane-1 -ol) (42.1 mg, 0.103 mmol) in 2 mL of anhydrous DMF was added N,N-diisopropylethylamine (35 pL, 0.188 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to give the title compound (45.0 mg, 0.0640 mmol, yield: 68.1%).

Step 2. Coupling with Peptide Pv1 (Compound 26)

The peptide Pv1 (25.0 mg, 7.37e-6), [trans-4-(2pyridyldisulfanyl)cyclohexyl] N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19 was placed in a vial. -methyl-5,9-dioxo8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11),12,14,16(24),17,19-heptaen -23-yl]carbamate (0.00777 g, 1.11e-5 mol), 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol).

151

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One-hundred-fifty-one The mixture was stirred for 65 h at RT. LC-MS indicated a complete reaction.

The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN/H2O+0.05% TFA, 20 min; retention time: 6.593 min) to yield Compound 26 (0.028 g , 7.23e-6 mol, yield: 98.2%). ESI (M+3H/3)³⁺: 1291.0.

Example 27: Synthesis of Compound 27

<img file="ECSP22010228A_D0243.tif" />

Step 1. Synthesis of [(2S)-3-methyl-2-(2-pyridyldisulfanyl)butyl] N-[(10S,23S)-10-ethyl18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11), 12,14,16(24),17,19-heptaen-23-yl]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol) and [(2S)-3-methyl-2-(2-pyridyldisulfanyl)butyl](4-nitrophenyl)carbonate (Synthesized from Lvaline, cf J. Org. Chem. 1990, 55, 2286-2288) (40.8 mg, 0.103 mmol) in 2 mL anhydrous DMF was added N,N-diisopropylethylamine (35 pL, 0.188 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to give the title compound (48.0 mg, 0.0695 mmol, yield: 73.9%).

Step 2. Coupling with Peptide Pv1 (Compound 27)

In a vial was placed the peptide Pv1 (25.0 mg, 7.37e-6 mol), [(2S)-3-methyl-2-(2pyridyldisulfanyl)butyl]N-[(10S,23S)-10-ethyl-18- fluoro-10-hydroxy-19-methyl-5,9-dioxo-8oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12,14,16(24 ),17,19-heptaen-23-yl]carbamate (0.00764 g, 1.11e-5 mol), 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol). The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN/H2O+0.05% TFA, 20 min; time

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One hundred fifty-two retention: 6.773 min) to produce Compound 27 (0.024 g, 6.22e-6 mol, yield: 84.4%). ESI (M+3H/3)³⁺: 1286.8.

Example 28: Synthesis of Compound 28

<img file="ECSP22010228A_D0244.tif" />

Step 1. Synthesis of [(2R)-3-methyl-2-(2-pyridyldisulfanyl)butyl]N-[(10S,23S)-10-ethyl18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate

To a mixture of exatecan mesylate [CAS: 169869-90-3] (50 mg, 0.0941 mmol) and (4-nitrophenyl)(2R)-3-methyl-2-(2-pyridyldisulfanyl)butyl]carbonate (Synthesized at From Dvalin, cf J. Org. Chem. 1990, 55, 2286-2288) (40.8 mg, 0.103 mmol) in 2 mL anhydrous DMF was added N,N-diisopropylethylamine (35 pL, 0.188 mmol). After stirring for 16 h at room temperature the mixture was diluted with EtOAc (50 mL), washed with 30 mL saturated NH4Cl, 30 mL water, and 20 mL brine. The organic layer was concentrated and the residue was purified by column chromatography (0-3% MeOH/DCM) to give the title compound (41.0 mg, 0.0594 mmol, yield: 63.1%).

Step 2. Coupling with Peptide Pv1 (Compound 28)

In a vial was placed the peptide Pv1 (25.0 mg, 7.37e-6), [(2R)-3-methyl-2-(2pyridyldisulfanyl)butyl]N-[(10S,23S)-10-ethyl-18-fluoro -10-hydroxy-19-methyl-5,9-dioxo-8oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracose1,6(11),12,14,16(24) ,17,19-heptaen-23-yl]carbamate (0.00764 g, 1.11e-5 mol), 1 mL ACN and 0.5 mL water. To this was added N-methylmorpholine (0.030 ml, 0.000273 mol). The mixture was stirred for 65h at RT. LC-MS indicated a complete reaction. The reaction mixture was directly purified by reverse phase HPLC (Waters SunfirePrep C18, PrepSlope_4 min, 20-95% ACN/H2O+0.05% TFA, 20 min; retention time: 6.708 min) to yield Compound 28 (0.012 g , 3.08e-6 mol, yield: 41.8%). ESI (M+3H/3)³⁺: 1287.8.

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One hundred and fifty three

Example 29: Synthesis of Compound 29

<img file="ECSP22010228A_D0245.tif" />

Analytical Methods: Chromatographic purities were determined on an Agilent 1200 Series, 1100 Series, or 6130 Series LC/MS system using a Merck Chromolith RP-18e Analytical HPLC column (monolithic, 50 χ 2 mm) and the following analytical HPLC method : injection volume 5 μΙ; flow rate 1 ml/min; 5>95% acetonitrile in water with 0.05% AcOH (Method A) or 0.05% TFA (Method B) for 5 minutes; Agilent diode array detector al = 254, 220, or 195 nm; room temperature.

Step 1. Preparation of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo[de]pyrano[3', 4 :67 7]indolizino[1,2b]quinolin-1-yl)-3-(pyridin-2-yldisulfaneyl)propanamide

A solution of 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfaneyl)propanoate (180 mg, 0.576 mmol), in DMF (4 mL) was added to solid exatecan mesylate [CAS: 169869-90- 3] (80 mg, 0.150 mmol) was then added aqueous PBS buffer (4 mL, pH=7.4, 50 mM) and sonicated ~5 min. The cloudy mixture was stirred at room temperature for 2 hours, and the reaction was determined to be approximately 25% complete. Ammonium acetate (11 mg, 0.143 mmol) was added with an additional 2 mL of DMF, and the resulting mixture was stirred at room temperature for 18 hours. The mixture was made acidic with TFA (80 mL, 0.98 mmol), and divided into 2 equal portions. Each individual portion was purified on a 50 g Redi-Sep C18 cartridge and eluted with a gradient of acetonitrile (5% to 95%) in water with TFA (0.05% v/v). The combined fractions were frozen and lyophilized to provide the title compound as a pale yellow solid (42 mg, 44%). Purity by HPLC at 254 nm: 97%. Retention time: 2.50 min (Method A). MS data, 633.2 (M+H)⁺.

Step 2. Coupling with Peptide Pv1 (Compound 29)

Solid Pv1 peptide (168.4 mg, 0.0480 mmol) was added to N-((1S,9S)-9-ethyl-5fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10 ,13,15-hexahydro-1H,12Hbenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-(pyridin-2-yldisulfaneyl) propanamide

154

154

One hundred fifty-four solid (30.5 mg, 0.0482 mmol) and dissolved in DMF (2 mL) with sonication (~1 minute) and flushed with nitrogen. 4-Methylmorpholine (20 mL, 0.182 mmol) was added and the solution was kept at room temperature for 18 h. The solution was made acidic with acetic acid (17 mL, 0.296 mmol), applied to a 25 g Biotage C18 300A reverse phase column, and eluted with a gradient of acetonitrile (25% to 95%) in water with TFA. (0.05% v/v). The combined fractions were frozen and lyophilized to provide a pale yellow solid. The product was dissolved in DMSO (3 mL) and 1 mL portions of the solution were individually purified on a 25 g Biotage C18 300A reverse phase column, eluted with a gradient (25% to 95%) of a solution ( acetonitrile/water/2-propanol, 3/2/1) in water with ammonium acetate (10 mM). The combined fractions were frozen and lyophilized to give a pale yellow solid, which was dissolved in water/acetonitrile (2/1) with 0.4% TFA, transferred into a tared vial, and lyophilized to a solid, Compound 29 (128 mg , 66%). Purity by HPLC at 254 nm: >95%. Retention time: 3.19 min (Method B) MS data: 1900.6 (M+2H/2)²⁺, 1267.3 (M+3H/3)³⁺.

Example A. Growth Retardation Assay

Cells were seeded in 96-well black-walled clear-bottom plates (Griener), DLD-1 WT cells at 2,500 cells per well, FaDu and HeLa cells at 5,000 cells per well, and HCT116 at 3,000 cells per well, in medium of growth containing 10% FBS. Cells were allowed to adhere at room temperature for 60 minutes before being returned to a 37C, 5% CO2 incubator. After 24 hours, the medium was removed and replaced with fresh culture medium containing various drug concentrations. Each drug concentration was added in triplicate. Non-drug treated controls contained only growth medium. The cells were returned to the incubator. Ninety-six hours after drug addition, cells were fixed with 4% paraformaldehyde for 20 minutes and stained with Hoechst at 1 ug/ml. Plates were imaged on a Cytation 5 automated imager (BioTek) and cells were counted using CellProfiler (http://cellprofiler.org). Percent cell growth retardation was calculated and data plotted using GraphPad Prism.

<td>Compound</td><td>DLD-1 (IC50, nM)</td><td>HCT116 (IC50, nM)</td><td>FaDu (IC50, nM)</td><td>HeLa (IC50, nM)</td>

<td>R.⁸H-5</td><td> 0.13</td><td> 0.05</td><td> 0.04</td><td> 0.12</td>

<td> 1</td><td> 1.8</td><td> 0.38</td><td> 0.21</td><td> 0.3</td>

<td> 2</td><td> 4.0</td><td> 0.66</td><td> 0.39</td><td> 0.51</td>

<td> 3</td><td>CI*</td><td> 8.33</td><td> 5.9</td><td> 7.6</td>

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One hundred fifty five

<td> 6</td><td> 13.9</td><td> 1.0</td><td> 0.76</td><td> 0.62</td>

<td> 5</td><td> 0.83</td><td> 0.12</td><td> 0.06</td><td> 0.07</td>

<td> 4</td><td> 0.80</td><td> 0.10</td><td> 0.06</td><td> 0.07</td>

*CI = Incomplete curve.

<td>Compound</td><td>HCT-116 (IC50, nM)</td>

<td> 11</td><td> 22.6</td>

<td> 12</td><td> 2.6</td>

<td> 13</td><td> 21.0</td>

<td> 14</td><td> 4.7</td>

<td> 15</td><td> 1.7</td>

<td> 16</td><td> 1.7</td>

<td> 17</td><td> 0.7</td>

<td> 18</td><td> 2.9</td>

<td> 19</td><td> 1.8</td>

<td> 20</td><td> 7.9</td>

<td> 21</td><td> 3.1</td>

<td> 22</td><td> 5.9</td>

<td> 23</td><td> 11.9</td>

<td> 24</td><td> 3.4</td>

<td> 25</td><td> 2.9</td>

<td> 26</td><td> 87.0</td>

<td> 27</td><td> 0.7</td>

<td> 28</td><td> 1.1</td>

<td> 29</td><td> 69.0</td>

Example B: Plasma Pharmacokinetics of Compound 11 in a Rat Model

Animal Dosage

Male Sprague Dawley rats underwent jugular vein cannulation and vascular access button insertion (VAB, Instech Labs Cat # VABR1B/22) at Envigo Labs prior to shipment. Aluminum magnetic caps (Instech Labs Cat # Cat #VABRC) were used to protect the access hole for the 10 jugular catheters, allowing animals to be housed 2 per cage on a bed of corn cobs for 4-5 days. before the study. Rats were administered a single intravenous dose of 5 mg/kg of Compound 11 prepared in a 5% mannitol vehicle in citrate buffer. At 1, 2, 4, 8, 24 and 30 hours after compound administration, blood (250 pl) was collected into microtainers filled with 15 K2EDTA from fed rats. Plasma was isolated by centrifugation and 100 µl aliquots transferred to 96-well polypropylene plates on dry ice. The

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One hundred fifty-six samples were stored at -80°C until processing for total peptide quantification by ELISA and exatecan released by LC-MS/MS.

ELISA Measurement of Total Peptide Plasma Concentrations

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

LC-MS/MS Measurement of Exatecan Plasma Concentrations

For exatecan quantification, a 20 µL plasma sample was added to a polypropylene autosampler vial. To each sample were added 20 μl of PPT-IS (ACN:H20 (50:50) + 0.5% FA containing 1000 ng/ml internal standard) and 20 μl of diluent (ACN:H20 (50:50) + 0.5 %FA). Followed by the addition of 120 µl of ACN + 5% FA. The vials were capped and vortexed for 2 minutes. Samples were centrifuged for 5-10 minutes at 3700 rpm and then analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The FIG. 1 shows a plot of the plasma concentration of Compound 11 and exatecan released after a single 5 mg/kg IV dose of Compound 11 in a rat (data expressed as mean ± SEM). As shown in FIG. 1, less than 0.002% of the exatecan warhead was released after 30 h in circulation. The FIG. 1 demonstrates that Compound 11 is stable in plasma for at least 30 h.

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Example C: Tumor and Bone Marrow Pharmacokinetics of Compound 11 in a Mouse Model

Animal Dosage

Foxn Mice^wildebeest Six week old female athymic nudes were obtained from Taconic Labs (Cat# NCRNU-F) and housed 5 per cage on AlphaDri bedding in a disposable cage system (Innovive). Human HCT116 cancer cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and implanted subcutaneously in the left flank of each mouse at a density of 2.5x10⁶ cells in 100 pl. When the xenografts reached a minimum volume of 300 mm³, mice were given a single intraperitoneal injection of 10 mg/kg of Compound 11 prepared in a 5% mannitol-citrate vehicle. Tumor and bone marrow samples were collected from fed and anesthetized mice 1, 2, 4, 8, 16, 24, 32, and 48 hours after compound administration. Total peptide concentrations in the tumor and bone marrow were determined via ELISA.

ELISA Measurement of Tissue Concentrations of Total Peptides

96-well plates were coated with 100 μl/well of 0.1 μΜ BSA-labeled peptide prepared in 0.2 M carbonate-bicarbonate buffer, pH 9.4 and incubated overnight at 4°C. Plates were washed 4x with ELISA washing buffer (PBS + 0.05% Tween 20), incubated for 2 hours at room temperature with blocking buffer (PBS + 5% dry milk + 0.05% Tween 20) (300 µl /well) and washed again 4x with ELISA wash buffer. Concurrently, 2x Compound 11 standards (in respective tissue matrix) or sample of tumor homogenates or bone marrow samples diluted with antibody diluent (PBS + 2% dry milk + 0.05% Tween 20) were preincubated with 1- 10 ng/ml of a primary antibody specific for the Pv1 peptide for 30 minutes at room temperature. Pre-incubated samples were added to pre-blocked, pre-coated assay plates at 100 µl/well and incubated for 1 hour at room temperature. Plates were washed 4x with ELISA wash buffer and incubated with 100 µl/well of a secondary goat anti-mouse IgG HRP antibody (1:5,000 in antibody diluent) for 1 hour at room temperature. Plates were washed 4x with ELISA wash buffer and incubated with 100 µl/well SuperSignal substrate at room temperature with gentle agitation for 1 minute. Luminescence was read from the plate on a BioTek Cytation 5 plate reader.

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158 one hundred fifty-eight FIG. 2 shows a plot of tumor and bone marrow peptide concentration after a single 10 mg/kg IP dose of Compound 11 in a mouse (data are expressed as mean ± SEM). The FIG. 2 demonstrates that Compound 11 effectively targets tumors.

Example D: Bone Marrow Toxicity Study in a Mouse Model

Animal Dosage

Foxn Mice^wildebeest Six week old female athymic nudes were obtained from Taconic Labs (Cat# NCRNU-F) and housed 5 per cage on AlphaDri bedding in a disposable cage system (Innovive). Human HCT116 cancer cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and implanted subcutaneously in the left flank of each mouse at a density of 2.5x10⁶ cells in 100 pl. When the xenografts reached a minimum volume of 200 mm³, mice were administered intraperitoneal doses of vehicle or 2.6 or 5.2 pmoles/kg of either unconjugated exatecan (equivalent to 1.15 or 2.3 mg/kg exatecan, respectively) or Compound 11 (equivalent to 10 or 20 mg/kg of Compound 11, respectively). Compounds were administered once daily for 4 days.

Bone Marrow Collection

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

The FIG. 3 shows a diagram of total bone marrow counts from the femurs of tumor-bearing nude mice after dosing at 2.6 and 5.2 pmoles/kg of either Compound 11 (equivalent to 10, 20 mg/kg conjugate) or

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One hundred fifty-nine free exatecan (equivalent to 1.15 and 2.3 mg/kg exatecan) dosed once daily for four days (data are expressed as mean ± SEM). Compound 11 did not exhibit the bone marrow toxicity that limits the clinical utility of exatecan.

Example E: Gastric Toxicity Study in a Mouse Model

Animal Dosing and Stomach Imaging

Foxn Mice^wildebeest Six week old female athymic nudes were obtained from Taconic Labs (Cat# NCRNU-F) and housed 5 per cage on Alpha-Dri bedding in a disposable cage system (Innovive). Human HCT116 cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and implanted subcutaneously in the left flank of each mouse at a density of 2.5x10⁶cells in 100 pl. When the xenografts reached a minimum volume of 300 mm³, mice were given intraperitoneal doses of vehicle or 5.2 pmoles/kg of either unconjugated exatecan (equivalent to 2.3 mg/kg exatecan) or Compound 11 (equivalent to 20 mg/kg Compound 11). Compounds were administered once daily for 4 days. At 6 hours after the last dose administration, the mice were sacrificed by cervical dislocation and gross necropsy was performed. Photographs of the stomachs were taken both in situ and ex vivo.

The FIG. 4A shows the stomachs of tumor-bearing nude mice, excised after dosing with vehicle or 5.2 pmoles/kg of Compound 11 (equivalent to 20 mg/kg conjugate) or free exatecan (equivalent to 2.3 mg/kg exatecan) dosed. QDx4. The FIG. 4B shows the stomachs of nude mice bearing tumors in situ after dosing with 5.2 pmoles/kg of Compound 11 (equivalent to 20 mg/kg conjugate) or free exatecan (equivalent to 2.3 mg/kg exatecan) dosed once. a day for four days. Compound 11 did not exhibit the gastric toxicity that limits the clinical utility of exatecan.

Example F: Efficacy of Compound 11 in an HCT116 Colorectal Cancer Model

Foxn Mice^wildebeest Six week old female athymic nudes were obtained from Taconic Labs (Cat# NCRNU-F) and housed 5 per cage on AlphaDri bedding in a disposable cage system. Human HCT116 cells derived from colorectal carcinoma were diluted 1:1 in phenol red-free Matrigel and implanted subcutaneously in the left flank of each mouse at a density of 2.5x10⁶cells in 100 pl. When the xenografts reached a mean volume of 100-200

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One hundred-sixty mm³, mice were randomized into groups and treated as detailed in the table below. Mice were given intraperitoneal (IP) doses of vehicle or 2.6 or 5.2 pmol/kg of either unconjugated exatecan (equivalent to 1.15 or 2.3 mg/kg exatecan, respectively) or Compound 11 (equivalent to 10 or 20 mg /kg of Compound 11, respectively). Doses were prepared by diluting 0.1 mg/pl DMSO stock solutions in 5% mannitol in citrate buffer and administered QDX4/week for three weeks at a volume of 12 ml/kg (300 pl per 25 g mouse). The xenograft tumors were measured by calipers and the volume was calculated using the equation for the volume of the ellipsoid: Volume = π/6 x (length) x (width)². Animals were withdrawn from the study due to death, tumor size exceeding 2000 mm³ or loss of >20% of body weight. The following table shows the dosing schedule for various treatment groups.

<td>Cluster</td><td>Treatment</td><td>Dose</td><td>Dosage Schedule</td><td>Route of Administration</td><td>Number of Mice</td>

<td> 1</td><td>Vehicle (5% mannitol in citrate buffer)</td><td>N/A</td><td>QDx4/week x 3</td><td>ip</td><td> 8</td>

<td> 2</td><td>compound 11</td><td>10mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 8</td>

<td> 3</td><td>compound 11</td><td>20mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 8</td>

<td> 4</td><td>exatecan</td><td>1.15mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 8</td>

<td> 5</td><td>exatecan</td><td>2.3mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 8</td>

The FIG. 5A shows a plot of the mean tumor volume resulting from dosing equimolar amounts of free exatecan or Compound 11 in nude mice bearing HCT116 colorectal flank tumors. Animals were dosed once a day four times a week intraparenterally for three weeks. The FIG. 5B shows a Kaplan Meier survival curve for dosing equimolar amounts of free exatecan or Compound 11 in nude mice bearing HCT116 colorectal flank tumors. Data are expressed as mean ± SEM. These data demonstrate that Compound 11 demonstrates potent antitumor activity in a preclinical colorectal cancer model.

Example G: Efficacy of Compound 11 in a MKN45 HER2 Negative Gastric Cancer Model

Foxn Mice^wildebeest Six week old female athymic nudes were obtained from Taconic Labs (Cat# NCRNU-F) and housed 5 per cage on AlphaDri bedding in a disposable cage system. Human MKN45 cells derived from gastric carcinoma were diluted 1:1 in phenol red-free Matrigel and implanted

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One-sixty-one subcutaneously in the left flank of each mouse at a density of 2x10⁶cells in 100 μΙ. When the xenografts reached a mean volume of 100-200 mm³, mice were randomized into groups and treated as detailed in the table below. Mice were administered intraperitoneal (IP) doses of vehicle or 2.6 or 5.2 µmol/kg of either unconjugated exatecan (equivalent to 1.15 or 2.3 mg/kg exatecan, respectively) or Compound 11 (equivalent to 10 or 20 mg /kg of Compound 11, respectively). Doses were prepared by diluting 0.1 mg/μl DMSO stock solutions in 5% mannitol in citrate buffer and administered QDX4/week for two weeks at a volume of 12 ml/kg (300 μl per 25 g mouse). The xenograft tumors were measured by calipers and the volume was calculated using the equation for the volume of the ellipsoid: Volume = π/6 x (length) x (width)². Animals were withdrawn from the study due to death, tumor size exceeding 2000 mm³ or loss of >20% of body weight. The following table shows the dosing schedule for the various treatment groups.

<td>Cluster</td><td>Treatment</td><td>Dose</td><td>Program of Dosage</td><td>Route of Administration</td><td>Number of Mice</td>

<td> 1</td><td>Vehicle (5% mannitol in citrate buffer)</td><td>N/A</td><td>QDx4/week x 2</td><td>ip</td><td> 8</td>

<td> 2</td><td>compound 11</td><td>2.5mg/kg</td><td>QDx4/week x 2</td><td>ip</td><td> 8</td>

<td> 3</td><td>compound 11</td><td>5mg/kg</td><td>QDx4/week x 2</td><td>ip</td><td> 8</td>

<td> 4</td><td>compound 11</td><td>10mg/kg</td><td>QDx4/week x 2</td><td>ip</td><td> 8</td>

<td> 5</td><td>compound 11</td><td>20mg/kg</td><td>QDx4/week x 2</td><td>ip</td><td> 8</td>

<td> 6</td><td>exatecan</td><td>1.15mg/kg</td><td>QDx4/week x 2</td><td>ip</td><td> 8</td>

<td> 7</td><td>exatecan</td><td>2.3mg/kg</td><td>QDx4/week x 2</td><td>ip</td><td> 8</td>

The FIG. 6A shows the single-agent efficacy of Compound 11 in nude mice bearing MKN45 HER2-negative gastric cancer flank tumors. Animals were dosed once a day four times a week intraparenterally for two weeks. The FIG. 6B shows a Kaplan Meier survival curve for dosing equimolar amounts of free exatecan or Compound 11 in nude mice bearing MKN45 HER2-negative gastric cancer flank tumors. Data are expressed as mean ± SEM. These data demonstrate that Compound 11 demonstrates potent antitumor activity in a preclinical gastric cancer model.

Figure 6B. Kaplan-Meier analysis was used to assess the survival rate based on death or removal from the study.

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One hundred sixty-two Example H: Efficacy of Compound 11 in a Breast Cancer Model

HER2 Intermediate JIMT-1

Five to six week old female NOD.SCID mice were obtained from Beijing Anikeeper Biotech Co., Ltd (Beijing, China). Human J1MT-1 cells derived from breast carcinoma were diluted 1:1 in phenol red-free Matrigel and implanted subcutaneously in the left flank of each mouse at a density of 5x10.⁶cells in 100 pl. When the xenografts reached a mean volume of 100 mm³, mice were randomized into groups and treated as detailed in the table below. Mice were given intraperitoneal (IP) doses of vehicle or 2.6 or 5.2 pmol/kg of Compound 11 (equivalent to 10 or 20 mg/kg of Compound 11, respectively). Doses were prepared by diluting 0.1 mg/pL DMSO stock solutions in 5% mannitol in citrate buffer and administered QDX4/week for three weeks at a volume of 12 mL/kg (300 pl per 25 g mouse). The xenograft tumors were measured by calipers and the volume was calculated using the equation for the volume of the ellipsoid: Volume = π/6 x (length) x (width)². The body weight of the animals was measured at the same time as the evaluation of the tumor volume. Animals were withdrawn from the study due to death, tumor size exceeding 2000 mm³or loss of >20% of body weight. The following table shows the dosing schedule for various treatment groups.

<td>Cluster</td><td>Treatment</td><td>Dose</td><td>Program of Dosage</td><td>Route of Administration</td><td>Number of Mice</td>

<td> 1</td><td>Vehicle (5% mannitol in citrate buffer)</td><td>N/A</td><td>QDx4/week x 3</td><td>ip</td><td> 8</td>

<td> 2</td><td>compound 11</td><td>10mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 8</td>

<td> 3</td><td>compound 11</td><td>20mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 8</td>

The FIG. 7A shows a plot of mean tumor volume resulting from dosing of Compound 11 in SCID mice bearing JIMT-1 intermediate HER2 breast cancer flank tumors. Animals were dosed once a day four times a week intraparenterally for three weeks. The FIG. 7B shows a plot of the percent change in body weight in SCID mice bearing JIMT-1 intermediate HER2 breast cancer flank tumors dosed with Compound 11. Data are expressed as mean ± SEM. These data demonstrate that Compound 11 demonstrates potent antitumor activity in a preclinical breast cancer model.

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one hundred sixty-three

Example I: Efficacy of Compound 11 in a Breast Cancer Model

MDA-MB-231 Triple Negative

Foxn Mice^wildebeest Three to four week old female athymic nudes were obtained from Envigo Labs. Human breast adenocarcinoma-derived MDA-MB-231 cells were diluted 1:1 in phenol red-free Matrigel and implanted subcutaneously into the left flank of each mouse at a density of 2x10⁶cells in 100 pl. When the xenografts reached a mean volume of 50-100 mm³, mice were randomized into groups and treated as detailed in the table below. Mice were given intraperitoneal (IP) doses of vehicle or 5, 10, or 20 mg/kg of Compound 11. Doses were prepared by diluting 0.1 mg/pl DMSO stock solutions in 5% mannitol in citrate buffer and were they administered QDX4/week for three weeks at a volume of 12 ml/kg (300 μΙ per 25 g mouse). The xenograft tumors were measured by calipers and the volume was calculated using the equation for the volume of the ellipsoid: Volume = π/6 x (length) x (width)². The body weight of the animals was measured at the same time as the evaluation of the tumor volume. Animals were withdrawn from the study due to death, tumor size exceeding 2000 mm³ or due to a loss of >20% of body weight. The following table shows the dosing schedule of various treatment groups.

<td>Cluster</td><td>Treatment</td><td>Dose</td><td>Dosage Schedule</td><td>Route of Administration</td><td>Number of Mice</td>

<td> 1</td><td>Vehicle (5% mannitol in citrate buffer)</td><td>N/A</td><td>N/A</td><td>ip</td><td> 8</td>

<td> 2</td><td>compound 11</td><td>5mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 9</td>

<td> 3</td><td>compound 11</td><td>10mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 9</td>

<td> 4</td><td>compound 11</td><td>20mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 9</td>

The FIG. 8A shows a plot of mean tumor volume in nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11. Animals were dosed once daily four times weekly intraparenterally. for three weeks. The FIG. 8B shows a plot of the percent change in body weight from day 0 in nude mice bearing MDAMB-231 triple negative breast cancer flank tumors dosed with Compound 11. Data are expressed as mean ± SEM. These data demonstrate that Compound 11 demonstrates potent antitumor activity in a preclinical breast cancer model.

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One hundred sixty-four Example J: Combined Efficacy of Compound 11 and Talazoparib in a

MDA-MB-231 Triple Negative Breast Cancer Model

Foxn Mice^wildebeest Three to four week old female athymic nudes were obtained from Envigo Labs. Human breast adenocarcinoma-derived MDA-MB-231 cells were diluted 1:1 in phenol red-free Matrigel and implanted subcutaneously into the left flank of each mouse at a density of 2x10⁶ cells in 100 pl. When the xenografts reached a mean volume of 50100 mm³, mice were randomized into groups and treated as detailed in the table below. Mice were given intraperitoneal (IP) doses of vehicle or 5 mg/kg of Compound 11 alone or in combination with an oral (PO) dose of 0.33 mg/kg talazoparib. Doses were prepared by diluting 0.1 mg/pl DMSO stock solutions in 5% mannitol in citrate buffer. Compound 11 was administered QDX4/week for three weeks at a volume of 12 mL/kg (300 µL per 25 g mouse) and talazoparib was administered once daily for 15 days. Xenograft tumors were measured with calipers and volume calculated using the equation for ellipsoid volume: Volume = π/6 x (length) x (width)². The body weight of the animals was measured at the same time as the evaluation of the tumor volume. Animals were withdrawn from the study due to death, tumor size exceeding 2000 mm³ or due to a loss of body weight >20%. The following table shows the dosing schedule of various treatment groups.

<td>Cluster</td><td>Treatment</td><td>Dose</td><td>Program of Dosage</td><td>Route of Administration</td><td>Number of Mice</td>

<td> 1</td><td>None</td><td>N/A</td><td>N/A</td><td>N/A</td><td> 9</td>

<td> 2</td><td>Talazoparib</td><td>0.33mg/kg</td><td>QDx15</td><td>po</td><td> 9</td>

<td> 3</td><td>compound 11</td><td>5mg/kg</td><td>QDx4/week x 3</td><td>ip</td><td> 10</td>

<td> 4</td><td>Talazoparib compound 11</td><td>0.33mg/kg 5mg/kg</td><td>QDx15 QDx4/week x 3</td><td>po ip</td><td> 8</td>

The FIG. 9A shows a plot of the mean tumor volume of nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11 and talazoparib. Animals were dosed once daily four times weekly intraparenterally for three weeks with Compound 11 and once daily for 18 days orally with talazoparib. The FIG. 9B shows a plot of the percent change in body weight relative to day 0 of nude mice bearing MDA-MB-231 triple negative breast cancer flank tumors dosed with Compound 11 and talazoparib.

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165 One-sixty-five Example K: Glutathione Cleavage Study

A 20 mM stock solution of conjugate was prepared in 100% DMSO. Stock solutions were further diluted in 100 mM Tris, pH 7.5, to produce an intermediate 500 μΜ dilution followed by a further 1:5 dilution in Tris.

100 mM, pH 7.5 to produce a final concentration of 100 μΜ of conjugate.

100 mM glutathione was prepared immediately before use in H20 and diluted 1:10 in challenge samples for a final glutathione challenge concentration of 10 mM. Samples were mixed by inversion and incubated at 37°C for up to 24 hours. Samples of 50 µl were aliquoted into siliconized microcentrifuge tubes at time 0, 4, and 24 hours and immediately frozen at -80°C.

Samples were thawed and extracted as follows: 8 µl of 25% phosphoric acid was added to each sample followed by 117 µl of 100% acetonitrile/0.1% TFA, mixed and centrifuged at 13000xG for 10 min. The supernatant was pipetted into 0.2 mL HPLC vials and placed in an automatic sampler.

HPLC Perkin Elmer Flexar. The following table summarizes the HPLC conditions:

<td>HPLC</td><td colspan="3">Perkin Elmer Flexar Binary Pump, Autosampler, UV Detector</td>

<td>Column</td><td colspan="3">Waters BioResolve RP mAb Polyphenyl Column, 450 A, 2.7 μm, 4.6 mm x 150 mm</td>

<td>Guard Column</td><td colspan="3">Waters BioResolve RP mAb Polyphenyl VanGuard Supported Cartridge, 450 A, 2.7 µm, 3.9 mm x 5 mm</td>

<td>wavelength of Detection</td><td colspan="3">217nm</td>

<td>column temperature</td><td colspan="3">37°C</td>

<td>Pressure Limits</td><td colspan="3">Min: 0 PSI, Max: 3050 PSI</td>

<td>mobile phase mobile phase A</td><td colspan="3">0.05% TFA in water</td>

<td>mobile phase B</td><td colspan="3">0.05% TFA in Acetonitrile</td>

<td>Flow rate</td><td colspan="3">0.8ml/min</td>

<td>Injection Volume</td><td colspan="3">10.0μl</td>

<td>run time</td><td colspan="3">14.0 minutes</td>

<td rowspan="6">gradient program</td><td>Time (minutes)</td><td>% of A</td><td>% of B</td>

<td> 0.0</td><td> 80</td><td> 20</td>

<td> 0.5</td><td> 80</td><td> 20</td>

<td> 10.0</td><td> 0</td><td> 100</td>

<td> 11.0</td><td> 80</td><td> 20</td>

<td> 14.0</td><td> 80</td><td> 20</td>

The data was analyzed by calculating the percentage reduction of the compound

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One hundred sixty six (peak area of ​​retention time of cleaved conjugate/peak area of ​​conjugate retention time at time 0) X 100.

The FIG. 10 shows a graph of the degradation of Compound 11 and Compound 29 resulting from treatment with 10 mM glutathione for 16 h. As shown in FIG. 10, Compound 29 is released much faster than compound under similar glutathione exposure.

The table below summarizes the degradation data for the 10mM glutathione exposure conditions described above for Compounds 11 through Compound 29 measured at 4h and 24h.

<td colspan="3">Challenge with glutathione (10 mM)</td>

<td>Compound</td><td>% Remaining at 4 h</td><td>% Remaining at 24 h</td>

<td> 11</td><td> 50.1</td><td> 10.2</td>

<td> 12</td><td>NA</td><td>NA</td>

<td> 13</td><td>NA</td><td>NA</td>

<td> 14</td><td> 14.9</td><td> 6.5</td>

<td> 15</td><td> 19.4</td><td> 8.3</td>

<td> 16</td><td> 3.4</td><td> 2.0</td>

<td> 17</td><td> 3.4</td><td> 1.7</td>

<td> 18</td><td> 60.1</td><td> 13.0</td>

<td> 19</td><td>NA</td><td>NA</td>

<td> 20</td><td> 5.2</td><td> 8.0</td>

<td> 21</td><td> 9.5</td><td> 12.6</td>

<td> 22</td><td> 65.8</td><td> 12.3</td>

<td> 23</td><td> 61.4</td><td> 13.2</td>

<td> 24</td><td> 20.8</td><td> 4.2</td>

<td> 25</td><td> 42.0</td><td> 4.1</td>

<td> 26</td><td> 75.8</td><td> 32.1</td>

<td> 27</td><td> 18.0</td><td> 0.0</td>

<td> 28</td><td> 40.5</td><td> 0.0</td>

<td> 29</td><td> 3.1</td><td> 5.9</td>

Example L: Stability Studies in Plasma

A 20 mM stock solution of conjugate was prepared in 100% DMSO. Stock solutions were further diluted in 100 mM Tris, pH 7.5 to produce an intermediate dilution of 500 pM and then diluted 1:5 directly in rat plasma to produce a final concentration of 100 pM conjugate. Samples were mixed by inversion and incubated at 37°C for up to 24 h. Samples of 50 pl were aliquoted into siliconized microcentrifuge tubes at time 0, 4, and 24 h and immediately frozen at -80°C.

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Samples were thawed and extracted as follows: 8 μΙ of 25% phosphoric acid was added to each sample followed by 117 μΙ of 100% acetonitrile/0.1%

TFA, mixed and centrifuged at 13000xG for 10 min. The supernatant was pipetted into 0.2 mL HPLC vials and placed in an automatic sampler.

HPLC Perkin Elmer Flexar. The following table summarizes the HPLC conditions:

<td>HPLC</td><td colspan="3">Perkin Elmer Flexar Binary Pump, Sampler automatic, UV detector</td>

<td>Column</td><td colspan="3">Waters BioResolve RP mAb Polyphenyl Column, 450 A, 2.7 μm, 4.6 mm x 150 mm</td>

<td>Guard Column</td><td colspan="3">Waters BioResolve RP mAb Polyphenyl VanGuard Supported Cartridge, 450 A, 2.7 µm, 3.9 mm x 5 mm</td>

<td>Detection Wavelength</td><td colspan="3">217nm</td>

<td>column temperature</td><td colspan="3">37°C</td>

<td>Pressure Limits</td><td colspan="3">Min: 0 PSI, Max: 3050 PSI</td>

<td>mobile phase mobile phase A</td><td colspan="3">0.05% TFA in water</td>

<td>mobile phase B</td><td colspan="3">0.05% TFA in Acetonitrile</td>

<td>Flow rate</td><td colspan="3">0.8ml/min</td>

<td>Injection Volume</td><td colspan="3">10.0μl</td>

<td>run time</td><td colspan="3">14.0 minutes</td>

<td rowspan="6">gradient program</td><td>Time (minutes)</td><td>% of A</td><td>% of B</td>

<td> 0.0</td><td> 80</td><td> 20</td>

<td> 0.5</td><td> 80</td><td> 20</td>

<td> 10.0</td><td> 0</td><td> 100</td>

<td> 11.0</td><td> 80</td><td> 20</td>

<td> 14.0</td><td> 80</td><td> 20</td>

Data were analyzed by calculating the percentage reduction of the compound (incubated conjugate retention time peak area/conjugate retention time peak area at time 0) X 100. The results of the study are shown in the table next.

<td></td><td colspan="2">Plasma Stability</td>

<td>Compound</td><td>% Remaining at 4 hours</td><td>% Remaining at 24 hours</td>

<td> 11</td><td> 108.2</td><td> 107.2</td>

<td> 12</td><td>NA</td><td>NA</td>

<td> 13</td><td>NA</td><td>NA</td>

<td> 14</td><td> 106.1</td><td> 105.9</td>

<td> 15</td><td> 104.9</td><td> 103.3</td>

<td> 16</td><td> 115.6</td><td> 106.1</td>

<td> 17</td><td> 106.8</td><td> 105.0</td>

<td> 18</td><td> 104.0</td><td> 93.0</td>

<td> 19</td><td>NA</td><td>NA</td>

168

168

One hundred sixty eight-

<td> 20</td><td> 98.7</td><td> 95.8</td>

<td> 21</td><td> 101.0</td><td> 98.2</td>

<td> 22</td><td> 106.0</td><td> 105.0</td>

<td> 23</td><td> 92.0</td><td> 92.0</td>

<td> 24</td><td> 87.0</td><td> 87.0</td>

<td> 25</td><td> 103.0</td><td> 104.0</td>

<td> 26</td><td> 105.0</td><td> 112.0</td>

<td> 27</td><td> 117.0</td><td> 109.0</td>

<td> 28</td><td> 106.0</td><td> 106.0</td>

<td> 29</td><td> 116.0</td><td> 110.0</td>

Various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including, without limitation, all patents, patent applications, and publications, cited in this application, is incorporated herein by reference in its entirety.