Exatecan-derived ADC linker-payload, pharmaceutical composition, and use thereof

Abstract

The present disclosure relates to linkers-payloads containing the structure of formula I, and salts thereof (including pharmaceutically acceptable salts), solvates or stereoisomers. The present disclosure also relates to pharmaceutical compositions containing these compounds and the use of these compounds, intermediates of the compounds, and compositions in the prevention or treatment of cancer and / or tumors. 【Chemical 1】 TIFF2025523332000151.tif57155

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority of U.S. Provisional Application No. 63 / 419,150, filed on October 25, 2022, the disclosure of which is incorporated herein by reference in its entirety.

Background Art

[0002] The present disclosure represents a linker - payload derived from linker exatecan (camptothecin) that is useful for conjugating to an antibody or other targeting moiety to generate antibody - drug conjugates (ADCs) and other target - ligand conjugates for tumor indications. The compound contains a topoisomerase - 1 inhibitor derived from an exatecan backbone connected to a novel linker structure that exhibits cytotoxicity when conjugated to a targeting moiety. Documents reporting the use of exatecan (chemical name: (1S,9S) - 1 - amino - 9 - ethyl - 5 - fluoro - 2,3 - dihydro - 9 - hydroxy - 4 - methyl - 1H,12H - benzo[de]pyrano[3,4,:6,7]imidazo[1,2 - b]quinoline - 10,13(9H,15H) - dione), a camptothecin derivative, are disclosed in WO2014057687, US11103593, US9808537, US7091186, US2010 / 0062008, WO2015057699, Clinical Cancer Research (2016) 22(20):5097 - 5108, and Cancer Sci (2016) 107:1039 - 1046. See also WO2022068878, WO2017062271, CN113816969, CN112125915, US20210353764.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Non-Patent Document

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0005] A class of linker camptothecin-derived payload compounds is disclosed, where the linker structure contains a maleimide attached to a peptide linker, has variations in the amino acid sequence, optionally incorporates PEG units, and terminates with a hemiaminal or p-aminobenzyl carbamate (PABC) connection to the camptothecin-derived payload. The utility of these linker camptothecin-derived payload compounds is demonstrated by binding to cysteine residues of various target moieties such as antibodies to generate antibody-drug conjugates (ADCs), which exhibit favorable physicochemical properties and high target-mediated efficacy. Accordingly, another embodiment of the disclosure is realized by the ADCs disclosed herein. The linker-payloads of the present disclosure provide potent and novel ADCs that are active across multiple cancer cell lines, thereby demonstrating broad utility in binding to several antibodies and other target moieties while maintaining favorable properties and efficacy. Exemplary ADCs from the linker payloads described herein are also detailed.

[0006] This compound is cytotoxic and can be applied, for example, as an antitumor agent and as a chemotherapeutic agent in tumor situations. This compound is potent, has a new structure, and is active against multiple cancer cell lines.

Embodiments for Carrying Out the Invention

[0007] In each of the following embodiments, variable elements not explicitly defined in the embodiment are as defined in formula (I). In each embodiment described herein, unless otherwise specified, each variable element is selected independently of the other variable elements.

[0008] The present disclosure relates to a linker camptothecin-derived payload compound, wherein the linker structure contains a maleimide bonded to a peptide linker, has a variation in the amino acid sequence, optionally incorporates a PEG unit, and terminates with a hemiaminal connection to the camptothecin-derived payload. One embodiment of the present disclosure relates to a linker camptothecin-derived payload compound, wherein the linker structure contains a maleimide bonded to a peptide linker, has a variation in the amino acid sequence, optionally incorporates a PEG unit, and terminates with a PABC connection to the camptothecin-derived payload. Another embodiment of the present disclosure relates to a compound in which the linker camptothecin-derived payload compound is bonded to a target moiety having a free cysteine group, including an antibody, a protein, a peptide, a polypeptide, and a modified antibody. Yet another embodiment of the present disclosure relates to a maleimide-containing linker group.

[0009] In another embodiment, the present disclosure provides a linker camptothecin-derived payload compound (also referred to as a linker payload), and a pharmaceutically acceptable salt, solvate or stereoisomer thereof, comprising the structure of Formula I below.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0010] Another aspect of this embodiment is realized when X 1 is PEG of 1 to 24 subunits. Another aspect of this embodiment is realized when X 1 is PEG of 1 to 12 subunits. Another aspect of this embodiment is realized when X 1 is PEG terminated with OH or Me group.

[0011] One embodiment of the present disclosure is realized when Z is hydrogen.

[0012] Another embodiment of the present disclosure is realized when Z is -CH2C(R x )(R y )CHF2-. Sub - embodiments of this aspect of the present disclosure are realized when R x and R y are independently selected from fluorine, chlorine, methyl, ethyl, and hydrogen. Another sub - embodiment of this aspect of the present disclosure is realized when R x and R y are both fluorine. Another sub - embodiment of this aspect of the present disclosure is realized when R x and R y are both methyl. Another sub - embodiment of this aspect of the present disclosure is realized when one of R x and R y is methyl and the other is fluorine.

[0013] Another embodiment of the present disclosure is realized when each R c is independently selected from hydrogen, OH, CH3, CH2OH, CHF2, CH2F, CF3, fluorine, and chlorine. Sub - embodiments of this aspect of the present disclosure are realized when one of the R c is hydrogen and the other is OH. Another sub - embodiment of this aspect of the present disclosure is realized when one of the Rc is realized when one is hydrogen and the other is CH3. Another sub - embodiment of this aspect of the present disclosure is that one R c is realized when one is hydrogen and the other is CH2OH. Another sub - embodiment of this aspect of the present disclosure is that one R c is realized when one is CH3 and the other is CHF2. Another sub - embodiment of this aspect of the present disclosure is that one R c is realized when one is hydrogen and the other is OH. A sub - embodiment of this aspect of the present disclosure is that both Rs c is realized when both are CH3. A sub - embodiment of this aspect of the present disclosure is that both Rs c is realized when both are fluorine. A sub - embodiment of this aspect of the present disclosure is that both Rs c is realized when both are hydrogen.

[0014] Another embodiment of the present disclosure is that both Rs c are combined to form C 3-6 spirocycloalkyl. A sub - embodiment of this aspect of the present disclosure is realized when it is spirocyclopropyl, spirocyclobutyl, spirocyclopentyl and spirocyclohexyl. Another sub - embodiment of this aspect of the present disclosure is realized when the spirocycloalkyl is cyclopropyl.

[0015] Another embodiment of formula I is realized when Q is C 1-6 alkyl, and the alkyl is selected from methyl, ethyl, propyl, butyl or hexyl. A sub - embodiment of this aspect of the present disclosure is that Q is C 1-6 alkyl, and the alkyl is methyl.

[0016] Another embodiment of formula V is realized when Q is OH.

[0017] Another embodiment of the present disclosure is realized when Z 2 is absent.

[0018] Another embodiment of the present disclosure is that Z 2 is - CRb R b - is realized when it is. Another embodiment of the present disclosure is Z 2 is -CH2CR b R b - is realized when it is. Another embodiment of the present disclosure is Z 2 is -CR b R b CH2- is realized when it is.

[0019] Another embodiment of the present disclosure is realized when each R b is independently selected from hydrogen, OH, and CH3. A sub - embodiment of this aspect of the present disclosure is realized when one R b is hydrogen and the other is OH. Another sub - embodiment of this aspect of the present disclosure is realized when one R b is hydrogen and the other is CH3. A sub - embodiment of this aspect of the present disclosure is realized when both Rs b are CH3. A sub - embodiment of this aspect of the present disclosure is realized when both Rs b are hydrogen.

[0020] Another embodiment of the present disclosure is realized when both Rs b are combined to form C 3-6 spirocycloalkyl. Another sub - embodiment of this aspect of the present disclosure is realized when both Rs b are combined to form spirocyclopropyl.

[0021] Another embodiment of the present disclosure is realized when Z 1 is -O-.

[0022] Another embodiment of the present disclosure is realized when Z 1 is -NH-.

[0023] Another embodiment of the present disclosure is realized when X is a linking group that is W1. A sub - embodiment of this aspect of the present disclosure is when W1 is

Chemical formula

[0024] Another embodiment of the present disclosure is realized when X is a linking group that is W2. A sub - embodiment of this aspect of the present disclosure is that when W2 is [Chemical formula] It is realized when it is. Another sub - embodiment of this aspect of the present disclosure is that when W2 is [Chemical formula] It is realized when it is. Another sub - embodiment of this aspect of the present disclosure is that when the R of W2 d is hydrogen. Another sub - embodiment of this aspect of the present disclosure is that when the R of W2 d is - CH2NHC(O)X 1 Q.

[0025] Another embodiment of the present disclosure is realized when X is a linking group that is W3.

[0026] Another embodiment of the present disclosure is realized when X is a linking group that is W4.

[0027] Another embodiment of the present disclosure is realized when X is a linking group that is W5.

[0028] Another embodiment of the present disclosure is realized when X is a linking group that is W6. A sub - embodiment of this aspect of the present disclosure is that when W6 is [Chemical formula] is realized when. Another sub - embodiment of this aspect of the present disclosure is that W6 is

Chemical formula

[0029] Another embodiment of the present disclosure is realized when X is a linking group that is W7. One sub - embodiment of the present disclosure is that W7 is

Chemical formula

Chemical formula

[0030] Another sub - embodiment of this aspect of the present disclosure is that X 3 is hydrogen or -C(O)NR a R z is realized when. Another sub - embodiment of this aspect of the present disclosure is that X 3 is hydrogen is realized when. Another sub - embodiment of this aspect of the present disclosure is that X 3 is -C(O)NR a R z and R a is selected from hydrogen and C 1-6 alkyl, and R z is X 1 is realized when. Another sub - embodiment of this aspect of the present disclosure is that X 3 is -C(O)NRa R z is, and R a and R z are combined to form C 3-10 cycloalkyl or a 3- to 10-membered heterocyclic ring. One aspect of this sub-embodiment is that R a and R z are combined to form a cycloalkyl selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Another aspect of this sub-embodiment is that the 3- to 10-membered heterocyclic ring formed by R a and R z is a cyclic amine selected from piperidine, piperazine, azetidine, aziridine, pyrrolidine, azepane, morpholine, pyridine, and imidazole. A further embodiment of the disclosure is when the cyclic amine is bonded to X 3 carbonyl by its nitrogen atom. A further embodiment of this aspect of the present invention is when the cyclic amine formed by R a and R z is piperidine. A further embodiment of this aspect of the present invention is when the cyclic amine formed by R a and R z is piperazine. A further embodiment of this aspect of the present invention is when the cyclic amine formed by R a and R z is azetidine. A further embodiment of this aspect of the present invention is when the cyclic amine formed by R a and R z is aziridine. A further embodiment of this aspect of the present invention is when the cyclic amine formed by R a and R z is pyrrolidine. A further embodiment of this aspect of the present invention is when the cyclic amine formed by R a and R z is azepane. A further embodiment of this aspect of the present invention is when the cyclic amine formed by R a and R zIt is realized when the cyclic amine formed by [is pyridine]. A further embodiment of this aspect of the present invention is R a and R z It is realized when the cyclic amine formed by [is imidazole]. A further embodiment of this aspect of the present invention is R a and R z The cyclic amine formed by [is

Chemical formula

[0031] Another embodiment of the present disclosure is realized when X is a linking group that is W8. A sub - embodiment of this aspect of the present disclosure is that X 4 is hydrogen. A sub - embodiment of this aspect of the present disclosure is that X 4 is

Chemical formula

[0032] One embodiment of the present disclosure is realized when each R a is independently selected from hydrogen and methyl. Another embodiment of the present disclosure is realized when R a is methyl. Another embodiment of the present disclosure is realized when R a is hydrogen.

[0033] One embodiment of the disclosure of formula I is represented by structural formula II

Chemical formula

[0034] Another embodiment of the disclosure of Formula II is that Z 2 is - CR b R b Another embodiment of the disclosure of Formula II is that Z 2 is - CH2CR b R b Another embodiment of the disclosure of Formula II is that Z 2 is - CR b R b CH2-. Sub - embodiments of this aspect of the present disclosure are realized when each R b is independently selected from hydrogen, OH, and CH3. Sub - embodiments of this aspect of the present disclosure are realized when Z 2 is selected from - CH2 -, - CH2CH2 -, - CH2CH(CH3)-, - CH2CH(OH)-, and - CH2 - spirocyclopropyl -. Sub - embodiments of this aspect of the present disclosure are realized when Z 2 is - CH2-. Sub - embodiments of this aspect of the present disclosure are realized when Z 2 is - CH2CH2-.

[0035] Another embodiment of the disclosure of Formula II is realized when X is a linking group that is W1. One lower embodiment of Formula II is when W1 is

Chemical formula

Chemical formula

[0036] Another embodiment of Formula II is realized when X is a linking group that is W2. One lower embodiment of Formula II is when W2 is

Chemical formula

Chemical formula

[0037] Another embodiment of Formula II is realized when X is a linking group that is W3.

[0038] Another embodiment of Formula II is realized when X is a linking group that is W4.

[0039] Another embodiment of Formula II is realized when X is a linking group that is W5.

[0040] Another embodiment of Formula II is realized when X is a linking group that is W6. One lower embodiment of Formula II is when W6 is

Chemical formula

Chemical formula

[0041] One embodiment of Formula II is realized when each R a is independently selected from hydrogen and methyl. Another embodiment of Formula II is when R a is methyl. Another embodiment of Formula II is when R a is hydrogen.

[0042] One embodiment of the disclosure of Formula I is the following structural formula III:

Chemical formula

[0043] Another embodiment of the disclosure of Formula III is realized when Z is hydrogen.

[0044] Another embodiment of the disclosure of Formula III is realized when Z is - CH2C(R x )(R y )CHF2. A sub - embodiment of this aspect of the present disclosure is realized when Z is CH2C(F2)CHF2.

[0045] Another embodiment of Formula III is realized when X is a linking group that is W2. One sub - embodiment of Formula III is that W2 is

Chemical formula

Chemical formula

[0046] Another embodiment of formula III is realized when X is a linking group that is W3.

[0047] Another embodiment of formula III is realized when X is a linking group that is W4.

[0048] Another embodiment of formula III is realized when X is a linking group that is W5.

[0049] Another embodiment of formula III is realized when X is a linking group that is W6. One lower embodiment of formula III is where W6 is

Chemical formula

Chemical formula

[0050] One embodiment of formula III is realized when each R a is independently selected from hydrogen and methyl. Another embodiment of formula III is where R a is methyl. Another embodiment of formula III is where R a is hydrogen.

[0051] Another embodiment of the disclosure of formula III is realized when X is a linking group that is W7. One lower embodiment of formula III is where W7 is

Chemical formula

Chemical formula

[0052] Another embodiment of Formula III is realized when X is a linking group that is W8. A first sub - embodiment of Formula III is realized when X is a linking group that is W8 and X 4 is hydrogen. A first sub - embodiment of Formula III is realized when X is a linking group that is W8 and X 4 is [Chemical formula] It is realized when it is as follows.

[0053] One embodiment of Formula III is realized when each R a is independently selected from hydrogen and methyl. Another embodiment of Formula III is realized when R a is methyl. Another embodiment of Formula III is realized when R a is hydrogen.

[0054] In another embodiment, it may be desirable to synthesize the linker and then attach it to the drug or target moiety to form an ADC. In such an embodiment, the linker compound can act as an intermediate compound. The intermediate of the present disclosure is represented by a compound of Formula V.

Chemical formula

Chemical formula

Chemical formula

[0055] One embodiment of Formula V is realized when R k is an amino acid residue of 10 or fewer amino acids. Another sub - embodiment of this aspect of Formula V is realized when R k is selected from 1 - 8, 1 - 6, 1 - 4, 1 - 2, 2 - 8, and 2 - 6 amino acid residues. Another sub - embodiment of Formula V is realized when the amino acid residue of R k is derived from one or more of the same or different amino acids selected from glycine, alanine, phenylalanine, valine, lysine, citrulline, and sarcosine. Another sub - embodiment of Formula V is realized when the amino acid residue of R k is selected from glucamine, glucosamine, and galactosamine. Another sub - embodiment of Formula V is realized when the amino acid residue of R k is glucamine. Another sub - embodiment of Formula V is realized when the amino acid residue of R k is glucosamine. Another sub - embodiment of Formula V is realized when the amino acid residue of R kis realized when the amino acid residue is galactosamine.

[0056] One embodiment of Formula V is realized when R j is OH.

[0057] One embodiment of Formula V is realized when R j is NH2.

[0058] One embodiment of Formula V is realized when R j is -NHR k R g is realized when.

[0059] One embodiment of Formula V is realized when R j is -NHR k NH(CH2) n OR q is realized when. Sub - embodiments of this aspect of the present disclosure are realized when R k is an amino acid residue of 10 or fewer amino acids. Sub - embodiments of this aspect of the present disclosure are realized when the amino acid residue R k is derived from one or more of the same or different amino acids selected from glycine, alanine, phenylalanine, valine, lysine, citrulline, and sarcosine. Another sub - embodiment of this aspect of the present disclosure is realized when R k is realized when the amino acid residue is selected from glucosamine, glucosamine, and galactosamine. Another sub - embodiment of this aspect of the present disclosure is realized when R k is realized when the amino acid residue is glucosamine. Another sub - embodiment of this aspect of the present disclosure is realized when R k is realized when the amino acid residue is glucosamine. Another sub - embodiment of this aspect of the present disclosure is realized when R k is realized when the amino acid residue is galactosamine. Another sub - embodiment of this aspect of the present disclosure is realized when R q is hydrogen. Another sub - embodiment of this aspect of the present disclosure is realized when R q is C 1-6 alkyl. Another sub - embodiment of this aspect of the present disclosure is realized when R qis realized when it is methyl, ethyl, or propyl.

[0060] One embodiment of Formula V is where R j is -NHR k is NHCH2OC(O)CH3. A sub - embodiment of this aspect of the present disclosure is where R k is an amino acid residue of 10 or fewer amino acids. A sub - embodiment of this aspect of the present disclosure is that the amino acid residue R k is derived from one or more of the same or different amino acids selected from glycine, alanine, phenylalanine, valine, lysine, citrulline, and sarcosine. Another sub - embodiment of this aspect of the present disclosure is where R k the amino acid residue is selected from glucamine, glucosamine, and galactosamine. Another sub - embodiment of this aspect of the present disclosure is where R k the amino acid residue is glucamine. Another sub - embodiment of this aspect of the present disclosure is where R k the amino acid residue is glucosamine. Another sub - embodiment of this aspect of the present disclosure is where R k the amino acid residue is galactosamine.

[0061] One embodiment of Formula V is where R j is -NHX 1a (CH2)2C(O)R k is NHCH2OC(O)CH3. A sub - embodiment of this aspect of the present disclosure is where R k is an amino acid residue of 10 or fewer amino acids. A sub - embodiment of this aspect of the present disclosure is that the amino acid residue R k is derived from one or more of the same or different amino acids selected from glycine, alanine, phenylalanine, valine, lysine, citrulline, and sarcosine. Another sub - embodiment of this aspect of the present disclosure is where R k the amino acid residue is selected from glucamine, glucosamine, and galactosamine. Another sub - embodiment of this aspect of the present disclosure is where R kis realized when the amino acid residue is glucosamine. Another sub - embodiment of this aspect of the present disclosure is that R k is realized when the amino acid residue is glucosamine. Another sub - embodiment of this aspect of the present disclosure is that R k is realized when the amino acid residue is galactosamine. Another sub - embodiment of this aspect of the present disclosure is that X 1a is realized when it is PEG of 1 to 24 subunits. Another aspect of this embodiment is that X 1a is realized when it is PEG of 1 to 12 subunits.

[0062] One embodiment of Formula V is realized when R j is - NHR k NHR L Another sub - embodiment of this aspect of the present disclosure is that R k is realized when it is an amino acid residue of 10 or fewer amino acids. Another sub - embodiment of this aspect of the present disclosure is that the amino acid residue R k is realized when it is derived from one or more of the same or different amino acids selected from glycine, alanine, phenylalanine, valine, lysine, citrulline, and sarcosine. Another sub - embodiment of this aspect of the present disclosure is that R k is realized when the amino acid residue is selected from glucosamine, glucosamine, and galactosamine. Another sub - embodiment of this aspect of the present disclosure is that R k is realized when the amino acid residue is glucosamine. Another sub - embodiment of this aspect of the present disclosure is that R k is realized when the amino acid residue is glucosamine. Another sub - embodiment of this aspect of the present disclosure is that R k is realized when the amino acid residue is galactosamine. Another sub - embodiment of this aspect of the present disclosure is that R L is realized when it is (a). Another sub - embodiment of this aspect of the present disclosure is that R L is realized when it is (b). Another sub - embodiment of this aspect of the present disclosure is that R L is realized when it is (c). Another sub - embodiment of this aspect of the present disclosure is that R L is (a), (b), or (c), then R pis realized when it is NH2. Another sub - embodiment of this aspect of the present disclosure is that R L is realized when R p is

Chemical formula

[0063] One embodiment of Formula V is realized when R j is - NHX 1a R k NHR L is realized when it is. A sub - embodiment of this aspect of the present disclosure is realized when R k is an amino acid residue of 10 or fewer amino acids. A sub - embodiment of this aspect of the present disclosure is realized when the amino acid residue R k is derived from one or more of the same or different amino acids selected from glycine, alanine, phenylalanine, valine, lysine, citrulline, and sarcosine. Another sub - embodiment of this aspect of the present disclosure is realized when the amino acid residue of R k is selected from glucosamine, glucosamine, and galactosamine. Another sub - embodiment of this aspect of the present disclosure is realized when the amino acid residue of R k is glucosamine. Another sub - embodiment of this aspect of the present disclosure is realized when the amino acid residue of R k is glucosamine. Another sub - embodiment of this aspect of the present disclosure is realized when the amino acid residue of R k is galactosamine. Another sub - embodiment of this aspect of the present disclosure is realized when R L is (a). Another sub - embodiment of this aspect of the present disclosure is realized when R L is (b). Another sub - embodiment of this aspect of the present disclosure is realized when R L is (c). Another sub - embodiment of this aspect of the present disclosure is realized when R p is, when R L is (a), (b), or (c), is NH2. Another sub - embodiment of this aspect of the present disclosure is that R L is realized when R L is (a), (b), or (c) and Rp is [Chem.] is realized when it is the case. Another sub - embodiment of this aspect of the present disclosure is X 1a is realized when it is PEG of 1 to 24 subunits. Another aspect of this embodiment is X 1a is realized when it is PEG of 1 to 12 subunits.

[0064] One embodiment of formula V is when R j is - NHCH2O(CH2)2CH(OH)C(O)OH.

[0065] Another embodiment of formula V is when R j is - NHR k H, and R k is an amino acid residue of 10 or fewer amino acids. A sub - embodiment of this aspect of the present disclosure is that the amino acid residue R k is derived from one or more of the same or different amino acids selected from glycine, alanine, phenylalanine, valine, lysine, citrulline, and sarcosine. Another sub - embodiment of this aspect of the present disclosure is when the amino acid residue of R k is selected from glucamine, glucosamine, and galactosamine. Another sub - embodiment of this aspect of the present disclosure is when the amino acid residue of R k is glucamine. Another sub - embodiment of this aspect of the present disclosure is when the amino acid residue of R k is glucosamine. Another sub - embodiment of this aspect of the present disclosure is when the amino acid residue of R k is galactosamine.

[0066] Another embodiment of formula V is when R d1 is hydrogen.

[0067] Another embodiment of formula V is when R d1 is CH2NHC(O)X 1a Q, and X 1aand is realized when Q is as described herein.

[0068] Another embodiment of Formula V is where R d1 is -CH2NHC(O)X 2l is Q; X 2l is PEG or PEG - amino sugar of 1 to 24 -CH2CH2O - subunits, and is realized when the PEG in the PEG - amino sugar is polyethylene glycol of 1 to 24 -CH2CH2O - subunits. A lower - level embodiment of this aspect of the present disclosure is realized when PEG is 4 to 12 -CH2CH2O - subunits. Another lower - level embodiment of this aspect of the present disclosure is realized when PEG is 4 -CH2CH2O - subunits. Another lower - level embodiment of this aspect of the present disclosure is realized when PEG is 6 -CH2CH2O - subunits. Another lower - level embodiment of this aspect of the present disclosure is realized when PEG is 8 -CH2CH2O - subunits. Another lower - level embodiment of this aspect of the present disclosure is realized when PEG is 10 -CH2CH2O - subunits. Another lower - level embodiment of this aspect of the present disclosure is realized when PEG is 12 -CH2CH2O - subunits.

[0069] Another embodiment of Formula V is realized when. The amino sugar of the PEG - amino sugar is an open - chain sugar - derived amino alcohol or glycamine, and the sugar is selected from glucose, galactose, sorbital, mannitol, xylitol, arabitol, ribitol, glycerol, ethylene glycol, galactitol, etc. A lower - level embodiment of this aspect of Formula V is realized when the amino sugar of the PEG - amino sugar is a closed - chain amino alcohol selected from glucosamine, glucamine, galactosamine, etc. A non - limiting example of the amino - sugar is represented by diamino sugar S1.

Chemical formula

[0070] Another embodiment of Formula V is realized when Q is H.

[0071] Exemplary intermediate linker compounds of the present disclosure or salts thereof are described herein. One embodiment of the present disclosure is realized when the linker compound is selected from Table 1, or is a solvate or stereoisomer.

[0072] Table 1

Table 1

[0073] The compounds of the present disclosure are useful for binding to an antibody or other target site to produce an antibody - drug conjugate (ADC) or other target ligand conjugate for tumor indications. Accordingly, an embodiment of the present disclosure is represented by the ADC of Structural Formula IV.

Chemical formula

[0074] The linker payload can bind to cysteine residues of various ligand conjugates such as antibodies to generate antibody-drug conjugates (ADCs). The cysteine residues of the antibody form a bond with the reactive maleimide on the linker group.

[0075] The ligand can be any moiety having a free cysteine group, such as (but not limited to) an antibody, protein, peptide, polypeptide, or modified antibody modified to provide a free cysteine. This one aspect is realized when the ligand is an antibody, preferably a full antibody. The ligand targets a specific population of target cells with which the ligand interacts and serves to present the drug thereto. Suitable ligands include, for example, antibodies such as full-length antibodies and antigen-binding fragments thereof, interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules (such as, but not limited to, transferrin), or any other cell-binding molecule or substance including small molecules and peptides. The ligand can be, for example, a non-antibody protein targeting agent.

[0076] When the complex contains a non-immunoreactive protein, polypeptide, or peptide ligand instead of an antibody, useful non-immunoreactive proteins, polypeptides, or peptide ligands include, but are not limited to, transferrin, epidermal growth factor (“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors (“TGF”), such as TGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin and insulin-like growth factors I and II, somatostatin, lectin, and apolipoproteins derived from low density lipoprotein.

[0077] Particularly preferred ligands are antibodies, including whole antibodies. In fact, in any of the embodiments described herein, the ligand can be an antibody. Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies against specific antigenic determinants (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) against a target antigen can be produced using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture.

[0078] Furthermore, recombinant antibodies such as chimeric antibodies and humanized monoclonal antibodies, which contain both human and non-human portions, can be created using standard recombinant DNA techniques and are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, for example, antibodies having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region (see, for example, U.S. Patent Nos. 4,816,567 and 4,816,397, which are hereby incorporated by reference in their entirety). Humanized antibodies are antibody molecules derived from non-human species and have one or more complementarity-determining regions (CDRs) from non-human species and a framework region from a human immunoglobulin molecule (see, for example, U.S. Patent No. 5,585,089, which is hereby incorporated by reference in its entirety). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using the methods described in International Publication No. WO87 / 02671, European Patent Publication No. 0184187 (each of which is hereby incorporated by reference in its entirety).

[0079] Fully human antibodies are particularly desirable and can be generated using transgenic mice that are unable to express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes.

[0080] Antibodies include analogs and derivatives modified by covalent attachment of any type of molecule, provided that the antibody retains its antigen-binding immunospecificity. For example, antibody derivatives and analogs include, but are not limited to, those further modified by, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to a cell antibody or another protein, etc. Any of a number of chemical modifications can be carried out by known techniques such as specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. (not limited to these). Furthermore, an analog or derivative can contain one or more non-natural amino acids.

[0081] In certain embodiments, known antibodies for cancer treatment can be used. Antibodies immunospecific for cancer cell antigens can be obtained commercially or can be produced by any method known to those skilled in the art, such as recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, from literature publications, or by routine cloning and sequencing.

[0082] In another particular embodiment, antibodies for the treatment of autoimmune diseases are used according to the compositions and methods of the present disclosure. Antibodies immunospecific for the antigens of cells that produce autoantibodies can be obtained from any source (e.g., university scientists or companies) or can be generated by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques.

[0083] In another embodiment, it may be desirable to attach the linker component to the ligand (such as an antibody) before attaching the camptothecin-derived drug component of the ADC. For example, in embodiments where a thiol-containing substituent (such as cysteine) is used to attach the camptothecin-derived drug component of the ADC, it may be desirable to attach the linker component to the ligand (such as an antibody) before attaching the camptothecin-derived drug component of the ADC.

[0084] One aspect of the present disclosure relates to a composition or pharmaceutical composition comprising a compound of formula I, II, III, IV, V or a salt, pharmaceutically acceptable salt, or solvate thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0085] Another aspect of the present disclosure relates to a composition or pharmaceutical composition comprising a compound of formula I, II, III, IV, or V described herein, or a tautomer, meso form, racemate, enantiomer, diastereomer, or mixture thereof, or a salt thereof, or a pharmaceutically acceptable salt, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0086] Another aspect of the present disclosure relates to compounds of Formulas I, II, III, IV, V described herein, or tautomers, meso forms, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof for use as a medicament or pharmaceutical ingredient.

[0087] Another aspect of the present disclosure relates to pharmaceutical compositions in the manufacture of a medicament for treating or preventing tumors, comprising a compound of Formulas I, II, III, IV, V described herein, or tautomers, meso forms, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof.

[0088] Another aspect of the present disclosure relates to intermediate linker compounds and compositions containing the same. Examples of intermediate linker compounds are represented by W1, W2, W3, W4, W5, W6, W7, W8 of X described herein.

[0089] In another embodiment, the compounds of the present disclosure include the compounds specifically identified as examples in the following table herein, and pharmaceutically acceptable salts thereof.

[0090] The compounds of the present disclosure may contain one or more chiral centers and can thus exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Depending on the nature of the various substituents on the molecule, additional chiral centers may be present. Since each such chiral center independently gives rise to two optical isomers, all possible optical isomers and diastereomers as mixtures and as pure or partially purified compounds are intended to be included within the scope of the present disclosure. Unless a specific stereochemistry is shown, the present disclosure is meant to embrace all such isomers of these compounds.

[0091] The independent synthesis or chromatographic separation of these diastereomers can be carried out as known in the art by appropriately modifying the methods disclosed herein. Their absolute stereochemistry can be determined, among other methods, by X-ray crystal structure analysis of crystalline products or crystalline intermediates derivatized with reagents containing chiral centers of known absolute configuration, if necessary.

[0092] If necessary, the racemic mixture of the compound can be separated to isolate the individual enantiomers. The separation can be carried out by methods well known in the art, for example, by coupling the racemic mixture of the compound to an enantiomerically pure compound to form a mixture of diastereomers, and then separating the individual diastereomers by standard methods such as fractional crystallization or chromatography. The coupling reaction is often the formation of a salt using an enantiomerically pure acid or base. Subsequently, the diastereomeric derivative can be converted to the pure enantiomer by cleavage of the added chiral residue. The racemic mixture of the compound can also be directly separated by a chromatographic method using a chiral stationary phase, which is a method known in the art.

[0093] Alternatively, any enantiomer of the compound can be obtained by stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods known in the art.

[0094] In the compounds of formula I, II, III, IV, or V, the atoms may exhibit the natural isotope abundance ratios, or one or more of the atoms may be artificially enriched with a specific isotope having the same atomic number but a different atomic mass or mass number than the atomic mass or mass number predominantly found in nature. The present disclosure can include all appropriate isotope variations of the compounds of general formula I, II, III, IV, V. For example, different isotopic forms of hydrogen (H) include protium ( 1 H) and deuterium ( 2There are, for example, protium (H). Protium is the dominant hydrogen isotope found in nature. Enriching deuterium can provide certain therapeutic advantages such as an increase in the in-vivo half-life or a reduction in the required dosage, or it can make it possible to provide compounds useful as standards for characterizing biological samples. For the purposes of this disclosure, when a compound is said to be "not deuterated", it means that it is not enriched in deuterium beyond the background state. Isotope-enriched compounds included in general formula I, II, III, IV, or V or II can be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the schemes and examples herein using appropriate isotope-enriched reagents and / or intermediates without performing unnecessary experiments.

[0095] If the compounds of the present disclosure can form tautomers, all such tautomers are also included within the scope of the present disclosure. For example, a compound containing a carbonyl-CH2C(O)- group (keto form) may undergo tautomerization to form a hydroxyl-CH=C(OH)- group (enol form). If both the keto form and the enol form are present, it is included within the scope of the present disclosure.

[0096] If any variable element (e.g., R 5 etc.) appears multiple times in any of the components, its definition in each occurrence is independent of any other occurrence. Also, combinations of substituents and variable elements are permitted only if such combinations result in stable compounds. A line drawn from a substituent to a ring system indicates that the indicated bond can be attached to any of the ring atoms that can be substituted. When the ring system is bicyclic, the bond is intended to be attached to any appropriate atom on either ring of the bicyclic moiety.

[0097] One of ordinary skill in the art would understand that by incorporating one or more silicon (Si) atoms in place of one or more carbon atoms into the compounds of the present disclosure, compounds can be provided that are chemically stable and can be readily synthesized by techniques known in the art from readily available starting materials. Since carbon and silicon have different covalent radii, when comparing similar C-element bonds and Si-element bonds, differences in bond distance and configuration occur. Due to these differences, subtle changes in the size and shape of silicon-containing compounds occur compared to carbon. One of ordinary skill in the art would understand that differences in size and shape can potentially result in subtle or dramatic changes in efficacy, solubility, lack of off-target activity, packaging properties, etc. (Diass, J. O. et al. Organometallics (2006) 5:1188-1198; Showell, G. A. et al. Bioorganic & Medicinal Chemistry Letters (2006) 16:2555-2558).

[0098] It is understood that the substituents and substitution patterns on the compounds of the present disclosure are selected by one of ordinary skill in the art, are chemically stable, and can be readily synthesized from readily available starting materials by techniques known in the art and the methods set forth below. When the substituent itself is substituted with a plurality of groups, it is understood that these plurality of groups may be on the same carbon or different carbons as long as a stable structure is obtained. The phrase "optionally substituted with one or more substituents" should be understood to mean that the group in question may be unsubstituted or optionally substituted with one or more substituents.

[0099] Absolute stereochemistry is shown by using hash line bonds and wedge-shaped solid line bonds as shown in Illus-I and Illus-II. Thus, the methyl group in Illus-I projects out of the page, the ethyl group in Illus-II is below the page, and the cyclohexene ring is within the page. It is assumed that the hydrogen on the same carbon as the methyl group in Illus-I is below the page and the hydrogen on the same carbon as the ethyl group in Illus-II projects out of the page. The rule is the same when both a hash line and a solid line rectangle are attached to the same carbon as in Illus-III, where the methyl group projects out of the page, the ethyl group is below the page, and the cyclohexene ring is in the page.

Chem.

[0100] As used herein, unless otherwise noted, the following terms have the following meanings.

[0101] The expression "at least one" when used with respect to a substituent on a compound or on a moiety attached to the core structure of a compound means that one substituent of the designated group of substituents is present and that multiple substituents can be attached to any chemically accessible bond point of that core.

[0102] Regardless of whether it is used with respect to a substituent on a compound or a component of a pharmaceutical composition, the expression "one or more" means the same as "at least one".

[0103] "Effective amount" or "therapeutically effective amount" refers to an amount of at least one compound of the present disclosure, or a composition comprising at least one compound of the present disclosure, that is effective in treating or inhibiting a disease or condition described herein, thereby producing a desired therapeutic, ameliorating, inhibiting, or prophylactic effect. For example, in treating a central nervous system disease or disorder with one or more of the compounds described herein, the "effective amount" (or "therapeutically effective amount") can be determined, for example, by analysis of pharmacodynamic markers or clinical evaluations of a patient suffering from the condition, and is suitable for managing, alleviating, ameliorating, or treating the condition, or alleviating, ameliorating, reducing, or eradicating one or more symptoms caused by the condition, and / or long-term stabilization of the condition, etc., and means providing an amount of a compound of Formula IV that produces a therapeutic response in a patient suffering from a central nervous system disease or disorder ("condition").

[0104] "Patient" and "subject" mean an animal, such as a mammal (e.g., human), preferably a human.

[0105] "Prodrug" means a compound that is rapidly converted to the parent compound, for example, by hydrolysis in blood in vivo, such as the conversion of a prodrug of Formula IV to a compound of Formula A or a salt thereof. Detailed discussions are in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol.14 of the A.C.S. Symposium Series and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference. The scope of the present disclosure includes prodrugs of the novel compounds of the present disclosure.

[0106] The term "substituted" means that one or more of the recited substituents can occupy one or more of the attachment positions on the substrate that are typically occupied by "-H", provided that such substitution does not exceed the normal valence rules for the atoms in the bonding arrangement represented by the substrate, and that the substitution provides a finally stable compound, i.e., such substitution does not provide a compound having geminal or adjacent mutually reactive substituents, and that the substitution provides a compound having sufficient robustness to withstand isolation to a useful purity from the reaction mixture.

[0107] When appropriate substitution of a moiety is described (e.g., "optionally substituted"), this term means that, when substitution is present, one or more of the recited ones (substituents for the designated substrate) can be present on the substrate at the attachment positions that are normally occupied by the initially set substituents in accordance with the definition of "substituted" presented herein. For example, the initially set substituent on a carbon atom of an alkyl moiety is a hydrogen atom, and any substituent can replace that initially set substituent.

[0108] As used herein, unless otherwise indicated, the terms used to describe a moiety, whether or not they include the overall definition of a variable portion of the structural representation of the compounds of the present disclosure or a substituent attached to the variable portion of the structural representation of a group of compounds of the present disclosure, have the following meanings, and unless otherwise indicated, when the term is used individually or as a component of another term, the definition of each term (i.e., moiety or substituent) applies (e.g., the definition of aryl is the same for aryl and for aryl moieties in arylalkyl, alkylaryl, arylalkynyl moieties, etc.). A moiety is equally described herein by structure, typographical representation, or chemical terms without any intention of a difference in meaning. For example, an "acyl" substituent is represented by the term "acyl", or by the typographical representation "R′-(C=O)-" or "R′-C(O)-", or by the structural representation:

Chemical formula

[0109] The PEG provided herein includes one or more polyethylene glycol chains containing repeating -CH2CH2O- subunits. The polyethylene glycol chains can be linked to each other, for example, in a linear, branched, or star configuration. Another embodiment of the present disclosure is realized when the PEG unit contains at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, at least 20 subunits, at least 21 subunits, at least 22 subunits, at least 23 subunits, or at least 24 subunits. A PEG moiety having four repeating -CH2CH2O- can be referred to as -PEG4-, and similarly, a PEG moiety having eight repeating -CH2CH2O- units may be referred to as -PEG8-.

[0110] As used herein, the term "antibody" is used in the broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity, provided that the antibody fragment has the required number of binding sites for the drug-linker. The native form of an antibody is a tetramer composed of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) together are primarily responsible for binding to the antigen. The light and heavy chain variable domains consist of framework regions interrupted by three hypervariable regions, also called "complementary determining regions" or "CDRs". The constant regions are recognized by and can interact with the immune system (see, e.g., Janeway et al., 2001, Immuno. Biology, 5th Ed., Garland Publishing, New York). Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Antibodies can be derived from any suitable species. In some embodiments, the antibodies are of human or murine origin. Antibodies can be, for example, human, humanized, or chimeric.

[0111] The term "alkyl" (including the alkyl portion of other moieties such as trifluoromethyl-alkyl- and alkoxy-) means a straight or branched aliphatic hydrocarbon moiety containing up to about 20 carbon atoms (e.g., the designation "C 1-20 -alkyl" indicates an aliphatic hydrocarbon moiety of 1 to 20 carbon atoms.). In some embodiments, for example, an alkyl moiety of 8 or fewer carbon atoms is herein referred to as "C 1-8Unless the term "alkyl" has been modified to indicate that shorter chains are contemplated, as may be the case when it is referred to as "-alkyl", alkyl preferably contains about 10 or fewer carbon atoms. When the term "alkyl" is shown with two hyphens (i.e., "-alkyl-"), this indicates that the attachment of the alkyl moiety is in such a form that the alkyl moiety is attached to a substituent on either side of it. For example, "-alkyl-OH" indicates an alkyl moiety to which a hydroxyl moiety is linked to a substrate.

[0112] As used herein, when the term "alkyl" is modified by "substituted" or "optionally substituted", this means that one or more CH bonds in the alkyl sub-group are replaced or may be replaced by substituents attached to the alkyl substrate specified in the definition of that moiety.

[0113] The term "cycloalkyl" means a moiety having a main hydrocarbon chain forming a monocyclic or bicyclic aliphatic moiety containing from at least 3 carbon atoms (the minimum number necessary to provide a monocyclic moiety) up to a specified number of carbon atoms, generally 8 for a monocyclic moiety and 10 for a bicyclic moiety including a spirocyclic moiety. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. The term "cycloalkyl" also includes non-aromatic fused polycyclic systems containing up to 20 carbon atoms which may be substituted as defined herein for "alkyl". Suitable polycyclic cycloalkyls are, for example, 1-decalin; norbornyl; adamantyl etc., but are not limited thereto.

[0114] As used herein, the term "alkylene" refers to a saturated straight-chain or branched-chain aliphatic having two residues obtained by removing two hydrogen atoms from the same or different carbon atoms of the parent alkane. Alkylene is a straight-chain or branched group having 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Non-limiting examples include methylene, ethylene, propylene, butylene, pentylene, and the like.

[0115] When the structural formula represents a bond between a moiety and a substrate using a bond line ending in the center of the structure, the following expression:

Chemical formula

[0116] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or polycyclic condensed ring having a conjugated π electron system (i.e., each ring in the system shares a pair of adjacent carbon atoms with another ring in the system), preferably 6- to 10-membered aryl, such as phenyl and naphthyl, and preferably phenyl.

[0117] The term "heteroaryl" refers to an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms in the monocyclic case, 1 to 6 heteroatoms in the bicyclic case, or 1 to 9 heteroatoms in the tricyclic case, wherein the heteroatoms are selected from O, N, or S (e.g., 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms in the carbon atoms and the monocyclic, bicyclic, or tricyclic cases, respectively). Non-limiting examples of heteroaryl include pyridyl, pyrazolyl, pyrimidinyl, furanyl, oxazolyl, triazolyl, oxadiazolyl, and thiophenyl. The heteroaryl groups described herein can also include fused rings sharing a common carbon-carbon bond.

[0118] "Heterocyclic ring" (or heterocyclic ring alkyl) means a non-aromatic saturated monocyclic or polycyclic ring system containing 3 to 10 ring atoms, preferably 5 to 10 ring atoms, of which one or more of the atoms in the ring system are elements other than carbon, such as nitrogen (e.g., piperidyl- or pyrrolidinyl), oxygen (e.g., furanyl and tetrahydropyranyl) or sulfur (e.g., tetrahydrothiophenyl and tetrahydrothiopyranyl), provided that the moiety does not contain adjacent oxygen and / or sulfur atoms present in the ring system; preferred heterocyclic moieties contain 5 to 6 ring atoms; the prefixes aza, oxa or thia before the heterocyclic stem name mean that at least one nitrogen, oxygen or sulfur atom is present as a ring atom respectively; the heterocyclic ring may be substituted by one or more independently selected substituents; the nitrogen or sulfur atom of the heterocyclic ring may be oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide (SO2); suitable monocyclic heterocyclic rings include piperidyl, pyrrolidinyl, piperazinyl, morpholinyl-

Chemical Structure

Chemical Structure

[0119] The term "solvate" refers to a pharmaceutically acceptable solvate formed by a compound of the present disclosure and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate, etc.

[0120] The term "halogen" means fluorine, chlorine, bromine or iodine; preferably, halogen is fluorine, chlorine and bromine when the term is used without further qualification, a substituent that is a halogen atom means -F, -Cl, -Br or -I, "halo" means a fluoro, chloro, bromo or iodo substituent attached to the defined moiety, for example "haloalkyl" means alkyl as defined above, where typically one or more of the bonding positions on the alkyl portion that are occupied by hydrogen atoms are instead occupied by halo groups, perhaloalkyl (or "fully halogenated" alkyl) means that all bonding positions not involved in attaching an alkyl substituent to the substrate are occupied by halogen, for example when alkyl is selected to be methyl, the term perfluoroalkyl means -CF3.

[0121] The terms "hydroxyl" and "hydroxy" mean an HO- group, "hydroxyalkyl" means a substituent of the formula: "HO-alkyl-" or equivalently "-alkyl-OH" (wherein the alkyl group is attached to the substrate and may or may not be substituted as defined above); preferred hydroxyalkyl moieties include lower alkyl; examples of suitable hydroxyalkyl groups include, but are not limited to, hydroxymethyl and 2-hydroxyethyl.

[0122] The bond order is indicated by a hyphen when the moiety is represented in the text, for example -alkyl indicates a single bond between the substrate and the alkyl moiety, -alkyl-X indicates that the alkyl group is attaching the "X" substituent to the substrate, and in a structural representation, the bond order is indicated by a wavy line terminating the bond representation, for example:

Chemical formula

[0123] The line of "-" as a bond usually indicates a mixture of possible isomers including, for example, (R)- and (S)-stereochemical configurations, or either of those isomers.

[0124] As used herein, the term "DAR" or "drug-antibody ratio" refers to the average number of linker / drug moieties bound to the antibodies present in the composition. In the case of a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is the average of all "p" of the individual antibody-drug conjugate molecules present in the composition, and this average is expressed as a decimal. Thus, in some embodiments for a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is a decimal from 0 to 24, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, and 0 to 1. In additional embodiments, in the case of a composition comprising an antibody-drug conjugate of the present invention, the DAR of the composition is a decimal from 1 to 4, 2 to 5, 3 to 6, 4 to 7, 5 to 8, and 6 to 8. In other embodiments, in the case of a composition comprising an antibody-drug conjugate of the present invention, the DAR of the composition is a decimal from 1 to 3, 2 to 4, 3 to 5, 4 to 6, 5 to 7, and 6 to 8. In further embodiments, in the case of a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is a decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, and 7 to 8. In certain embodiments, the DAR of the composition is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. The term "composition" as used above is understood to include pharmaceutical compositions. The average DAR can be measured by various conventional means such as UV spectroscopy, mass spectrometry, ELISA assay, radiometric methods, hydrophobic interaction chromatography (HIC), electrophoresis, HPLC, and the like.

[0125] Further, in structures containing multiple stereocenters, in order to represent known relative configurations, boldface that is not wedge-shaped or dashed lines that are not wedge-shaped are used. For example, [Chemistry] and, on the one hand [Chemistry] is as follows.

[0126] In all cases, the compound name is associated with the depicted structure and is intended to capture each possible stereochemical permutation for a given structural isomer based on the synthetic operations used in its preparation. A list of the individual stereoisomers used in combination indicates that the presented compound (e.g., "Example Number") was isolated as a single stereoisomer and that the identity of that stereoisomer corresponds to one of the listed possible configurations. A list of the individual stereoisomers used in combination indicates that the presented compound was isolated as a racemic mixture or a mixture of diastereomers.

[0127] A specific absolute configuration is indicated using a wedge-shaped boldface or a wedge-shaped slash. Unless a specific absolute configuration is indicated, this disclosure is meant to encompass all such stereoisomeric forms of these compounds.

[0128] In this specification, when a ring system has a plurality of oxygen atoms and / or sulfur atoms, adjacent oxygen atoms and / or sulfur atoms shall not be present in the ring system.

[0129] As is well known in the art, a bond drawn from a particular atom with no portion drawn at the end of the bond indicates a methyl group attached to that atom via that bond, unless otherwise indicated. For example, it is as follows. [Chemistry] In the text, diagrams, examples, structural formulas, and any tables in this specification, valences that are not satisfied are assumed to have the number of hydrogen atoms or multiple hydrogen atoms necessary to satisfy the valence.

[0130] The compounds of the present disclosure above 1 may exist as solvates or may be converted into solvates. The production of solvates is known. Thus, for example, M. Caira et al, J. Pharmaceutical Sci., 93(3), 601-611 (2004) describes the production of solvates of the antifungal agent fluconazole from ethyl acetate and from water. Similar production of solvates and hemisolvates, such as hydrates (where the solvent is water or is aqueous), etc. are described in E. C. van Tonder et al, AAPS PharmSciTech., 5(1), article 12(2004); and A. L. Bingham et al, Chem. Commun., 603-604(2001). In a representative non-limiting process, the compound of the present invention is dissolved in a desired amount of a desired solvent (e.g., an organic solvent, an aqueous solvent, water or a mixture of two or more thereof) at a temperature higher than ambient temperature, and the solution is cooled at a rate sufficient to form crystals, in the presence or absence of an antisolvent, and then isolated by standard methods. The presence of the solvent (such as water) in the crystals as a solvate (or, if water is incorporated into the crystal form, a hydrate) is indicated by analytical techniques such as IR spectroscopy measurement.

[0131] The present disclosure also includes the compounds of the present disclosure in isolated and purified forms obtained by conventional techniques. Compounds of Formula I, Formula II, Formula III, Formula IV, and Formula V, as well as polymorphic forms of salts, solvates, and prodrugs of the compounds of Formula I, Formula II, Formula III, Formula IV, and Formula V are intended to be included in the present disclosure. Certain compounds of the present disclosure may exist in different isomeric forms (e.g., enantiomers, diastereomers, atropisomers). The compounds of the present invention include all of their isomeric forms, both in pure form and in mixtures of two or more such as racemic mixtures.

[0132] Similarly, unless otherwise indicated, the provision of a structural representation of any tautomeric form of a compound that exhibits tautomerism includes all such tautomers of that compound. Thus, if the compounds of the present disclosure can exist in different tautomeric forms or in an equilibrium form between such forms, all such forms of the compound are encompassed by and within the scope of the present disclosure. Examples of such tautomers include, but are not limited to, keto / enol tautomers, imine-enamine tautomers, and heteroaromatic forms such as, for example, the following moieties.

Chemical formula

[0133] The salts of the disclosed compounds can be pharmaceutically acceptable salts or non-pharmaceutically acceptable salts that are useful in the preparation of the compounds according to the present disclosure.

[0134] As used herein, "pharmaceutically acceptable salt" refers to derivatives in which the parent compound is modified by making its acid or base salts. Salts in solid form can exist in multiple crystal structures and may be in the form of hydrates. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids such as formic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, etc. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, iron(III), iron(II), lithium, magnesium, manganese salts, manganous, potassium, sodium, zinc, etc.

[0135] When the compounds of the present disclosure are basic, the salts can be prepared from pharmaceutically acceptable non-toxic acids such as inorganic and organic acids. Such acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc. In one embodiment of the present disclosure, the salts are citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, fumaric acid, and tartaric acid. Similarly, salts of acidic compounds are formed by reaction with appropriate inorganic or organic bases.

[0136] The term "adjacent" means being bonded to the same carbon atom.

[0137] The term "chemotherapeutic agent" refers to compounds that can be used in the treatment of tumors. This definition includes antihormonal agents that have the effect of regulating, reducing, blocking, or inhibiting the effects of hormones that promote cancer growth, and these hormonal agents are often in the form of systemic therapy or holistic therapy. These can be hormones. Examples of chemotherapeutic agents include alkylating agents such as thiotepa; cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benaodopa, carbocone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and aziridine and methylamelamine including trimethylolomelamine; nitrogen mustards such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, nitrobine hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosureas such as carmustine, chloroozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, calicheamicin, carabicin, chromomycin, caminomycin, cardinophilin, chromomycins, daunorubicin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptomycin, tubercidin, ubenimex, dinostatin, zorubicin, etc.; antimetabolites, such as methotrexate, 5-fluorouracil (5-FU), etc.; folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate, etc.; pterin analogs, such as fludarabine, 6-mercaptopterin, thiomethopterin, thioguanopterin, etc.; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, 5-FU, etc.; androgens, such as calusterone, drostanolone propionate, epitioestanol, mepitiostane, testolactone, etc.; antiadrenalines, such as aminoglutethimide, mitotane, trilostane, etc.; folic acid replenishers, such as frolinic acid, etc.; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diacontin; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine;Pintostatin; Phenamet; Pirarubicin; Podophyllinic acid; 2-Ethylhydrazine; Procarbazine; PSK (registered trademark); Razoxane; Sizofiran; Spirogermanium; Tenazone acid; Triazicone; 2,2′,2″-Trichloroethylamine; Urethane; Vin desine; Dacarbazine; Mannomustine; Mitobronitol; Dibromodulcitol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxanes, such as paclitaxel (TAXOL (registered trademark), Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE (registered trademark), Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, such as cisplatin and carboplatin, etc.; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunorubicin; Aminopterin; Zoladex; Ibandronate; CPT-11; Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoic acid, esperamicins; Capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances, etc. This definition also includes hormonal agents that can regulate or inhibit the effect of hormones on tumors, such as anti-estrogen agents, such as tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and fairston; and anti-androgen agents, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin, etc.; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

[0138] As used herein, the terms "treating" or "treatment" (e.g., of a disease, disorder, or condition or related symptoms, which may be collectively or individually referred to as "indications") mean suppressing a disease, disorder or condition, i.e., arresting or reducing the development or progression of a disease or its physiological processes or clinical symptoms, or alleviating a disease, i.e., causing regression of a disease or its physiological processes or progression and / or clinical symptoms.

[0139] As would be apparent to one of ordinary skill in the art, subjects treated by the methods described herein are generally mammals, including humans and non-human animals (e.g., laboratory animals and pet animals). The term "therapeutically effective amount" means the amount of a compound of interest that elicits the physiological or medical response of a tissue, system, animal or human that is sought by a researcher, veterinarian, physician or other clinical personnel.

[0140] As used herein, the term "composition" is intended to encompass a product comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof and a specified amount of one or more additional specified components, as well as any product directly or indirectly resulting from a combination of specified amounts of the specified components. Such terms with respect to pharmaceutical compositions encompass products comprising an active ingredient, which comprises a compound of the present disclosure or a pharmaceutically acceptable salt thereof and optionally one or more additional active ingredients, and an inert ingredient which constitutes a carrier, as well as any product directly or indirectly resulting from a combination, complex formation or aggregation of two or more of the foregoing components, or any product directly or indirectly resulting from dissociation of one or more of the foregoing components, or any product directly or indirectly resulting from other types of reaction or interaction of one or more of the foregoing components. Thus, pharmaceutical compositions of the present disclosure encompass any composition produced by mixing a compound of the present disclosure or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the carrier, diluent, or excipient is compatible with the other ingredients of the formulation and not harmful to the recipient thereof.

[0141] As described above, additional embodiments of the present disclosure each relate to a method of treating a disease, disorder, or condition, or one or more symptoms thereof (a “condition”), comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising said compound or a salt thereof.

[0142] In another embodiment, the invention relates to a method of manufacturing a medicament for use in a subject, comprising combining a compound of the present disclosure or a pharmaceutically acceptable salt thereof with a pharmaceutical carrier or diluent.

[0143] One such embodiment is a method of treating or preventing cancer selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or recurrent anaplastic large cell lymphoma) in a subject in need of treatment, comprising administering to the subject in need of treatment a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising said compound, a salt or solvate thereof. In one such embodiment, the subject is human.

[0144] Another aspect of the present disclosure relates to a method of treating and / or preventing a tumor, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a compound according to the present disclosure.

[0145] Combination with additional therapeutic agents is also contemplated in the methods of the present invention. For example, the combination of a compound of Formula IV of the present disclosure with a PPAR-G (i.e., PPAR-gamma) agonist and a PPAR-D (i.e., PPAR-delta) agonist is useful for the treatment of certain malignancies. PPAR-G and PPAR-D are nuclear peroxisome proliferator-activated receptors G and D, respectively. PPAR-G agonists have been shown to inhibit the angiogenic response to VEGF in vitro, and both troglitazone and rosiglitazone maleate inhibit the development of retinal angiogenesis in mice (Arch. Ophthamol. 2001;119:709-717). Examples of PPAR-D agonists and PPAR-G / A agonists include thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, pioglitazone, etc.), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid (disclosed in USSN09 / 782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchroman-2-carboxylic acid (disclosed in USSN60 / 235,708 and 60 / 244,697), or their pharmaceutically acceptable salts, etc., but are not limited thereto.

[0146] Another embodiment of the present disclosure is the use of a compound of Formula IV in combination with gene therapy for the treatment of cancer. For an overview of gene strategies for the treatment of cancer, see Hall et al., (Am. J. Hum. Genet. 61:785-789, 1997) and Kufe et al., (Cancer Medicine, 5th Ed, pp876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressor gene. Examples of such genes include p53 (see, for example, U.S. Patent No. 6,069,134), which can be delivered by recombinant virus-mediated gene transfer, uPA / uPAR antagonists (″Adenovirus-Mediated Delivery of a uPA / uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice,″ Gene Therapy, August 1998;5(8):1105-13), and interferon gamma (J. Immunol. 2000;164:217-222), J.Immunol.2000, 164:217-222), but are not limited thereto.

[0147] The compounds of Formula IV of the present disclosure can also be administered in combination with inhibitors of intrinsic multidrug resistance (MDR), particularly MDR associated with high-level expression of transport proteins. Such MDR inhibitors include inhibitors of p-glycoprotein (P-gp) such as LY335979, XR9576, OC144-093, R101922, VX853, PSC833 (valspodar), or their pharmaceutically acceptable salts, and the like.

[0148] The compounds of Formula IV of the present disclosure can also be administered together with immunopotentiators such as levamisole, isoprinosine, zidaxine, or their pharmaceutically acceptable salts.

[0149] The compounds of formula IV of the present disclosure may also be useful in treating or preventing cancer in combination with P450 inhibitors such as foreign substances, quinidine, tyramine, ketoconazole, testosterone, quinine, methylapone, caffeine, phenelzine, doxorubicin, troleandomycin, cyclobenzaprine, erythromycin, cocaine, frafilin, cimetidine, dextromethorphan, ritonavir, indinavir, amprenavir, diltiazem, terfenadine, verapamil, cortisol, itraconazole, mibefradil, nefazodone and nelfinavir, or pharmaceutically acceptable salts thereof.

[0150] The compounds of formula IV of the present disclosure may also be useful in treating or preventing cancer in combination with Pgp and / or BCRP inhibitors such as cyclosporin A, PSC833, GF120918, Cremophor EL, fumitremorgin C, Ko132, Ko134, Iressa, imatinib mesylate, EKI-785, Cl1033, novobiocin, diethylstilbestrol, tamoxifen, reserpine, VX-710, triptolide A, flavonoids, ritonavir, saquinavir, nelfinavir, omeprazole, quinidine, verapamil, terfenadine, ketoconazole, nifidepine, FK506, amiodarone, XR9576, indinavir, amprenavir, cortisol, testosterone, LY335979, OC144-093, erythromycin, vincristine, digoxin and talinolol, or pharmaceutically acceptable salts thereof.

[0151] The compounds of formula IV of the present disclosure may also be useful in treating or preventing cancers such as osteosarcoma in combination with bisphosphonates such as (but not limited to) etidronate (Didronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or simadronate, clodronate, EB-1053, minodronate, neridronate, pyridronate and tiludronate.

[0152] The compounds of formula IV of the present disclosure may also be useful in treating or preventing breast cancer when used in combination with an aromatase inhibitor. Examples of aromatase inhibitors include, but are not limited to, anastrozole, letrozole, and exemestane, or pharmaceutically acceptable salts thereof.

[0153] The compounds of formula IV of the present disclosure may also be useful in treating or preventing cancer when used in combination with an siRNA therapeutic agent.

[0154] The compounds of formula IV of the present disclosure can also be administered in combination with a γ-secretase inhibitor and / or an inhibitor of NOTCH signaling. Such inhibitors include the compounds described in WO01 / 90084, WO02 / 30912, WO01 / 70677, WO03 / 013506, WO02 / 36555, WO03 / 093252, WO03 / 093264, WO03 / 093251, WO03 / 093253, WO2004 / 039800, WO2004 / 039370, WO2005 / 030731, WO2005 / 014553, USSN10 / 957,251, WO2004 / 089911, WO02 / 081435, WO02 / 081433, WO03 / 018543, WO2004 / 031137, WO2004 / 031139, WO2004 / 031138, WO2004 / 101538, WO2004 / 101539, and WO02 / 47671 (including LY-450139), or pharmaceutically acceptable salts thereof.

[0155] 1. In one embodiment, specific anticancer agents useful in the combination therapy of the present invention include pembrolizumab (Keytruda®), abarelix; (Plenaxis Depot®); aldesleukin (Prokine®); aldesleukin (Proleukin®); alemtuzumab (Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole (Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine (Vidaza®); bevacizumab (Avastin®); bexarotene capsule (Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); busulfan intravenous injection (Busulfex®); busulfan oral (Myleran®); calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®); carmustine + Polifeprosan 20 implant (Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (Cytoxan injection®); cyclophosphamide tablets (Cytoxan tablets®); cytarabine (Cytosar-U®); cytarabine liposome (DepoCyt®);Dacarbazine (DTIC-Dome®); Dactinomycin, Actinomycin D (Cosmegen®); Darbepoetin alfa (Aranesp®); Daunorubicin Liposome (DanuoXome®); Daunorubicin, Daunomycin (Daunorubicin®); Daunorubicin, Daunomycin (Cerubidine®); Denileukin diftitox (Ontak®); Dexrazoxane (Zinecard®); Docetaxel (Taxotere®); Doxorubicin (Adriamycin PFS®); Doxorubicin (Adriamycin®, Rubex®); Doxorubicin (Adriamycin PFS Injection®); Doxorubicin Liposome (Doxil®); Dromostanolone Propionate (Dromostanolone®); Dromostanolone Propionate (Masterone Injection®); Elliot B Solution (Elliot B Solution®); Epirubicin (Ellence®); Epoetin alfa (epogen®); Erlotinib (Tarceva®); Estramustine (Emcyt®); Etoposide Phosphate (Etopophos®); Etoposide, VP-16 (Vepesid®); Exemestane (Aromasin®); Filgrastim (Neupogen®); Floxuridine (arterial administration) (FUDR®); Fludarabine (Fludara®); Fluorouracil, 5-FU (Adrucil®); Fulvestrant (Faslodex®); Gefitinib (Iressa®); Gemcitabine (Gemzar®); Gemtuzumab ozogamicin (Mylotarg®); Goserelin Acetate (Zoladex Implant®); Goserelin Acetate (Zoladex®); Histrelin Acetate (Histrelin Implant®); Hydroxyurea (Hydrea®); Ibritumomab tiuxetan (Zevalin®);Idarubicin (Idamycin®); Ifosfamide (IFEX®); Imatinib Mesylate (Gleevec®); Interferon α-2a (Roferon A®); Interferon α-2b (Intron A®); Irinotecan (Camptosar®); Lenalidomide (Revlimid®); Letrozole (Femara®); Leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate (Eligard®); Levamisole (Ergamisol®); Lomustine, CCNU (CeeBU®); Mechlorethamine, Nitrogen Mustard (Mustargen®); Megestrol Acetate (Megace®); Melphalan, L-PAM (Alkeran®); Mercaptopurine, 6-MP (Purinethol®); Mesna (Mesnex®); Mesna (Mesnextabs®); Methotrexate (Methotrexate®); Methoxsalen (Uvadex®); Mitomycin C (Mutamycin®); Mitotane (Lysodren®); Mitoxantrone (Novantrone®); Nandrolone Phenylpropionate (Durabolin-50®); Nelarabine (Arranon®); Nofetumomab (Verluma®); Oprelvekin (Neumega®); Oxaliplatin (Eloxatin®); Paclitaxel (Paxene®); Paclitaxel (Taxol®); Paclitaxel Protein-Bound Particles (Abraxane®); Palifermin (Kepivance®); Pamidronate (Aredia®); Pegademase (Adagen (Pegademase Bovine)®); Pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®); Pemetrexed Disodium (Alimta®); Pentostatin (Nipent®);Vercyte®; Plicamycin, Mithracin®; Porfimer Sodium, Photofrin®; Procarbazine, Matulane®; Quinacrine, Ata Brine®; Rasburicase, Elite®; Rituximab, Rituxan®; Lenalidomide; Sargramostim, Leukine®; Sargramostim, Prokine®; Sorafenib, Nexavar®; Streptozocin, Zanosar®; Sunitinib Malate, Sutent®; Talc, Sclerosol®; Tamoxifen, Nolvadex®; Temozolomide, Temodar®; Teniposide, VM-26, Vumon®; Testolactone, Teslac®; Thioguanine, 6-TG, Thioguanine®; Thiotepa, Thioplex®; Topotecan, Hycamtin®; Toremifene, Fareston®; Tositumomab, Bexxar®; Tositumomab / I-131 Tositumomab, Bexxar®; Trastuzumab, Herceptin®; Tretinoin, ATRA, Vesanoid®; Uracil Mustard Capsule®; Valrubicin, Valstar®; Vinblastine, Velban®; Vincristine, Oncovin®; Vinorelbine, Navelbine®; Olaparib, Lynparza®, Vorinostat, Zolinza® and Zoledronic Acid, Zometa®, or pharmaceutically acceptable salts thereof, among others, but not limited thereto.;

[0156] Accordingly, the scope of the present disclosure encompasses the use of a compound of Formula IV of the present disclosure in combination with a second compound selected from estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents, cytotoxic / cell growth inhibitory agents, anti-proliferative agents, prenyl protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, PPAR-γ agonists, PPAR-δ agonists, multidrug resistance inhibitors, antiemetics, agents useful for the treatment of anemia, agents useful for the treatment of neutropenia, immunostimulants, inhibitors of cell growth and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase and / or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), agents that interfere with cell cycle checkpoints, and any of the therapeutic agents listed above.

[0157] Yet another example of the present disclosure is a method of treating cancer, comprising administering a therapeutically effective amount of a compound of Formula IV of the present disclosure in combination with paclitaxel or trastuzumab.

[0158] The therapeutic combinations disclosed herein can be used in combination with other anti-cancer agents (but not limited to these) used for the prevention, treatment, suppression, amelioration or risk reduction of a particular disease or condition (e.g., a cell proliferation disorder). In one embodiment, a compound of Formula IV of the present disclosure is combined with one or more other anti-cancer agents used in the prevention, treatment, suppression, amelioration or risk reduction of a particular disease or condition for which the compound of Formula IV of the present disclosure is useful. Such other active agents can be administered before, simultaneously or sequentially with the compound of the present disclosure, by the routes and in the amounts generally used therefor.

[0159] The present disclosure also includes a pharmaceutical composition useful for the treatment or prevention of cancer, comprising a therapeutically effective amount of a compound of Formula IV of the present disclosure and a second compound selected from an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic and cytostatic agent, an anti-proliferative agent, a prenyl protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δ agonist, an inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic agent, a γ-secretase and / or NOTCH inhibitor, an agent that interferes with a receptor tyrosine kinase (RTK), an agent that interferes with a cell cycle checkpoint, and any of the therapeutic agents listed herein.

[0160] The present disclosure further relates to a method of treating cancer in a human patient, comprising administering a PD-1 antagonist to the patient. The compounds of the present disclosure and the PD-1 antagonist can be administered simultaneously or sequentially.

[0161] In certain embodiments, the PD-1 antagonist is an anti-PD-1 antibody or an antigen-binding fragment thereof. In alternative embodiments, the PD-1 antagonist is an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some embodiments, the PD-1 antagonist is independently an anti-PD-1 antibody selected from pembrolizumab, nivolumab, semipramab, sintilimab, tislelizumab, atezolizumab (MPDL3280A), camrelizumab, and toripalimab. In other embodiments, the PD-L1 antagonist is independently an anti-PD-L1 antibody selected from atezolizumab, durvalumab, and avelumab.

[0162] In one embodiment, the PD-1 antagonist is pembrolizumab. In certain sub-embodiments, the method comprises administering 200 mg of pembrolizumab to the patient every about 3 weeks. In other sub-embodiments, the method comprises administering 400 mg of pembrolizumab to the patient every about 6 weeks.

[0163] In a further lower-level embodiment, this method includes administering pembrolizumab to the patient at 2 mg / kg every about three weeks. In a particular lower-level embodiment, the patient is a pediatric patient.

[0164] In some embodiments, the PD-1 antagonist is nivolumab. In a particular lower-level embodiment, this method includes administering 240 mg of nivolumab to the patient every about two weeks. In other lower-level embodiments, this method includes administering 480 mg of nivolumab to the patient every about four weeks.

[0165] In some embodiments, the PD-1 antagonist is semiprimab. In a particular embodiment, this method includes administering 350 mg of semiprimab to the patient every about three weeks.

[0166] In some embodiments, the PD-1 antagonist is atezolizumab. In a particular lower-level embodiment, this method includes administering 1200 mg of atezolizumab to the patient every about three weeks.

[0167] In some embodiments, the PD-1 antagonist is durvalumab. In a particular lower-level embodiment, this method includes administering 10 mg / kg of durvalumab to the patient every about two weeks.

[0168] In some embodiments, the PD-1 antagonist is avelumab. In a particular lower-level embodiment, this method includes administering 800 mg of avelumab to the patient every about two weeks.

[0169] When a compound of Formula IV of the present disclosure is administered in combination with an anti-human PD-1 antibody (or an antigen-binding fragment thereof), the anti-human PD-1 antibody (or an antigen-binding fragment thereof) can be administered simultaneously with, before, or after the compound of Formula IV of the present disclosure. Either the anti-human PD-1 antibody (or an antigen-binding fragment thereof) and / or the compound of Formula IV of the present disclosure, or a pharmaceutically acceptable salt thereof, can be administered separately, by the same or different routes of administration, or together in the same pharmaceutical composition with other agents. The weight ratio of the anti-human PD-1 antibody (or an antigen-binding fragment thereof) to the compound of Formula IV of the present disclosure can vary and is determined by the therapeutically effective dosage of each agent. Generally, the therapeutically effective dosage of each is used. Combinations comprising at least one anti-human PD-1 antibody (or an antigen-binding fragment thereof), the compound of Formula IV of the present disclosure, and optionally other active agents usually contain the therapeutically effective dosage of each active agent. In such combinations, the anti-human PD-1 antibody (or an antigen-binding fragment thereof), the compound of Formula IV, and the other active agents can be administered separately or in combination. Further, the administration of one element can be carried out before, simultaneously with, or after the administration of the other agents.

[0170] In one embodiment, the present disclosure provides a combination formulation for the simultaneous, separate, or sequential use of an anti-human PD-1 antibody (or an antigen-binding fragment thereof), and / or a compound of Formula IV, and at least one other active agent in the treatment of cancer.

[0171] The present disclosure also provides the use of a compound of Formula IV of the present disclosure for the treatment of cancer when the patient has been previously treated (e.g., within 24 hours) with an anti-human PD-1 antibody (or an antigen-binding fragment thereof). The present disclosure also provides the use of an anti-human PD-1 antibody (or an antigen-binding fragment thereof) for the treatment of a cell proliferative disorder when the patient has been previously treated (e.g., within 24 hours) with a compound of Formula IV of the present disclosure that is an antibody-linker-payload compound (ADC).

[0172] The present invention further relates to a method for treating cancer, which comprises administering to a subject in need of treatment a combination therapy agent comprising (a) a compound of formula IV of the present invention, and (b) an anti-human PD-1 antibody (or an antigen-binding fragment thereof), and the anti-human PD-1 antibody (or an antigen-binding fragment thereof) is administered once every 21 days.

[0173] Furthermore, the present disclosure relates to a method for treating cancer, which comprises administering to a subject in need of treatment a combination therapy agent comprising (a) a compound of formula IV of the present disclosure, and (b) an anti-human PD-1 antibody (or an antigen-binding fragment thereof). In certain embodiments, the cancer occurs as one or more solid tumors or lymphomas. In further specific embodiments, the cancer is selected from the group consisting of progressive or metastatic solid tumors and lymphomas. In even further specific embodiments, the cancer is selected from the group consisting of melanoma, head and neck squamous cell carcinoma, MSI-H cancer, MMR-deficient cancer, non-small cell lung cancer, urothelial cancer, gastric or gastroesophageal junction adenocarcinoma, breast adenocarcinoma, and lymphoma. In additional embodiments, the lymphoma is selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, mediastinal large B-cell lymphoma, splenic marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, lymphoplasmacytic lymphoma, primary effusion lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma (primary cutaneous type), anaplastic large cell lymphoma (systemic type), peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, adult T-cell lymphoma / leukemia, nasal-type extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, gamma / delta hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, and Hodgkin lymphoma. In certain embodiments, the cell proliferative disorder is a metastatic cancer, such as liver metastasis from colorectal cancer. In additional embodiments, the cell proliferative disorder is a cancer classified as stage III cancer or stage IV cancer. In the case of these embodiments, the cancer is not surgically resectable.

[0174] In an embodiment of the method disclosed herein, the anti-human PD-1 antibody (or antigen-binding fragment thereof) is administered by intravenous injection or subcutaneous injection.

[0175] In one embodiment, the present disclosure provides a composition comprising a compound of formula IV, a pharmaceutically acceptable carrier, and an anti-human PD-1 antibody (or antigen-binding fragment thereof).

[0176] In another embodiment, the present disclosure provides a composition comprising a compound of formula IV, a pharmaceutically acceptable carrier, and pembrolizumab.

[0177] In one embodiment, the present disclosure provides a composition comprising a compound of formula IV, a pharmaceutically acceptable carrier, and two additional therapeutic agents, one of which is an anti-human PD-1 antibody (or antigen-binding fragment thereof) and the other is independently selected from the group consisting of anti-cancer agents.

[0178] The compounds of the present disclosure can be used in combination with an antiemetic for treating nausea or vomiting such as acute, delayed, delayed-phase, and anticipatory vomiting that may occur by the use of the compounds of the present disclosure alone or in combination with radiotherapy. For the prevention or treatment of vomiting, the compounds of the present disclosure can be used in combination with other antiemetics, particularly neurokinin-1 receptor antagonists, 5HT3 receptor antagonists (such as ondansetron, granisetron, tropisetron, and zatisetron), GABAB receptor agonists (such as baclofen), corticosteroids (such as decadron (dexamethasone), kenalog, Aristocort, Nasalide, Preferid, Benecorten, or other agents such as those disclosed in U.S. Pat. Nos. 2,789,118; 2,990,401; 3,048,581; 3,126,375; 3,929,768; 3,996,359; 3,928,326; and 3,749,712), antidopaminergic drugs (such as phenothiazines (e.g., prochlorperazine, fluphenazine, thioridazine, and mesoridazine), metoclopramide, aprepitant, fosaprepitant, or dronabinol). In another example, combination therapy with an antiemetic selected from a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist, and a corticosteroid is disclosed for the treatment or prevention of vomiting that may occur upon administration of a compound of formula IV.

[0179] The compounds of formula IV can also be administered with agents useful in the treatment of anemia. Such anemia-treating agents are, for example, continuous erythropoietin receptor activators (such as epoetin alpha).

[0180] The compounds of formula IV can also be administered with agents useful in the treatment of neutropenia. Such neutropenia-treating agents are, for example, hematopoietic growth factors that regulate the production and function of neutrophils, such as human granulocyte colony-stimulating factor (G-CSF). Examples of G-CSF include filgrastim.

[0181] The compounds of formula IV may be useful when combined with other therapies such as, but not limited to, radiation therapy, surgery, and gene therapy. Thus, in one embodiment, the cancer treatment methods described herein may optionally include administration of an effective amount of radiation therapy, unless otherwise contraindicated. Gamma rays are preferred for radiation therapy.

[0182] The cancer treatment methods described herein may include administration of an effective amount of radiation (i.e., the cancer treatment methods described herein may include the implementation of radiation therapy).

[0183] The cancer treatment methods described herein include, in combination with radiation therapy, and / or an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic / cell growth inhibitor, an anti-proliferative agent, a prenyl protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δ agonist, a multidrug resistance inhibitor, an antiemetic, a drug useful for the treatment of anemia, a drug useful for the treatment of neutropenia, an immunopotentiator, an inhibitor of cell growth and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic agent, a γ-secretase and / or NOTCH inhibitor, a drug that interferes with receptor tyrosine kinase (RTK), a drug that interferes with cell cycle checkpoints, and a cancer treatment method including administering a therapeutically effective amount of a compound of formula IV in combination with a second compound selected from the group consisting of additional therapeutic agents listed herein.

[0184] Further embodiments of the disclosure include the pharmaceutical compositions, combinations, uses, and methods described above, and it should be understood that each embodiment can be combined with one or more other embodiments, provided that such combinations are consistent with the description of the embodiment. It should be further understood that the embodiments provided above include all embodiments, including embodiments arising from combinations of the embodiments.

[0185] Kit In one aspect, there is provided a kit comprising a therapeutically effective amount of a compound of Formula IV of the present disclosure or a pharmaceutically acceptable salt, solvate or ester of the compound, and a pharmaceutically acceptable carrier, medium or diluent.

[0186] In another aspect, there is provided a kit comprising an amount of a compound of Formula IV of the present disclosure and an amount of at least one additional therapeutic agent listed above, wherein the amounts of the two or more active ingredients are those that provide a desired therapeutic effect. In one embodiment, the compound of Formula IV of the present disclosure and one or more additional therapeutic agents are provided in the same container. In one embodiment, the compound of Formula IV of the present disclosure and one or more additional therapeutic agents are provided in separate containers.

[0187] The present disclosure includes prodrugs of the compounds of the present disclosure within its scope. Generally, such prodrugs are functional derivatives of the compounds of the present disclosure that are readily convertible in vivo to the required compound. Thus, in the therapeutic methods of the present disclosure, the terms "administer" or "administering" a compound include treatment of the various conditions described by the specifically disclosed compound, or a compound that is not specifically disclosed but is converted in vivo to the designated compound after administration to a patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985. Metabolites of these compounds include the active species generated when the compounds of the present disclosure are introduced into a physiological environment.

[0188] The compounds of Formula IV can be administered orally, parenterally (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracapsular injection or infusion, subcutaneous injection or implantation), by inhalation spray, nasal, vaginal, rectal, sublingual, buccal or topical routes of administration, alone or together, in suitable unit dosage formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration. In addition to the treatment of warm-blooded animals, the compounds of the present disclosure are effective for use in humans.

[0189] The pharmaceutical compositions for administering the compounds of the present disclosure can be conveniently provided in unit dosage forms and can be prepared by any of the methods known in the pharmaceutical arts. All methods include the step of combining the active ingredient with a carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical compositions, the active compound is included in an amount sufficient to produce the desired effect in the course and condition of the disease. As used herein, the term "composition" shall include the product containing the specified amount of the specified ingredients and any product directly or indirectly obtained as a result of the combination of the specified amounts of the specified ingredients.

[0190] The pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, solutions, hard or soft capsules, or syrups or elixirs. The composition intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically elegant and palatable formulation. Tablets contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients can be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch, gelatin or acacia; and lubricants such as magnesium stearate, stearic acid or talc. Tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a long period. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used. It can also be coated by the techniques described in U.S. Patent Nos. 4,256,108, 4,166,452, and 4,265,874 to form osmotic therapeutic tablets for controlled release. Oral tablets can also be formulated for immediate release, such as fast melt tablets or wafers, rapid dissolve tablets or fast dissolve films.

[0191] Formulations for oral administration may be provided as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with a water or oil medium, such as arachis oil, liquid paraffin, or olive oil.

[0192] The aqueous suspension contains an active substance mixed with an excipient suitable for the production of an aqueous suspension. Such excipients are, for example, suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, tragacanth gum, and acacia gum, and the dispersing agent or wetting agent is, for example, a natural phospholipid such as lecithin, or a condensation product of an alkylene oxide and a fatty acid such as polyoxyethylene stearate, or a condensation product of ethylene oxide and a long-chain aliphatic alcohol such as heptadecaethylene oxide cetanol, or a condensation product of a partial ester derived from a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate and ethylene oxide, or a condensation product of a partial ester derived from a fatty acid and a hexitol anhydride such as polyethylene sorbitan monooleate and ethylene oxide. The aqueous suspension may also contain one or more preservatives, for example, ethyl p-hydroxybenzoate or n-propyl, one or more colorants, one or more flavoring agents, and one or more sweetening agents, for example, sucrose or saccharin.

[0193] The oily suspension can be formulated by suspending the active ingredient in a vegetable oil, for example, peanut oil, olive oil, sesame oil, coconut oil, or a mineral oil, for example, liquid paraffin. The oily suspension may contain a thickening agent, for example, beeswax, hard paraffin, acetyl alcohol. By adding the above-mentioned sweeteners and flavoring agents, an oral formulation with a good taste can be provided. These compositions can be preserved by adding an antioxidant such as ascorbic acid.

[0194] The dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide an active ingredient mixed with a dispersing agent or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing agents or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweeteners, flavoring agents, and colorants may also be present.

[0195] The pharmaceutical composition of the present invention may be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil such as olive oil or peanut oil, or a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifiers may be natural gums such as gum acacia or tragacanth gum, natural phospholipids such as soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweeteners and flavorings.

[0196] Syrups and elixirs can be formulated using sweeteners such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain analgesics, preservatives and flavorings and colorants.

[0197] The pharmaceutical composition may be in the form of a sterile injectable aqueous suspension or an oily suspension. This suspension can be formulated according to known techniques using the suitable dispersing or wetting agents and suspending agents described above. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent such as, for example, a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixed oils have conventionally been used as solvents or suspending media. In this regard, any non-irritating fixed oil containing synthetic monoglycerides or diglycerides can be used. Furthermore, fatty acids such as oleic acid are also used in the preparation of injectables.

[0198] The compounds of the present disclosure can also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

[0199] For local use, creams, ointments, gels, solutions, suspensions, etc. containing the compounds of the present invention are used. Similarly, transdermal patches can also be used for local administration.

[0200] The pharmaceutical compositions and methods of the present disclosure can further contain other therapeutically active compounds commonly applied to the treatment of the above-mentioned pathological conditions, as described herein.

[0201] In the treatment, prevention, control, improvement, or risk reduction of the symptoms disclosed herein, the appropriate dosage level of the compounds of the present disclosure is generally about 0.01 to 500 mg per kg of the patient's body weight per day, which can be administered in a single dose or multiple doses. Suitable dosage levels can be about 0.01 to 250 mg / kg per day, about 0.05 to 100 mg / kg per day, or about 0.1 to 50 mg / kg per day. Within this range, the dosage can be 0.05 to 0.5, 0.5 to 5, or 5 to 50 mg / kg per day. In the case of oral administration, the composition can be provided in the form of tablets containing 1.0 to 1000 mg of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the adjustment of the dosage for the patient to be treated. The compound can be administered by a dosing method of 1 to 4 times a day, or can also be administered once or twice a day.

[0202] However, the specific dosage levels and frequencies of administration for a particular patient can vary, and it will be understood that they are determined by various factors such as the activity of the specific compound used, the metabolic stability and duration of action of that compound, age, body weight, general health status, gender, diet, method and time of administration, excretion rate, concomitant medications, severity of the specific condition, and the host being treated.

[0203] The manufacturing methods of the compounds of the present disclosure are illustrated in the following schemes and examples. The starting materials are manufactured according to procedures known in the art or procedures exemplified herein.

[0204] Production Example The compounds of the present disclosure can be manufactured according to the following schemes and specific examples, or modifications thereof, using readily available starting materials, reagents, and conventional synthetic procedures. It is also possible to utilize variations known to those skilled in the art but not specifically mentioned in detail. The general procedures for manufacturing the compounds claimed in the present disclosure can be readily understood by those skilled in the art by referring to the following schemes and descriptions. The abbreviations used in the experiments can include, but are not limited to, the following.

Table 2

[0205] Synthetic schemes, intermediates, and examples The compounds of the present disclosure can be prepared by methods known in the field of organic synthesis, which are partially described in the following general synthetic schemes and specific manufacturing examples. The starting materials are commercially available or can be prepared by known methods Preparation of Intermediate I-1

Chemical formula

[0206] Step B-: Synthesis of Compound I-1d To tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycinate (I-1c, 60 g, 146 mmol) in DMF (250 mL) was added diethylamine (78 g, 1.1 mol) at 20 °C, and the mixture was stirred at 20 °C for 1 hour. The excess Et2NH was distilled off under reduced pressure, and the solution of crude tert-butyl glycylglycinate (I-1d) in DMF was used directly as it was

[0207] Step C-: Synthesis of Compound I-1g To a solution of ((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (I-1e, 65 g, 183 mmol) in DMF (650 mL) was added HATU (77 g, 202 mmol), and the reaction mixture was stirred at 20 °C for 5 minutes. Then, DIPEA (50 g, 385 mmol) and tert-butyl L-phenylalaninate (I-1f, 52 g, 202 mmol) were added. The mixture was stirred at 20 °C for 2 hours. The mixture was diluted with EtOAc (500 mL) and water (200 mL), and stirred until the product precipitated. The solid was collected by filtration. The filter cake was slurried with MTBE (500 mL) and filtered to obtain tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalaninate (I-1g) as a solid, which was used directly in the next reaction.

[0208] Step D: Synthesis of compound I-1g To a solution of tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalaninate (I-1g, 95 g, 170 mmol) in DCM (200 mL) was added TFA (200 mL), and the mixture was stirred at 25 °C for 16 hours. The mixture was concentrated under reduced pressure, and the crude product was triturated with MTBE (1 liter) at room temperature for 1 hour. The solid was collected by filtration, washed with MTBE, and concentrated under reduced pressure to obtain ((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (I-1h) as a solid, which was used directly in the next reaction.

[0209] Step E: Synthesis of compound I-1i A solution of ((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (I-1h, 76 g, 152 mmol) in DMF (350 mL) was added to PyBOP (83 g, 159 mmol), and the mixture was stirred at 20 °C for 30 minutes. Next, DIPEA (21 g, 159 mmol) was added, and then a solution of tert-butyl glycylglycinate (I-1d, 27 g, 144 mmol, in 250 mL DMF) was added while controlling the temperature within 10 - 20 °C in an ice bath during the addition. The reaction mixture was stirred at 10 - 20 °C for 2 hours. The mixture was quenched with ice water (1 liter) and EtOAc (500 mL), and the product precipitated from the solution. The solid was collected by filtration. The filter cake was slurried with EtOAc (1 liter) at room temperature for 30 minutes. The solid was filtered and dried in vacuo to obtain tert-butyl ((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycylglycinate (I-1i) as a solid, which was used directly in the next reaction.

[0210] Step F: Synthesis of Compound I-1j To a solution of tert-butyl ((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycylglycinate (I-1i, 65 g, 97 mmol) in DCM (120 mL) was added TFA (120 mL). The mixture was stirred at 45 °C for 2 hours. The mixture was concentrated under reduced pressure. The crude product was triturated with MTBE (1 liter) at room temperature for 20 minutes. The solid was collected by filtration and concentrated under reduced pressure to obtain ((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycylglycine (I-1j) as a solid, which was used directly in the next reaction.

[0211] Step G: Synthesis of Compound I-1- A mixture of ((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycylglycine (I-1j, 56 g, 91 mmol) in THF (560 mL) and AcOH (56 mL) was heated to 40 °C and stirred for 30 minutes, then Pb(OAc)4 (170 g, 364 mmol, purity 95%) was added to the mixture. The mixture was stirred at 60 °C for 2 hours. The reaction mixture was cooled to 20 °C and filtered. The filter cake was diluted with MTBE (500 mL) and water (500 mL). Next, the two layers were separated. The aqueous phase was extracted with EtOAc (500 mL). Next, THF (500 mL) was added to the combined organic extracts. The organic layer was washed with brine (twice with 800 mL), dried over anhydrous MgSO4, filtered through Celite® and concentrated under reduced pressure. The residue was purified by preparative HPLC (40 - 57% MeCN / water). The desired fractions were concentrated under reduced pressure at 30 °C. The mixture was extracted with EtOAc, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure at 30 °C to give (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazapentadecan-17-yl acetate (I-1) as a solid. 11H NMR (400 MHz, DMSO-d6) δ 8.84 (t, J = 6.9 Hz, 1H), 8.33 (br t, J = 5.9 Hz, 1H), 8.15 (br d, J = 8.3 Hz, 1H), 8.01 (br t, J = 5.5 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 7.5 Hz, 2H), 7.59 (br t, J = 6.0 Hz, 1H), 7.47 - 7.38 (m, 2H), 7.36 - 7.30 (m, 2H), 7.30 - 7.09 (m, 5H), 5.11 (dd, J = 7.0, 1.3 Hz, 2H), 4.53 (td, J = 8.8, 4.4 Hz, 1H), 4.36 - 4.12 (m, 3H), 3.90 - 3.50 (m, 6H), 3.06 (dd, J = 13.8, 4.3 Hz, 1H), 2.79 (dd, J = 13.8, 9.8 Hz, 1H), 2.00 (s, 3H). Production of Intermediate I-2 [Chemical formula] Step A: Synthesis of Compound I-2c A solution of ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (I-2a, 1.46 kg, 4.69 mol), DIPEA (1.82 kg, 1.41 mol), and tert-butyl L-alaninate hydrochloride (I-2b, 852 g, 4.69 mol) in THF (15 liters) was stirred while adding PyBOP (2.68 kg, 5.16 mol) portionwise thereto at 10 °C under a nitrogen atmosphere. The resulting mixture was stirred at 20 °C for 1 hour under nitrogen. The mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (30 liters). The resulting mixture was filtered and the filter cake was washed with water (twice with 5 liters). The recovered solid was dried in a dryer under vacuum to obtain the crude tert-butyl ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alaninate (I-2c) as a solid, which was used directly in the next step.

[0212] Step B: Synthesis of Compound I-2d While stirring a solution of tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alaninate (I-2c, 2.06 kg, 4.70 mol) in DCM (6.0 liters), trifluoroacetic acid (6.0 liters) was added thereto little by little at 15 °C under a nitrogen atmosphere. The resulting mixture was stirred at 20 °C for 3 hours under nitrogen. The resulting mixture was concentrated under reduced pressure and diluted with water (20 liters). The resulting mixture was filtered and the filter cake was washed with water (twice with 5 liters). The recovered solid was dried in a dryer under vacuum to obtain crude ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (I-2d) as a solid, which was used directly in the next step.

[0213] Step C-: Synthesis of Compound I-2e While stirring a solution of crude ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (I-2d, 1.59 kg, 3.88 mol), DIPEA (1.51 kg, 11.6 mol) and tert-butyl glycinate hydrochloride (I-1b HCl, 509 g, 3.88 mol) in THF (16 liters), PyBOP (2.22 kg, 4.27 mol) was added thereto little by little at 10 °C under a nitrogen atmosphere. The resulting mixture was stirred at 20 °C for 0.5 hour under nitrogen. The resulting mixture was concentrated under reduced pressure, diluted with water (20 liters) and filtered. The filter cake was washed with water (twice with 2 liters). The recovered solid was dried in a dryer under vacuum to obtain crude tert-butyl ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alaninylglycinate (I-2e) as a solid, which was used directly in the next step.

[0214] Step D: Synthesis of Compound I-2f A solution of tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanylglycinate (I-2e, 1.80 kg, 1.21 mol) in DCM (3.6 liters) was stirred, and thereto, TFA (3.6 liters) was added little by little at 15 °C under a nitrogen atmosphere. The resulting mixture was stirred at 20 °C for 0.5 h under nitrogen. The resulting mixture was concentrated under reduced pressure, diluted with MTBE (20 liters), and filtered. The filter cake was washed with MTBE (2 times with 5 liters) to obtain (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanylglycine (I-2f) as a solid, which was used directly in the next step.

[0215] Step E: Synthesis of Compound I-2- A solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanylglycine (I-2f, 430 g, 978 mmol) in DMF (4.3 liters) was treated with Cu(OAc)2 (67 g, 367 mmol) at room temperature under a nitrogen atmosphere, and then acetic acid (13 g, 215 mmol) was added dropwise at room temperature. Next, lead tetraacetate (651 g, 1.47 mol) was added little by little at room temperature. The reaction solution was stirred at 60 °C for 3 h. The mixture was cooled and purified by silica gel flash column chromatography (5:1 DCM:MeOH) to obtain (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazoundecane-11-yl acetate (I-2) as a solid. MS: m / z = 476 [M+Na]. 11H NMR (300 MHz, DMSO-d6) δ 8.89 (t, J = 7.1 Hz, 1H), 8.06 (d, J = 7.4 Hz, 1H), 7.91 (d, J = 7.5 Hz, 2H), 7.75 (t, J = 6.3 Hz, 2H), 7.56 (d, J = 7.6 Hz, 1H), 7.44 (td, J = 7.5, 1.3 Hz, 2H), 7.36 (td, J = 7.4, 1.3 Hz, 2H), 5.13 (dd, J = 7.0, 2.0 Hz, 2H), 4.32 - 4.19 (m, 4H), 4.11 (q, J = 7.2 Hz, 1H), 1.99 (s, 3H), 1.22 (d, J = 2.1 Hz, 2H), 1.20 (d, J = 2.0 Hz, 3H). Production of Intermediate I-3 [Chemical formula] Step A: Synthesis of Compound I-3b For a mixture of dicyclohexylamine (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (I-3a, 1.50 g, 3.22 mmol) and formic acid (124 μL, 3.22 mmol) in DCM, direct silica gel flash column chromatography (0 - 40% MeOH / DCM) was performed to obtain (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (I-3b). 1 1H NMR (500 MHz, DMSO-d6) δ 13.27 (s, 1H), 7.09 (s, 2H), 6.97 (t, J = 6.2 Hz, 1H), 4.59 (dd, J = 10.5, 3.8 Hz, 1H), 3.59 - 3.52 (m, 1H), 3.45 - 3.38 (m, 1H), 1.32 (s, 9H). Step B: Synthesis of Compound I-3c (S)-3-((tert-Butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (I-3b, 350 mg, 1.2 mmol) was dissolved in DCM (820 μL) and TFA (410 μL, 5.3 mmol).

[0216] The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure to give an oil. The product was precipitated with Et2O (20 mL) and then sonicated. The precipitate was collected by vacuum filtration, redissolved in MeCN, and reconcentrated under reduced pressure to give (S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid 2,2,2-trifluoroacetate (I-3c) as a solid. 1 1H NMR (500 MHz, DMSO-d6) δ 13.93 (s, 1H), 7.99 (s, 3H), 7.18 (s, 2H), 4.86 (dd, J = 10.0, 4.7 Hz, 1H), 3.47 (dd, J = 13.3, 4.6 Hz, 1H), 3.34 (dd, J = 13.1, 10.3 Hz, 2H, overlapping with the signal of water). Step C: Synthesis of Compound I-3 A solution of (S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid 2,2,2-trifluoroacetate (I-3c, 49 mg, 0.16 mmol) and DIPEA (0.057 mL, 0.33 mmol) in DMF (0.70 mL) was added to m-dPEG®8-NHS ester (I-3d, 100 mg, 0.20 mmol). The reaction mixture was stirred at room temperature for about 10 min (monitored by LCMS). The product was directly subjected to reverse-phase flash column chromatography (0 - 95% MeCN / H2O + 0.1% FA) to give (S)-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-oic acid (I-3) as an oil. 11H NMR (500 MHz, DMSO-d6) δ 13.35 (s, 1H), 7.99 (t, J = 5.9 Hz, 1H), 7.09 (s, 2H), 4.62 - 4.56 (m, 1H), 3.81 - 3.75 (m, 1H), 3.55 - 3.49 (m, 24H), 3.48 - 3.46 (m, 3H), 3.44 - 3.41 (m, 4H), 3.24 (s, 3H), 2.24 - 2.17 (m, 2H). Using a method similar to that described in Example I-3 with slight modifications to the reaction time and / or solvent, and using appropriate reactants and / or reagents instead (I-3d using other commercially available NHS esters), the following compounds of the present disclosure in Table 3 were prepared.

[0217] Table 3 [Table 3] Preparation of Intermediate I-5 [Chemical formula] Step A: Synthesis of Compound I-5 To a solution of 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane-38-carboxylic acid (0.095 g, 0.16 mmol) and HATU (0.061 g, 0.16 mmol) in DMF (2 mL) was added N-methylmorpholine (0.050 mL, 0.46 mmol). The reaction solution was stirred at room temperature for about 5 minutes, and then (S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid, TFA (I-3c, 0.048 g, 0.16 mmol) was added. The reaction solution was stirred at room temperature for about 2.5 hours. The mixture was concentrated under reduced pressure. The resulting (S)-41-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azatetratetracontane-42-carboxylic acid (I-5) oil was used directly as it was.

[0218] Production of Intermediate I-6

Chem.

[0219] The obtained crude (29S,38S,48R)-38-benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-48-hydroxy-26,30,33,36,39,42-hexaoxo-2,5,8,11,14,17,20,23,45-nonaoxo-27,31,34,37,40,43-hexaazanonatetracosane-49-oic acid (I-6) was dried and used directly in the next reaction.

[0220] Production of Intermediate I-7

Chem.

[0221] Step B - Synthesis of Compound I-7d Glycylglycine (I-7c, 12 g, 93 mmol) and sodium bicarbonate (7.8 g, 93 mmol) were dissolved in water (150 mL). This aqueous solution was added to a solution of 2,5-dioxopyrrolidin-1-yl (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycinate (I-7b, 35 g, 78 mmol) in 1,4-dioxane (380 mL). The mixture was stirred at 25 °C for 18 h. The solution was concentrated under reduced pressure. At the same time, the pH value was adjusted to pH 2 - 3 by adding a 10% aqueous citric acid solution. The resulting white solid precipitated, was filtered, and washed with cold water. The solid was dried in vacuo to obtain (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycylglycylglycine (I-7d), which was used directly in the next step.

[0222] Step C: Synthesis of Compound I-7 A solution of ((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycylglycylglycine (I-7d, 5.0 g, 11 mmol) in DMF (50 mL) was added with copper(II) acetate (0.78 g, 4.3 mmol), acetic acid (1.4 mL, 25 mmol) and lead(IV) tetraacetate (24 g, 53 mmol). The reaction solution was heated to 60 °C and allowed to stand for 1 hour. The mixture was filtered at 60 °C, the filtrate was added to water (100 mL), filtered, the obtained cake solid was dissolved again in DMF (50 mL), heated to 60 °C and filtered again. The filtrate was added to ice water (150 mL), then the precipitate was filtered, and the filter cake was dried in vacuo to obtain 1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazatetradecan-14-yl acetate (I-7) as a solid. MS: m / z = 505 [M+Na]. 1 H NMR (400 MHz, DMSO-d6) δ 8.90 (br t, J = 6.9 Hz, 1H), 8.16 (td, J = 5.7, 11.8 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 7.4 Hz, 2H), 7.58 (br t, J = 6.0 Hz, 1H), 7.45 - 7.39 (m, 2H), 7.36 - 7.30 (m, 2H), 5.09 (d, J = 7.0 Hz, 2H), 4.33 - 4.27 (m, 2H), 4.25 - 4.21 (m, 1H), 3.78 - 3.72 (m, 4H), 3.70 - 3.65 (m, 2H), 1.98 (s, 3H). Preparation of Intermediate I-8

Chemical Structure

[0223] Step B: Synthesis of compound I-8c L-Alanyl-L-alanine (I-8b, 19 g, 120 mmol) and sodium bicarbonate (9.87 g, 118 mmol) were dissolved in water (40 mL). The aqueous solution was added to a solution of 2,5-dioxopyrrolidin-1-yl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alaninate (I-8a, 40 g, 98 mmol) in 1,4-dioxane (160 mL). The mixture was stirred at 25 °C for 17 h. The solution was concentrated to a small volume (10 mL), and at the same time, the pH value was adjusted to near pH 2 - 3 by adding 10% aqueous citric acid solution. The resulting gelatinous precipitate was filtered and washed with cold water. The filter cake was dried in vacuo to obtain ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alanine (I-8c) as a solid.

[0224] Step C: Synthesis of compound I-8d A solution of ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alanine (I-8c, 40 g, 88 mmol) and 1-hydroxypyrrolidine-2,5-dione (12 g, 110 mmol) in 1,4-dioxane (500 mL) was stirred at 0 °C. Next, a solution of DCC (22 g, 110 mmol) in 1,4-dioxane (100 mL) was added slowly. The mixture was stirred at 0 °C for 0.5 h and then at 25 °C for an additional 18 h. After cooling in the refrigerator for 1 h, the precipitate was removed to obtain a solution of 2,5-dioxopyrrolidin-1-yl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alaninate (I-8d) in 1,4-dioxane (600 mL), which was used directly in the next step.

[0225] Step D: Synthesis of Compound I-8e Glycine (I-1b, 7.5 g, 100 mmol) and sodium bicarbonate (8.4 g, 100 mmol) were dissolved in water (300 mL). This aqueous solution was added to a solution of 2,5-dioxopyrrolidin-1-yl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alaninate (I-8d, 46 g, 84 mmol) in 1,4-dioxane (800 mL) over 0.5 h at 0 °C. The mixture was stirred at 25 °C for 18 h. The solution was concentrated under reduced pressure while adjusting the pH value to pH 2 - 3 by adding 10% aqueous citric acid solution. The resulting gelatinous precipitate was filtered and washed with cold water. By vacuum drying, ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alanylglycine (I-8e) was obtained as a solid, which was used directly in the next step.

[0226] Step E: Synthesis of Compound I-8 A solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alanylglycine (I-8e, 4.0 g, 7.8 mmol) in DMF (100 mL) was treated with copper(II) acetate (0.57 g, 3.1 mmol), acetic acid (1.0 mL, 18 mmol), and lead(IV) tetraacetate (17 g, 39 mmol). The reaction mixture was heated to 60 °C and allowed to stir for 1 h. The mixture was filtered at 60 °C and the filtrate was added to water (100 mL) and filtered. The cake solid was redissolved in DMF (100 mL), heated to 60 °C, filtered again, and the filtrate was added to ice water (300 mL). The precipitate was filtered and the filtrate was added to DCM / MeOH (4:1, 100 mL). The resulting precipitate was centrifuged and the filter cake was concentrated under reduced pressure to give (5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazatetradecan-14-yl acetate (I-8) as a solid. MS: m / z = 547 [M+Na]. 1 H NMR (400 MHz, DMSO-d6) δ 8.86 (br t, J = 6.8 Hz, 1H), 7.98 (br dd, J = 7.3, 16.1 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H), 7.72 (br t, J = 7.0 Hz, 2H), 7.54 (br d, J = 7.5 Hz, 1H), 7.44 - 7.39 (m, 2H), 7.36 - 7.31 (m, 2H), 5.14 - 5.03 (m, 2H), 4.28 - 4.19 (m, 5H), 4.13 - 4.01 (m, 1H), 1.98 (s, 3H), 1.22 - 1.18 (m, 9H). [Chemical Structure] Preparation of Intermediate I-9 Step A: Synthesis of Compound I-9c A mixture of 2-(4-nitrophenyl)-2-oxoacetic acid (I-9a, 0.55 g, 2.8 mmol), HATU (1.03 g, 2.7 mmol), and NMM (0.66 mL, 6.0 mmol) in DMF (6.0 mL) was stirred at room temperature for 2 minutes, and at that time, 2,5,8,11,14,17,20,23-octaoxapentacosane-25-amine (I-9b, 770 mg, 2.0 mmol) in DMF (1.0 mL) was added. The reaction mixture was stirred at room temperature for 20 minutes and concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography (0-70% MeCN / H2O + 0.1% formic acid modifier) to obtain 2-(4-nitrophenyl)-2-oxo-N-(2,5,8,11,14,17,20,23-octaoxapentacosane-25-yl)acetamide (I-9c) as an oil. MS: m / z = 561 [M+H].

[0227] Step B: Synthesis of Compound I-9d A mixture of 2-(4-nitrophenyl)-2-oxo-N-(2,5,8,11,14,17,20,23-octaoxapentacosane-25-yl)acetamide (I-9c, 760 mg, 1.36 mmol), iron (380 mg, 6.78 mmol), and ammonium chloride (435 mg, 8.13 mmol) in ethanol (5.8 mL) and water (1.9 mL) was stirred at 70 °C for 3 hours. The reaction mixture was filtered, washed with MeOH, and concentrated under reduced pressure. After redissolving in MeCN, the mixture was filtered and concentrated again under reduced pressure. The residue was dissolved in MeOH (5.8 mL), cooled to 0 °C, and treated portionwise with sodium borohydride (260 mg, 6.78 mmol) over 2 minutes. After gas evolution ceased, the mixture was concentrated under reduced pressure, suspended in water, and purified by reverse-phase column chromatography (0-90% MeCN / H2O + 0.1% formic acid modifier) to obtain 2-(4-aminophenyl)-2-hydroxy-N-(2,5,8,11,14,17,20,23-octaoxapentacosane-25-yl)acetamide (I-9d) as a viscous oil. MS: m / z = 533 [M+H].

[0228] Step C - Synthesis of Compound I-9e 2-(4-Aminophenyl)-2-hydroxy-N-(2,5,8,11,14,17,20,23-octaoxapentacos-25-yl)acetamide (I-9d, 200 mg, 0.38 mmol), (((9H-Fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (I-2d, 170 mg, 0.45 mmol) and EEDQ (185 mg, 0.75 mmol) in a mixture of DCM (1.67 mL) and MeOH (0.83 mL) was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and purified by reverse phase column chromatography (10-100% MeCN / H2O + 0.1% formic acid modifier) to give (9H-Fluoren-9-yl)methyl ((2S)-1-(((2S)-1-((4-(28-hydroxy-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azaoctacos-28-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-9e) as a solid. MS: m / z = 879 [M+H-H2O].

[0229] Synthesis of Step D - Compound I-9 A solution of ((9H-fluoren-9-yl)methyl ((2S)-1-(((2S)-1-((4-(28-hydroxy-27-oxo-2,5,8,11,14,17,20,23-octaoxo-26-azaoctacos-28-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-9e, 200 mg, 0.22 mmol) in DMF (1.1 mL) was added with bis(4-nitrophenyl) carbonate (135 mg, 0.45 mmol). The reaction mixture was stirred at room temperature for 26 h. The mixture was purified directly by reverse-phase column chromatography (10 - 95% MeCN / H2O + 0.1% formic acid regulator) to obtain ((9H-fluoren-9-yl)methyl ((2S)-1-(((2S)-1-((4-(1-(4-nitrophenoxy)-1,4-dioxo-2,8,11,14,17,20,23,26,29-nonaoxo-5-azatriacontan-3-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-9). MS: m / z = 1084 [M+Na]. 1 H NMR (500 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.48 (t, J = 5.5 Hz, 1H), 8.33 (d, J = 9.2 Hz, 2H), 8.13 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.72 (t, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.59 - 7.52 (m, 3H), 7.45 (d, J = 8.7 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 5.89 (s, 1H), 4.45 - 4.37 (m, 1H), 4.32 - 4.17 (m, 3H), 4.14 - 4.06 (m, 1H), 3.52 - 3.37 (m, 30H), 3.26 - 3.20 (m, 5H), 1.31 (d, J = 7.1 Hz, 3H), 1.23 (d, J = 7.1 Hz, 3H). Preparation of Intermediate I-10

Chem.

[0230] Step B: Synthesis of Compound I-10 A mixture of 1-morpholino-2-(4-nitrophenyl)ethane-1,2-dione (I-10b, 1.25 g, 4.7 mmol) and 10 wt% Pd / C (0.25 g, 0.24 mmol Pd) in DCM (12 mL) and MeOH (12 mL) was stirred under H2 (1.0 atm) for 2 days. After adding an additional 10 wt% Pd / C (0.25 g, 0.24 mmol Pd), the reaction mixture was stirred under H2 (1.0 atm) for 1 day. The mixture was filtered through Celite, washed with MeOH and DCM, concentrated under reduced pressure, and purified by silica gel column chromatography (0 - 15% MeOH / DCM) to obtain 2-(4-aminophenyl)-2-hydroxy-1-morpholinoethan-1-one (I-10). MS: m / z = 219 [M+H-H2O]. 1 H NMR (500 MHz, DMSO-d6) δ 6.97 (d, J = 8.3 Hz, 2H), 6.52 (d, J = 8.3 Hz, 2H), 5.17 (s, 3H), 3.61 - 3.34 (m, 7H), 3.25 - 3.11 (m, 2H). Intermediate I-11 Production of Intermediate I-11 [Chemical] Step A: Synthesis of Compound I-11b A mixture of 2-(4-aminophenyl)-2-hydroxy-1-morpholinoethan-1-one (I-10, 0.68 g, 2.9 mmol), (tert-butoxycarbonyl)-L-alanyl-L-alanine (I-11a, 1.12 g, 4.3 mmol) and EEDQ (1.42 g, 5.8 mmol) in DCM (6.4 mL) and MeOH (3.2 mL) was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (0 - 15% MeOH / DCM) to obtain tert-butyl ((2S)-1-(((2S)-1-((4-(1-hydroxy-2-morpholino-2-oxoethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-11b) as a solid. MS: m / z = 461 [M+H-H2O].

[0231] Step B: Synthesis of Compound I-11 [1] A mixture of tert-butyl ((2S)-1-(((2S)-1-((4-(1-hydroxy-2-morpholino-2-oxoethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-11b, 1.08 g, 2.3 mmol), bis(4-nitrophenyl) carbonate (1.37 g, 4.5 mmol) and DIPEA (0.39 mL, 2.3 mmol) in MeCN (4.5 mL) and DMF (2.0 mL) was stirred at room temperature for 3 hours. The reaction mixture was directly purified by reverse phase column chromatography (10 - 90% MeCN / H2O + 0.1% formic acid modifier) to obtain tert-butyl ((2S)-1-(((2S)-1-((4-(2-morpholino-1-(((4-nitrophenoxy)carbonyl)oxy)-2-oxoethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-11) as a solid. MS: m / z = 666 [M+Na]. 1 H NMR (500 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.33 (d, J = 9.1 Hz, 2H), 8.04 (d, J = 7.1 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 9.1 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 7.3 Hz, 1H), 6.46 (s, 1H), 4.44 - 4.34 (m, 1H), 4.04 - 3.93 (m, 1H), 3.57 - 3.40 (m, 6H), 3.24 - 3.16 (m, 1H), 3.13 - 3.06 (m, 1H), 1.38 (s, 9H), 1.31 (d, J = 7.1 Hz, 3H), 1.18 (d, J = 7.1 Hz, 3H). By slightly changing the reaction time and / or the solvent and using appropriate reactants and / or reagents instead, the following compounds of the present disclosure in Table 4 were prepared using the same method as described in Example I-11.

[0232] Table 4

Table 4

Chemical formula

[0233] Step B: Synthesis of Compound I-14c A mixture of methyl 2-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanamido)phenyl)-2-hydroxyacetate (I-14b, 870 mg, 2.05 mmol) and lithium hydroxide (98 mg, 4.1 mmol) in MeOH (12 mL) and water (6 mL) was stirred at room temperature overnight. The mixture was then neutralized with 1 M aqueous HCl (5 mL) and shaken with DCM (100 mL). The aqueous layer was back-extracted with 10% MeOH / DCM. The combined organic layers were washed twice with brine and the second brine layer was back-extracted with IPA / DCM. The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure to give 2-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanamido)phenyl)-2-hydroxyacetic acid (I-14c) as a solid. MS: m / z = 432 [M+Na].

[0234] Step C: Synthesis of Compound I-14e A solution of 2-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanamido)phenyl)-2-hydroxyacetic acid (I-14c, 580 mg, 1.4 mmol), N-methyl-2,5,8,11,14,17,20,23-octaoxapentacosane-25-amine (I-14d, 730 mg, 1.8 mmol) and NMM (0.31 mL, 1.8 mmol) in DMF (2.8 mL) was stirred, and HATU (700 mg, 1.8 mmol) was added thereto. The mixture was stirred at room temperature for 20 minutes and purified directly by reverse phase column chromatography (10 - 60% MeCN / H2O + 0.1% formic acid modifier) to give tert-butyl ((2S)-1-(((2S)-1-((4-(28-hydroxy-26-methyl-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azaoctacosane-28-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-14e) as an oil. MS: m / z = 771.6 [M+H-H2O].

[0235] Step D: Compound I-14 tert-Butyl ((2S)-1-(((2S)-1-((4-(1-hydroxy-2-morpholino-2-oxoethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-14e, 720 mg, 0.91 mmol), bis(4-nitrophenyl) carbonate (550 mg, 1.8 mmol) and DIPEA (0.16 mL, 0.91 mmol) in DMF (2.3 mL) were stirred at room temperature for 2 h. At this point, additional bis(4-nitrophenyl) carbonate (270 mg, 0.89 mmol) and DIPEA (0.16 mL, 0.91 mmol) were added. After stirring for a further 3 h, additional bis(4-nitrophenyl) carbonate (270 mg, 0.89 mmol) and DIPEA (0.16 mL, 0.91 mmol) were added. After 30 min, the reaction mixture was purified directly by reverse-phase column chromatography (10 - 90% MeCN / H2O + 0.1% formic acid modifier) to give tert-butyl ((2S)-1-(((2S)-1-((4-(5-methyl-1-(4-nitrophenoxy)-1,4-dioxo-2,8,11,14,17,20,23,26,29-nonaoxo-5-azatriacontan-3-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-14) as a viscous gel. MS: m / z = 976 [M+Na].

[0236] Preparation of Intermediate I-15

Chemical formula

[0237] Intermediates I-16 and I-17 Preparation of Intermediates I-16 and I-17

Chemical formula

[0238] To obtain the free acid, the diethylamine salt of enantiomer 1 was dissolved in EtOAc and shaken with 1 M aqueous HCl. The organic layer was back-extracted twice with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give 2-(((benzyloxy)carbonyl)amino)-3,3-difluoro-2-methylpropanoic acid (I-16b, enantiomer 1, absolute configuration unknown). 1 1H NMR (500 MHz, DMSO-d6) δ 13.20 (s, 1H), 8.10 (s, 1H), 7.42 - 7.27 (m, 5H), 6.28 (t, J = 56.1 Hz, 1H), 5.03 (s, 2H), 1.34 (s, 3H).

[0239] The free acid of 2-(((benzyloxy)carbonyl)amino)-3,3-difluoro-2-methylpropanoic acid (I-16c, enantiomer 2, absolute configuration unknown) was obtained in the same manner.

[0240] Step A: Synthesis of Intermediates I-16 and I-17 A mixture of 2-(((benzyloxy)carbonyl)amino)-3,3-difluoro-2-methylpropanoic acid (I-16b, enantiomer 1, 290 mg, 1.1 mmol), 10 wt% Pd / C (430 mg, 0.41 mmol Pd), and MeOH (20 mL) was stirred overnight under H2 (1.0 atm). The reaction mixture was filtered through Celite® and washed with MeOH and DCM, and then the solvent was removed under reduced pressure to give 2-amino-3,3-difluoro-2-methylpropanoic acid (I-16, enantiomer 1, absolute configuration unknown) as a solid. 1 1H NMR (500 MHz, DMSO-d6) δ 7.71 (br s, 3H), 6.18 (t, J = 54.4 Hz, 1H), 1.27 (s, 3H). 2-Amino-3,3-difluoro-2-methylpropanoic acid (I-17, enantiomer 2, absolute configuration unknown) was obtained from I-16c in the same manner.

[0241] Production of Intermediate I-18

Chemical Structure

[0242] Step B: Synthesis of Compound I-18 A mixture of tert-butyl (1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-methyl-1-oxopropan-2-yl)carbamate (I-18b, 250 mg, 0.40 mmol), TFA (0.31 mL, 4.0 mmol) and DCM (1.3 mL) was stirred at room temperature for 6 h. The mixture was homogenized by adding MeOH and concentrated under reduced pressure to give crude 2-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-methylpropanamide bis(2,2,2-trifluoroacetate) (I-18) as a solid, which was used directly without further purification.

[0243] Intermediate I-19 Preparation of Intermediate I-19

Chemical Structure

[0244] The following compounds of the present disclosure in Table 5 were prepared using a method similar to that described in Example I-19 with slight variations in reaction time and / or solvent and using appropriate reactants and / or reagents in place.

[0245] Table 5 [Table 5] TIFF2025523332000077.tif Preparation of Intermediate I-27

Chemical formula

[0246] Step B: Synthesis of Compound I-27c (5S,8S,13R)-13-((Benzyloxy)methyl)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecanoic acid (I-27b, 60 mg, 0.10 mmol) and HATU (46 mg, 0.12 mmol) in DMF (0.30 mL) were stirred, and thereto were added (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-aminium methanesulfonate (I-15a, 0.33 mL, 0.12 - 0.13 mmol, about 0.35 - 0.40 M in DMF, containing 3 equivalents of TEA) and DIPEA (18 μL, 0.10 mmol). After stirring for 10 minutes at room temperature, the mixture was treated with 4-methylpiperidine (60 μL, 0.51 mmol) and stirred for an additional 2 hours. The reaction was quenched with formic acid (20 μL), and the product was purified by reverse-phase column chromatography (10 - 60% MeCN / H2O + 0.1% formic acid modifier) to give (R)-2-(((S)-2-((S)-2-aminopropanamido)propanamido)methoxy)-3-(benzyloxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizin-1-yl)propanamide formate (I-27c) as a solid. MS: m / z = 785 [M+H].

[0247] Step C: Synthesis of Compound I-27 (R)-2-(((S)-2-((S)-2-Aminopropanamide)propanamide)methoxy)-3-(benzyloxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)propanamide formate (I-27c, 31 mg, 0.037 mmol) and 10 wt% Pd / C (8.0 mg, 0.0075 mmol Pd) in MeOH (1.0 mL) were stirred at room temperature under H2 (1.0 atm). After 3 h, DCM (1.0 mL) was added and stirring under H2 was continued for 3 h. At that point, the reaction mixture was concentrated to dryness under reduced pressure and the residue was redissolved in DMSO (1.0 mL) and MeOH (1.0 mL). Additional 10 wt% Pd / C was added and the reaction mixture was stirred under H2 (1.0 atm) and evaluated by LCMS until the progress of the reaction stopped. Next, the mixture was syringe filtered and purified directly by reverse phase column chromatography (10 - 60% MeCN / H2O + 0.1% formic acid modifier) to afford (R)-2-(((S)-2-((S)-2-aminopropanamide)propanamide)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxypropanamide formate (I-27). MS: m / z = 695 [M+H].

[0248] Preparation of Intermediate I-28 [Chemical Structure] Step A - Synthesis of Compound I-28b A solution of (R)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)butanoic acid (I-28a, 200 mg, 0.45 mmol) in DCM (3.0 mL) was added to diethylamine (0.14 mL, 1.4 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, and the resulting crude (R)-4-amino-3-((tert-butoxycarbonyl)amino)butanoic acid (I-28b) was used directly in the next reaction.

[0249] Step B - Synthesis of Compound I-28c To a solution of (R)-4-amino-3-((tert-butoxycarbonyl)amino)butanoic acid (I-28b, 0.099 g, 0.45 mmol) and N-methylmorpholine (0.050 mL, 0.45 mmol) in DMF (3.0 mL) was added 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23-octaoxahexacosane-26-oate (0.23 g, 0.45 mmol). The reaction mixture was stirred at room temperature for about 3 hours. Using a small amount of MeOH, the mixture was transferred to a new vial and concentrated under reduced pressure. The remaining product was washed with ether. The resulting crude (R)-29-((tert-butoxycarbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azapentatriacontan-31-oic acid (I-28c) was dried under vacuum and used directly in the next reaction.

[0250] Step C - Synthesis of Compound I-28d (R)-29-((tert-Butoxycarbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azapentatriacontan-31-oic acid (I-28c, 0.28 g, 0.45 mmol) and TFA (0.15 mL, 1.9 mmol) were added to a mixture of DCM (5.0 mL). The reaction mixture was stirred overnight at room temperature. Next, additional TFA was added and the reaction mixture was stirred at room temperature for about 5.5 h. The mixture was concentrated under reduced pressure, and the resulting (R)-29-amino-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azapentatriacontan-31-oic acid, TFA (I-28d) was used directly as a crude product in the next reaction.

[0251] Step D: Synthesis of Compound I-28 (R)-29-Amino-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azapentatriacontan-31-oic acid, TFA (I-28d, 280 mg, 0.45 mmol) and maleic anhydride (45 mg, 0.45 mmol) were added to pure ethanol (3.0 mL), followed by triethylamine (0.20 mL, 1.4 mmol). The mixture was stirred at room temperature for about 4 h 20 min, and then additional maleic anhydride (14 mg) was added. Next, the reaction mixture was stirred overnight at room temperature. The solution was concentrated under reduced pressure, then acetic anhydride (2.0 mL, 21 mmol) was added, followed by sodium acetate (75 mg, 0.91 mmol). The reaction mixture was heated to 65 °C and allowed to proceed for about 2 h 20 min. The obtained product was cooled to room temperature, then 1 M HCl (0.50 mL) was added, and then silica gel flash column chromatography (0 - 100% MeOH / DCM) was performed on the obtained product to obtain (R)-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azapentatriacontan-31-oic acid (I-28). MS: m / z = 593.6 [M + H].

[0252] Production of Intermediate I-29 [Chemical formula] Step A: Synthesis of Compound I-29c Cbz-N-amido-PEG4-acid (I-29a, 300 mg, 0.76 mmol) was added to a vial containing DMF (13 mL), HATU (240 mg, 0.63 mmol), (S)-2-amino-N 1 ,N 5 ,N-bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)pentanediamide (I-29b, 300 mg, 0.63 mmol) and Hunig's base (0.11 mL, 0.63 mmol). The reaction mixture was stirred at room temperature overnight and concentrated under reduced pressure. The resulting residue was purified by C18 flash column chromatography (0-50% MeCN / water) to afford benzyl ((17S,23S,24R,25R,26R)-23,24,25,26,27-pentahydroxy-15,20-dioxo-17-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-3,6,9,12-tetraoxa-16,21-diazaheptacocyl)carbamate (I-29c). MS: m / z = 855.6 [M+H].

[0253] Step B: Synthesis of Compound I-29d Benzyl ((17S,23S,24R,25R,26R)-23,24,25,26,27-pentahydroxy-15,20-dioxo-17-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-3,6,9,12-tetraoxa-16,21-diazaheptacocyl)carbamate (I-29c, 1.0 g, 1.2 mmol) was added to a flask containing water (3.9 mL) and EtOH (20 mL). Palladium / carbon (0.12 g, 1.2 mmol) was added, and then triethylsilane (1.9 mL, 12 mmol) was added slowly (gas evolution). The resulting emulsion was stirred at room temperature for 1 hour, then filtered and washed with EtOH. The filtrate was concentrated under reduced pressure to afford (S)-2-(1-amino-3,6,9,12-tetraoxapentadecan-15-amide)-N 1 ,N 5-Bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)pentanediamide (I-29d) was directly used in the following reaction.

[0254] Step C: Synthesis of compound I-29e (S)-2-(1-Amino-3,6,9,12-tetraoxapentadecan-15-amide)-N 1 ,N 5 -Bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)pentanediamide (I-29d, 200 mg, 0.28 mmol) was added to a vial containing DMF (2.0 mL), N-ethyl-N-isopropylpropan-2-amine (72 mg, 0.56 mmol), and 16-((2,5-dioxopyrrolidin-1-yl)oxy)-16-oxo-4,7,10,13-tetraoxahexadecanoic acid (110 mg, 0.28 mmol). The reaction mixture was stirred at room temperature for 2 hours and then purified by C18 flash column chromatography (0 - 40% MeCN / water + 0.1% TFA modifier) to obtain (34S,40S,41R,42R,43R)-40,41,42,43,44-pentahydroxy-16,32,37-trioxo-34-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-4,7,10,13,20,23,26,29-octaoxa-17,33,38-triazatetratetracontanoic acid (I-29e). MS: m / z = 997.4 [M+H].

[0255] Step D: Synthesis of compound I-29 Using I-29e and I-3c, purification was carried out by C18 flash column chromatography (0 - 60% MeCN / water) according to the same HATU protocol as described in step A to obtain (2S,38S,44S,45R,46R,47R)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-44,45,46,47,48-pentahydroxy-5,20,36,41-tetraoxo-38-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-8,11,14,17,24,27,30,33-octaoxa-4,21,37,42-tetraazatetracosanoic acid (I-29) as a solid. MS: m / z = 1164.6 [M+H].

[0256] One of ordinary skill in the art can understand the method for manufacturing other PEG-sugar intermediates by making necessary modifications and following the intermediate-29 manufacturing procedure.

[0257] Example 1 Manufacture of Example 1 [Chemical formula] Step A: Synthesis of Compound 1b To a solution of (R)-3-hydroxydihydrofuran-2(3H)-one (1a, 2.2 g, 21.6 mmol) in MeOH (20 mL) was added Dowex 50W X8, hydrogen form, strongly acidic, 200 - 400 mesh resin (1.1 g). The reaction mixture was stirred at room temperature for 2 hours and 20 minutes. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure. The resulting methyl (R)-2,4-dihydroxybutanoate (1b) was used directly in the next reaction as an oil.

[0258] Step B: Synthesis of Compound 1c (R)-Methyl 2,4-dihydroxybutanoate (1b, 3.1 g, 23 mmol) and (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazoundecane-11-yl acetate (I-2, 1.5 g, 3.3 mmol) in DCM (15 mL) were added trifluoroacetic acid (0.80 mL, 10 mmol). The reaction mixture was stirred at room temperature for about 2.5 h (monitored by LCMS). For that solution, direct silica gel flash column chromatography (0-10% MeOH / DCM) was performed to obtain methyl (5S,8S,15R)-1-(9H-fluoren-9-yl)-15-hydroxy-5,8-dimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecane-16-oate (1c) as a solid. MS: m / z = 550 [M+Na].

[0259] Step C: Synthesis of Compound 1d To a solution of methyl (5S,8S,15R)-1-(9H-fluoren-9-yl)-15-hydroxy-5,8-dimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecane-16-oate (1c, 0.41 g, 0.79 mmol) in DMSO (10 mL) was added a solution of Novozym 51032 (5.0 mL) in 0.1 M K3PO4 pH 8 buffer (100 mL). The reaction mixture was heated to 30 °C and allowed to proceed overnight. The mixture was cooled and diluted with 3:1 CHCl3:IPA (100 mL) and 1 M HCl (12 mL). After extraction, the mixture was passed through a hydrophobic membrane phase separator. The aqueous layer was washed again with 3:1 CHCl3:IPA mixture (100 mL) and the mixture was passed through the phase separator. The combined organic layers were passed through the phase separator again and concentrated under reduced pressure. For the crude product, column chromatography (0-60% MeOH / DCM, 100% MeOH flash) was performed to obtain (5S,8S,15R)-1-(9H-fluoren-9-yl)-15-hydroxy-5,8-dimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecane-16-oic acid (1d) as a solid. MS: m / z = 536 [M+Na].

[0260] Synthesis of step D - compound 1e (5S,8S,15R)-1-(9H - Fluoren - 9 - yl)-15 - hydroxy - 5,8 - dimethyl - 3,6,9 - trioxo - 2,12 - dioxo - 4,7,10 - triazapentadecan - 16 - oic acid (1d, 0.28 g, 0.55 mmol) in DMF (6.0 mL) was added with diethylamine (0.18 mL, 1.7 mmol). The reaction solution was stirred at room temperature for about 1 hour and 45 minutes. The mixture was concentrated under reduced pressure to remove the excess diethylamine. The obtained solution of (R)-4 - (((S)-2 - ((S)-2 - aminopropanamido)propanamido)methoxy)-2 - hydroxybutanoic acid diethylamine salt (1e) was directly used in the next reaction as a crude product.

[0261] Step E: Synthesis of compound 1g (R)-4 - (((S)-2 - ((S)-2 - aminopropanamido)propanamido)methoxy)-2 - hydroxybutanoic acid, diethylamine salt (1e, 0.044 g, 0.15 mmol) in DMF (1.0 mL) was added with N - methylmorpholine (0.020 mL, 0.18 mmol), and then 2,5 - dioxopyrrolidin - 1 - yl 3-(2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl)propanoate (1f, 0.040 g, 0.15 mmol). The reaction solution was stirred at room temperature for about 1 hour and 45 minutes. Next, the solution was concentrated under reduced pressure. The residue was washed three times with ether. The obtained (5S,8S,15R)-1-(2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl)-15 - hydroxy - 5,8 - dimethyl - 3,6,9 - trioxo - 12 - oxa - 4,7,10 - triazapentadecan - 16 - oate (1g) was dried in vacuo and directly used in the next reaction.

[0262] Step F: Synthesis of compound 1 (5S,8S,15R)-1-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-hydroxy-5,8-dimethyl-3,6,9-trioxo-12-oxa-4,7,10-triazapentadecan-16-oate (1 g, 66 mg, 0.15 mmol) and N-methylmorpholine (0.020 mL, 0.18 mmol) in DMF (1.0 mL) were added HATU (57 mg, 0.15 mmol). The reaction mixture was stirred at room temperature for about 5 minutes, then a solution of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-aminium methanesulfonate (I-15a, 0.30 mL, 0.11 - 0.12 mmol, solution in about 0.35 - 0.40 M DMF containing 3.0 equivalents of Et3N) was added. The reaction mixture was stirred at room temperature for about 4 hours. The reaction mixture was filtered and purified by reverse-phase column chromatography (25 - 65% MeCN / water + 0.1% FA modifier) to give (R)-4-(((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propanamido)propanamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizin-1-yl)-2-hydroxybutanamide, formic acid (1) as an oil. MS: m / z = 860 [M+H]. 11H NMR (500 MHz, DMSO-d6) δ 8.56 (t, J = 6.5 Hz, 1H), 8.49 (d, J = 9.0 Hz, 1H), 8.30 (s, 2H), 8.18 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 7.4 Hz, 1H), 7.77 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 6.99 (s, 2H), 5.59 - 5.52 (m, 1H), 5.46 - 5.37 (m, 2H), 5.22 (d, J = 18.9 Hz, 1H), 5.11 (d, J = 18.8 Hz, 1H), 4.61 - 4.55 (m, 1H), 4.54 - 4.47 (m, 1H), 4.26 - 4.18 (m, 2H), 4.05 (dd, J = 9.2, 3.4 Hz, 1H), 3.61 - 3.57 (m, 3H, overlapping with water signal), 3.54 - 3.50 (m, 3H, overlapping with water signal), 3.26 - 3.18 (m, 2H, overlapping with water signal), 3.17 - 3.09 (m, 2H, overlapping with water signal), 2.89 (s, 1H), 2.73 (s, 1H), 2.41 - 2.34 (m, 5H), 2.21 - 2.12 (m, 2H), 2.08 - 1.99 (m, 1H), 1.91 - 1.78 (m, 3H), 1.23 (d, J = 7.1 Hz, 3H), 1.15 (d, J = 7.1 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H). With a slight change in the reaction time and using appropriate reactants and / or reagents (using I-2 and / or 1f (other suitable commercially available NHS esters) instead), the following compounds of the present disclosure in Table 6 were prepared using the same method as described in Example 1.

[0263] Table 6

Table 6

[0264] Step B: Synthesis of Compound 13c (5S,8S,13R)-1-(9H-Fluoren-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecan-15-oic acid (13b, 200 mg, 0.40 mmol) in DMF (2.7 mL) was added to (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione (I-15a, 1.5 mL, 0.53 - 0.60 mmol, solution in about 0.35 - 0.40 M DMF containing 3.0 equivalents of Et3N), HATU (200 mg, 0.52 mmol), and DIPEA (210 μL, 1.2 mmol). The resulting reaction mixture was stirred at room temperature for 1 hour and purified by reverse-phase flash column chromatography (20 - 95% MeCN / water + 0.1% formic acid) to give (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((((R)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-4-oxobutan-2-yl)oxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (13c). MS: m / z = 915 [M+H].

[0265] Step C: Synthesis of Compound 13 A solution of ((9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((((R)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-4-oxobutan-2-yl)oxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (13c, 60 mg, 0.066 mmol) in DMF (1.0 mL) was added with 4-methylpiperidine (1.6 μL, 0.013 mmol), and the reaction solution was stirred at 40 °C for 30 minutes. The reaction mixture was cooled to room temperature, and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (1f, 21 mg, 0.079 mmol) was added. The resulting mixture was stirred at room temperature for 10 minutes and purified by reverse-phase column chromatography (20 - 60% MeCN / water + 0.1% formic acid) to obtain (R)-3-(((R)-2-((R)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide)propanamide)propanamide)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)butanamide (13). MS: m / z = 844 [M+H]. 11H NMR (500 MHz, DMSO-d6) δ 8.53 (t, J = 6.5 Hz, 1H), 8.43 (d, J = 8.7 Hz, 1H), 8.32 (s, 1H), 8.17 (d, J = 7.2 Hz, 1H), 7.98 (d, J = 7.4 Hz, 1H), 7.79 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 7.00 (s, 2H), 5.58 - 5.53 (m, 1H), 5.42 (s, 1H), 5.20 (q, J = 18.8 Hz, 2H), 4.59 (dd, J = 10.2, 6.9 Hz, 1H), 4.48 (dd, J = 10.2, 6.3 Hz, 1H), 4.23 - 4.12 (m, 2H), 4.03 - 3.95 (m, 1H), 3.61 - 3.56 (m, 1H), 3.21 - 3.13 (m, 1H), 2.46 - 2.34 (m, 6H), 2.22 (dd, J = 14.1, 6.3 Hz, 1H), 2.17 - 2.08 (m, 2H), 1.93 - 1.80 (m, 2H), 1.20 (d, J = 7.1 Hz, 3H), 1.15 - 1.10 (m, 5H), 0.87 (t, J = 7.3 Hz, 3H). The reaction time was slightly changed, and the following compounds of the present disclosure in Table 7 were prepared using a method similar to that described in Example 13, instead using appropriate reactants and / or reagents (I-2, 13a (using other commercially available acids), and / or 1f (using other appropriate commercially available NHS esters)).

[0266] Table 7

Table 7

Chemical formula

[0267] Table 8

Table 8

Chemical formula

[0268] Step B: Synthesis of Compound 28 1-(4-((S)-2-((S)-2-Aminopropanamido)propanamido)phenyl)-2-morpholino-2-oxoethyl (1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-methyl-1-oxopropan-2-yl)carbamate (28a, 5.9 mg, 0.0061 mmol), DIPEA (2.1 μL, 0.012 mmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (1f, 4.9 mg, 0.018 mmol) and DMF (0.50 mL) were stirred at room temperature for 20 minutes. The reaction mixture was purified directly by reverse phase column chromatography (10 - 60% MeCN / water + 0.1% formic acid modifier) to give 1-(4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propanamido)propanamido)phenyl)-2-morpholino-2-oxoethyl (1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-methyl-1-oxopropan-2-yl)carbamate formate (28). MS: m / z = 1076 [M+H]. 11H NMR (500 MHz, DMSO-d6) δ 9.88 (d, J = 10.5 Hz, 1H), 8.53 (s, 1H), 8.23 - 8.18 (m, 1H), 8.13 (d, J = 7.1 Hz, 1H), 8.08 - 8.00 (m, 1H), 7.79 (d, J = 11.1 Hz, 1H), 7.66 - 7.48 (m, 3H), 7.39 - 7.28 (m, 2H), 7.02 - 6.94 (m, 3H), 6.76 (s, 1H), 6.58 - 6.51 (m, 1H), 6.12 (s, 0.5H), 6.03 (s, 0.5H), 5.54 - 5.12 (m, 5H), 4.40 - 4.30 (m, 1H), 4.25 - 4.17 (m, 1H), 3.60 (t, J = 7.2 Hz, 2H), 3.53 - 3.39 (m, 3H), 3.19 - 3.03 (s, 4H), 2.99 - 2.82 (m, 1H), 2.43 - 2.37 (m, 5H), 2.23 - 2.12 (m, 1H), 2.10 - 1.97 (m, 1H), 1.93 - 1.76 (m, 2H), 1.48 - 1.25 (m, 9H), 1.16 (d, J = 7.1 Hz, 3H), 0.91 - 0.80 (m, 3H). The reaction time was slightly changed and appropriate reactants and / or reagents (I-19, and / or 1f (using other appropriate commercially available NHS esters)) were used instead, and the following compounds of the present disclosure in Table 9 were prepared using the same method as described in Example 28.

[0269] Table 9 [Table 9] Example 32 Preparation of Example 32 [Chemical formula] Step A: Synthesis of Compound 32 Mixture A: A mixture of ((9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-methyl-1-oxopropan-2-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (I-21, 31 mg, 0.030 mmol) and NMM (13 μL, 0.12 mmol) in DMF (0.30 mL) was stirred at 50 °C overnight. Then it was cooled to 0 °C.

[0270] Mixture B: In a separate vial, a mixture of (S)-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-oic acid (I-3, 47 mg, 0.082 mmol) and COMU (23 mg, 0.054 mmol) in DMF (0.30 mL) was cooled to 0 °C, and at that time 2,6-lutidine (19 μL, 0.17 mmol) was added. The mixture was stirred at 0 °C for 30 minutes and then added to the pre-cooled mixture A.

[0271] The combined reaction mixture was stirred at 0 °C for 40 minutes and then purified directly by reverse-phase column chromatography (10 - 60% MeCN / H2O + 0.1% formic acid modifier) to give 4-((32S,35S)-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-32,35-dimethyl-26,30,33-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,31,34-triazaoctatriacontan-36-amido)benzyl (1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-methyl-1-oxopropan-2-yl)carbamate (32). Assuming a 4:1 dr at the maleimide stereocenter by NMR. MS: m / z = 687 [M+2H]. 11H NMR (500 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.30 (d, J = 7.1 Hz, 1H), 8.17 (d, J = 7.3 Hz, 1H), 8.12 (d, J = 7.1 Hz, 1H), 7.90 (t, J = 6.2 Hz, 1H), 7.79 (d, J = 11.1 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.31 (s, 1H), 7.20 (s, 1H), 7.11 - 7.05 (m, 2H), 7.02 (s, 2H), 6.72 (br s, 2H), 6.50 (s, 1H), 5.52 - 5.45 (m, 1H), 5.45 - 5.34 (m, 2H), 5.31 - 5.17 (m, 2H), 4.86 - 4.74 (m, 2H), 4.58 (dd, J = 10.2, 4.4 Hz, 1H), 4.42 - 4.34 (m, 1H), 4.31 - 4.23 (m, 1H), 3.82 - 3.73 (m, 1H), 3.55 - 3.40 (m, 30H), 3.23 (s, 3H), 3.12 - 3.02 (m, 2H), 2.38 (s, 3H), 2.24 - 2.07 (m, 3H), 2.07 - 1.96 (m, 1H), 1.89 - 1.76 (m, 2H), 1.38 (s, 6H), 1.31 (d, J = 7.0 Hz, 3H), 1.14 (d, J = 7.2 Hz, 3H), 0.81 (t, J = 7.2 Hz, 3H). Example 33 Preparation of Example 33

Chemical Structure

[0272] Table 10

Table 10

[0273] Example 36 The antibody (siltuximab, 20 mg) was exchanged with PBS (10 mM pH 7.4) and made to a concentration of 10 mg / mL with PBS (1500 μL) and Tris / EDTA (400 μL) (10 volume % 500 mM pH 8 TRIS / 90% 25 mM EDTA). An aqueous solution of 3,3′,3″-phosphanetriyltripropionate hydrochloride (6 equivalents, 81 μL, 0.810 μmol) was added, and the mAb decreased at room temperature for 2 hours. DMSO (100 μL) was added, and then a solution of 10 mM 1 (135 μL, 10 equivalents) in DMSO was added. The reaction solution was mixed overnight at room temperature.

[0274] The mixture was purified and exchanged with 20 mM sodium acetate buffer at pH 5.5 using a desalting column (AKTA (trademark) chromatography system, monitored at 280 nm), and then 9% sucrose was added to obtain ADC Example 36. The solution was characterized by LCMS (Agilent PLRP-S column, 1000 Å, 5 μm, 15 - 90% MeCN / H2O + 0.1% formic acid, column temperature 80 °C) and SEC (Acquity UPLC Protein BEH SEC, 200 Å, 1.7 μm, 100 mM sodium phosphate, 200 mM NaCl, 0.02% azide, 5% IPA was added to the mobile phase in the case of hydrophobic ADC).

[0275] R0R1 ADC data Table 11 below shows the average DAR and percent aggregation of ROR1 ADCs using the exemplary compounds of the present disclosure and the binding protocol described above.

[0276] Table 11

Table 11

[0277] The EMT6 cells were washed once with PBS (without calcium or magnesium), then 5 mL of 0.25% trypsin-EDTA (from Thermo Fisher, catalog number 25200056) was added, and the flask was incubated at 37 °C for about 3 minutes. Next, 10 mL of cell culture medium (RPMI 1640 (Gibco™ 72400-047) + 10% FBS (Gibco™ 26140-079)) was added, and the cells were separated by pipetting up and down several times. The cells were transferred to a 15 mL conical tube and centrifuged at 300 g for 5 minutes. Next, the cell pellet was resuspended in 2 mL of cell culture medium, and the cells were counted with Vi-CELL (the viability of all cell lines was >95%). Next, the cells were seeded into a 96-well plate (Corning™ 3904), and 200 live cells were placed in 90 μL of cell culture medium per well and cultured overnight in a cell culture incubator.

[0278] The next day, serial dilutions of the ADC were prepared in cell culture medium (the starting concentration of the ADC was 1 μM and the dilution factor was 1 / 5), and 10 μL of the dilution was added to each well (the total volume was 100 μL per well). Only the inner 60 wells on the plate were used for drug treatment and untreated controls. Next, the plate was incubated in a cell culture incubator for an additional 96 hours.

[0279] After 4 days of treatment, the plate and its contents were equilibrated at room temperature for about 30 minutes. Further, the CellTiter-Glo™ buffer was thawed and equilibrated at room temperature, and an appropriate volume of the CellTiter-Glo™ buffer was transferred to an amber bottle containing the CellTiter-Glo™ substrate to regenerate the lyophilized enzyme / substrate mixture (Promega™ catalog number G7573). Next, 100 μL of the CellTiter-Glo™ reagent was added to each well, and the contents were mixed on an orbital shaker for 2 minutes to induce cell lysis. The plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. Luminescence was recorded with a PerkinElmer Multimode Plate Reader EnVision™. The data was analyzed with GraphPad Prism8 for EC50 was obtained.

[0280] An exemplary R0R1 ADC of the present disclosure was tested in the above EMT6 cytotoxicity assay, and the results are provided in Table 12 below.

[0281] Table 12 [Table 12] Example 64 The antibody (sacituzumab, 20 mg) was exchanged with PBS (10 mM pH 7.4) and made to a concentration of 10 mg / mL with PBS (1500 μL) and Tris / EDTA (400 μL) (10 volume % 500 mM pH 8 TRIS / 90% 25 mM EDTA). An aqueous solution of 3,3′,3″-phosphanetriyltripropionate hydrochloride (6 equivalents, 81 μL, 0.810 μmol) was added and the mAb was reduced at room temperature for 2 hours. DMSO (100 μL) was added, followed by addition of a solution of 10 mM 1 (135 μL, 10 equivalents) in DMSO. The reaction solution was mixed overnight at room temperature. If the reaction did not appear to be complete, up to an additional 4 equivalents of linker-payload were added.

[0282] The mixture was purified and exchanged with either pH 5.5 20 mM sodium acetate buffer or pH 6.5 10 mM histidine buffer on a desalting column (AKTA™ chromatography system, monitored at 280 nm), and then 9% sucrose was added to obtain ADC Example 64. The solution was characterized by LCMS (Agilent PLRP-S column, 1000 Å, 5 μm, 15-90% MeCN / H2O + 0.1% formic acid, column temperature 80° C.) and SEC (Acquity UPLC Protein BEH SEC, 200 Å, 1.7 μm, 100 mM sodium phosphate, 200 mM NaCl, 0.02% azide, 5% IPA was added to the mobile phase in the case of hydrophobic ADCs).

[0283] Trop2 ADC data Table 13 below shows the average DAR and percent aggregation of Trop2 ADCs using the exemplary compounds of the present disclosure and the binding protocols described above.

[0284] Table 13 [Table 13] *Note: The binding procedure for DAR is as follows.

[0285] Trastuzumab having two modified Cys residues was decapped using literature procedures (WO2017072662) and diluted to 5 mg / mL in PBS. Next, 9% DMSO was added, and 28-(4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propanamido)propanamido)phenyl)-26-methyl-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azaoctacosan-28-yl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)(2,2,3,3-tetrafluoropropyl)carbamate (31, 27 μL, 0.27 μmol) was added as a 10 mM DMSO solution, and the reaction mixture was mixed at room temperature for 2 hours. The ADC was purified by AKTA (trademark) (desalting column, histidine pH 6.5 buffer, monitored at 280 nm) and characterized by LCMS and SEC (as described above).

[0286] Trop2 N87 Cytotoxicity Assay Protocol For the exemplary Trop2 ADCs of the present disclosure, a cell-based cytotoxicity assay (NCI-N87 cells) was performed using the following protocol.

[0287] Step 1: Seed on a 384-well plate for the assay on Day 0 (45 μL per well) Cells (NCI-N87) were incubated in a 37 °C water bath for less than 1 minute until a little ice remained in the vial to quickly thaw in the cryovial. The vial was immediately taken out and wiped with 70% ethanol. The cells were transferred from the vial to a sterile centrifuge tube containing 8 mL of pre-warmed cell culture medium (RPMI-1640 (catalog number 30-2001) + 10% FBS + 1% P / S). The vial was rinsed with an additional 1 mL of medium to ensure that all the cells were transferred to the centrifuge tube. Then, the cells were centrifuged at 150 g for 5 minutes. The supernatant was aspirated, and the cell pellet was resuspended in 10 - 20 mL of cell culture medium (RPMI-1640 (catalog number 30-2001) + 10% FBS + 1% P / S). The cells were counted using Vi-cell and adjusted to 1,500 cells / 45 μL per well. Next, using a Standard Cassette Combi, 45 μL / well of the cells were added to a Corning® 384-well low-flange white flat-bottom polystyrene TC-treated microplate (Corning, catalog number 3570) (if necessary, one dummy plate was dispensed with 20 μL to standardize the Combi at a moderate speed). The plate was centrifuged at 150 g for 30 seconds.

[0288] Step 2: Addition of ADC on Day 1 The ADC vial and reference stock were taken out and thawed at room temperature. The tube was centrifuged at 2000 g for 30 seconds. A 10-fold intermediate assay plate (Waters plate, catalog number 186002632) was prepared using a Bravo liquid handler. Serial dilutions were performed using an appropriate buffer (10 mM pH 6.5 histidine 9% sucrose buffer). Medium (without cells) was used for Max_E. Next, using a Bravo liquid handler, 5 μL of the 10-fold stock was added from the intermediate plate to the assay plate at a very low speed so as not to disrupt the cell monolayer. The plate was centrifuged at 150 g for 30 seconds.

[0289] Step 3: CellTiter-Glo 2.0 assay on Day 7 (Promega, catalog number G9242) (CellTiter-Glo kit stored at -70 °C) The CellTiter-Glo® 2.0 reagent was thawed overnight at 4°C (the reagent was not exposed to temperatures exceeding 25°C). The kit was equilibrated to room temperature for approximately 30 minutes. Using a Standard Cassette Combi, 20 μL of the CellTiter-Glo® 2.0 reagent was added to 50 μL of the medium containing cells. The contents were mixed on an orbital shaker for 2 - 3 minutes to induce cell lysis. The plate was centrifuged at 150 g for 30 seconds. The plate was incubated at room temperature for 5 minutes to stabilize the luminescence signal. Luminescence was recorded and the EC 50 values were calculated using an integration time of 0.25 - 1 second per well as a guideline.

[0290] An exemplary Trop2 ADC of the present disclosure was tested in the above NCI-N87 cytotoxicity assay, and the results are provided in Table 14 below.

[0291] Table 14

Table 14

Claims

1. A compound having Structural Formula I or a salt of the compound. 【Chemical Formula 1】 [Wherein, Z is hydrogen and -CH ２ C(R ｘ )(R ｙ )(CHF ２ selected from; Z １ is selected from -NH- and -O-; Z ２ is non-existent or -CR ｂ R ｂ -, -CH ２ CR ｂ R ｂ -, and -CR ｂ R ｂ CH ２ is selected from; Each R ｂ is independently selected from hydrogen, -C １－６ alkyl, and hydroxyl; or two adjacent Rs ｂ are combined to form a spirocycloalkyl; Each R ｃ is independently selected from hydrogen, -C １－６ alkyl, halogen, and hydroxyl; or two adjacent Rs ｃ are combined to form a spirocycloalkyl; R ｘ and R ｙ are independently selected from hydrogen, C １－６ alkyl, C １－３ haloalkyl, halogen, hydroxyl, and -C １－６ alkyl-OH; or R ｘ and R ｙ are combined to form C ３－６ cycloalkyl or spirocycloalkyl; X is [Chemical 2] 【Chem.】 [Chemical] a linking group selected from; [Chemical Formula 3] represents the bonding position to Structural Formula I; X １ is polyethylene glycol (PEG); X ３ is hydrogen or -C(O)NR ａ R ｚ ; X ４ is hydrogen or [Chemical Formula 4] and R ａ and R ｚ are independently selected from hydrogen, C １－６ alkyl, and X １ or R ａ and R ｚ combine to form C ３－１０ cycloalkyl, or a 3- to 10-membered heterocyclic ring; R ｄ is hydrogen, -CH ２ NHCOX １ or -CH ２ NHCOX １ Q; Q is C １－６ alkyl or hydrogen.]

2. Z １ The compound according to claim 1, or a salt thereof, wherein Z is -O-.

3. Z １ The compound according to claim 1, or a salt thereof, wherein Z is -NH-.

4. R ｄ The compound or a salt thereof according to any one of claims 1 to 3, wherein R is hydrogen.

5. R ｄ is -CH ２ NH C(O)X １ Q, a compound according to any one of claims 1 to 3 or a salt of the compound.

6. Each R ａ The compound according to any one of claims 1 to 5, or a salt of the compound, wherein each R is independently selected from hydrogen and methyl.

7. Each R ｃ is independently selected from hydrogen, hydroxyl, methyl, fluorine, and CHF ２ or two adjacent Rs ｃ are combined to form spirocyclopropyl, the compound according to any one of claims 1 to 6, or a salt of the compound.

8. The compound or a salt of the compound according to any one of Claims 1 to 7, wherein Z is hydrogen.

9. Z is -CH ２ C(F ２ )CHF ２ The compound according to any one of claims 1 to 7, or a salt of the compound.

10. Z ２ is -CH ２ CH ２ -, -CH ２ -, -CH(CH ３ ), -CH ２ CH(CH ３ )-spirocyclopropyl(CH ２ ) ２ -, and -(CH ２ ) ２ The compound according to any one of claims 1 to 9, or a salt of the compound, which is selected from -CH

11. Z ２ The compound according to any one of claims 1 to 9, or a salt thereof, wherein Z is absent.

12. X is W1: [Chemical Formula 5] The compound or a salt of the compound according to any one of Claims 1 to 11.

13. X is W2: 【Chemical Formula 6】 The compound or a salt of the compound according to any one of Claims 1 to 11.

14. X is W3: 【Chemical Formula 7】 The compound or a salt of the compound according to any one of Claims 1 to 11.

15. X is W4: [Chemical Formula 8] The compound or a salt of the compound according to any one of Claims 1 to 11.

16. X is W5: 【Chemical Formula 9】 The compound or a salt of the compound according to any one of Claims 1 to 11.

17. X is W6: 【Chemical Formula 10】 The compound or a salt of the compound according to any one of Claims 1 to 11.

18. X is W7: 【Chemical 11】 The compound or a salt of the compound according to any one of Claims 1 to 11.

19. X is W8: 【Chemical 12】 The compound or a salt of the compound according to any one of Claims 1 to 11.

20. The compound or a salt of the compound according to any one of Claims 1, 4 to 7 and 10 to 18, represented by the following Structural Formula II: 【Chemical 13】

21. The compound or a salt of the compound according to any one of Claims 1, 4 to 7 and 10 to 18, represented by the following Structural Formula III: 【Chemical 14】

22. A linker-drug compound selected from the following, or a salt of the compound. 【Table 1】

23. The compound of the following Structural Formula IV or a pharmaceutically acceptable salt of the compound. 【Chemical Formula 15】 [Wherein, Ab is an antibody; p is an integer from 1 to 24; Z is hydrogen and -CH ２ C(R ｘ )(R ｙ ).CHF ２ selected from; Z １ is selected from -NH- and -O-; Z ２ is non-existent or -CR ｂ R ｂ -, -CH ２ CR ｂ R ｂ -, and -CR ｂ R ｂ CH ２ is selected from; Each R ｂ is independently selected from hydrogen, -C １－６ alkyl, and hydroxyl; or two adjacent Rs ｂ are combined to form a spirocycloalkyl; Each R ｃ is independently selected from hydrogen, -C １－６ alkyl, halogen, and hydroxyl; or two adjacent Rs ｃ are combined to form a spirocycloalkyl; R ｘ and R ｙ are independently selected from hydrogen, C １－６ alkyl, C １－３ haloalkyl, halogen, hydroxyl, and -C １－６ alkyl-OH; or R ｘ and R ｙ are combined to form C ３－６ cycloalkyl or spirocycloalkyl; X is 【Chemical 16】 【Chem.】 a linking group selected from; 【Chemical 17】 represents the bonding position to Structural Formula I; X １ is polyethylene glycol (PEG); X ２ is -NR ａ X １ -, or 【Chemical Formula 18】 and X ３ is hydrogen or -C(O)X ２ ; X ４ is hydrogen or 【Chemical Formula 19】 and R ａ is selected from hydrogen and C １－６ alkyl; R ｄ is hydrogen or -CH ２ NHCOX １ Q.]

24. The compound or a pharmaceutically acceptable salt of the compound according to Claim 23, which is the following. 【Table 2】 [In the above, Ab is an antibody and p is an integer from 1 to 12.]

25. The compound of the following Structural Formula V or a pharmaceutically acceptable salt or solvate of the compound. 【Chemical 20】 [Wherein, R ｊ is OH, -NH ２ , -NHR ｋ R ｇ , -NHR ｋ NH(CH ２ ) ｎ OR ｑ , -NHR ｋ NH(CH ２ ) ｎ OC(O)CH ３ , -NHX １ａ (CH ２ ) ｎ C(O)R ｋ NHCH ２ OC(O)CH ３ , -NHR ｋ NHR Ｌ , -NHX １ａ R ｋ NHR Ｌ , and -NHCH ２ O(CH ２ ) ｎ CH(OH)C(O)OH selected from; R ｇ is COOH or CONH ２ ; R ｋ is an amino acid residue of 10 or fewer amino acids; R Ｌ is 【Chemical 21】 selected from; R ｐ is NH ２ and 【Chemical 22】 selected from; R ｑ is hydrogen or C １－６ alkyl; X １ａ is PEG of 1-24 -CH ２ CH ２ O-subunit; R ｄ１ is hydrogen, -CH ２ NHCOX １ａ Q or -CH ２ NHCOX ２ｌ Q; Q is C １－６ alkyl or H; X ２ｌ is selected from PEG, PEG - amino sugar, and p - aminobenzylcarbonyl of 1 - 24 - CH ２ CH ２ O - sub - units; n is 1, 2, 3, or 4.]

26. The compound according to claim 25 selected from the following, or a pharmaceutically acceptable salt or solvate of the compound. 【Chemical 23】 【Chem.】 [wherein, X １ is polyethylene glycol (PEG); X ２ is -NR ａ X １ -, or 【Chemical 24】 is; X ３ is hydrogen or -C(O)X ２ ; X ４ is hydrogen or 【Chemical 25】 is; R ａ is selected from hydrogen and C １－６ alkyl; R ｄ is hydrogen or -CH ２ NH C(O)X １ Q.] [

27. ] The compound according to any one of claims 25 to 26 selected from the following, or a salt of the compound. 【Table 3】 [wherein, X １ａ is PEG of a 1 to 24 - CH ２ CH ２ O - subunit.] [

28. ] A pharmaceutical composition comprising the compound according to any one of claims 1 to 27, or a pharmaceutically acceptable salt of the compound, and a pharmaceutically acceptable carrier. [

29. ] Use of the compound according to any one of claims 1 to 27, or a pharmaceutically acceptable salt of the compound, or the pharmaceutical composition according to claim 28, for the manufacture of a medicament for the treatment or prevention of cancer or tumor. [

30. ] A method for treating or preventing cancer selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma in a subject in need of treatment, comprising administering to the subject in need of such treatment a therapeutically effective amount of the compound according to any one of claims 1 to 24, or a pharmaceutically acceptable salt of the compound, or a pharmaceutical composition comprising the compound, a salt or solvate of the compound. [

31. ] A method for treating and / or preventing a tumor, comprising administering to a patient in need of treatment a therapeutically effective amount of the compound according to any one of claims 1 to 24, or a pharmaceutically acceptable salt of the compound, or a pharmaceutical composition comprising the compound.

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