Azotomycin prodrug with preferential tumor delivery

Abstract

The present application discloses azotomycin prodrugs and their use in the treatment of diseases, disorders, or conditions associated with excessive and / or abnormal glutamine utilization, including cancer.

Description

[Technical Field]

[0001] (Statement of Government Support) This invention was made with government support under Grant CA229451 awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention. [Background technology]

[0002] Azotomycin is a diazo analog of L-glutamine isolated from Streptomyces ambofaciens. It is a potent glutamine antagonist that broadly inhibits glutamine metabolism, utilizing a reaction important for the generation of α-ketoglutarate, which is required for nucleic acid, amino acid, and protein synthesis and energy metabolism. Azotomycin has demonstrated strong efficacy in multiple preclinical cancer models and exploratory clinical trials. Despite its promise, development was discontinued due to toxicities, including vomiting, anorexia, bloody stool, hepatotoxicity, nephrotoxicity, and central nervous system toxicity (tremors, convulsions, ataxia, and lethargy). Summary of the Invention

[0003] In some aspects, the subject of the present disclosure is a compound of formula (I): [ka] R1 and R2 each independently represent -OR4, -NR5R6, -O - M + , and -O-(CH2CH2O) n -R7 is selected from n is an integer selected from 1, 2, 3, and 4; R4 is selected from H and C1-C4 substituted or unsubstituted branched or unbranched alkyl; R5 and R6 are each independently H, C1-C4 substituted or unsubstituted branched or unbranched alkyl, -AA-COOR7, where R7 is C1-C4 substituted or unsubstituted branched or unbranched alkyl and AA is an amino acid; M + Na + , K. + , Li + and in some embodiments, NH4 + metal cations, including but not limited to: R3 is H, -(C=O)-R8, -C(=O)-CH2-(NH-C(=O)-CH2-NR9R 10 )-CH2-R 11 , -C(=O)-CH-(NH-C(=O)-CH(NH2)-CH-(CH3)2)-((CH2)3NH-C(=O)NH2, -C(=O)-CH((CH2)4-NH-C(=O)-CH3)(NH-C(=O)-adamantane), -C(=O)-AA-R 12 , acetyl (Ac), dimethylglycine, dimethylglycyl (DMG), L-Lys(Ac), D-Val-L-Leu-L-Lys(Ac), D-Val-L-Leu-L-Lys(H), D-Val-L-Leu-L-Lys(DMG), L-Val-L-Cit, para-aminobenzoic acid (PABA), L-Cit-PABA, L-Val-L-Cit-PABA, Val-Cit-OH, dimethylglycyl-L-tryptophan, acetoxymethyl pivalate, 1-acetoxyethyl pivalate, acetoxy(phenyl)methyl pivalate, and 2-acetoxypropan-2-yl pivalate, wherein AA is an amino acid, and the amino acids of R3, R5, and R6 may be selected from the amino acids or amino acid-related substituents listed in Table 2 or Table 3, and combinations thereof; R8 is a C1-C4 substituted or unsubstituted branched or unbranched alkyl and -CH2-NR 14 R 15 is selected from R9, R 10 , R 14 , and R 15 are each independently selected from H and C1-C4 substituted or unsubstituted branched or unbranched alkyl; R 11 is selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; R 12is H, -(C=O)-R 13 and -NH-dimethylglycyl; R 13 is a C1-C4 substituted or unsubstituted branched or unbranched alkyl; provided that when R3 is H, R1 and R2 cannot both be -OH; stereoisomers and pharmaceutically acceptable salts thereof, A compound of formula (I) is provided.

[0004] In some aspects, the presently disclosed subject matter provides methods for treating a disease, disorder, or condition associated with excessive and / or abnormal glutamine utilization, the methods comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of formula (I) or a pharmaceutical formulation thereof.

[0005] In certain embodiments, the disease, disorder or condition associated with excessive and / or abnormal glutamine utilization is selected from infectious diseases, cancer, autoimmune diseases, inflammatory diseases, and neurodegenerative or neurological diseases.

[0006] In certain embodiments, the cancer is selected from a newly diagnosed cancer, a recurrent cancer, a refractory cancer, and combinations thereof.

[0007] In more particular embodiments, the cancer is selected from (i) cancer of the central nervous system, (ii) cancer associated with transplantation and / or immunosuppression, (iii) cancer resistant to chemotherapy, (iv) cancer resistant to photodynamic therapy, (v) cancer resistant to proton beam therapy, (vi) cancer resistant to radiation therapy, and (vii) cancer resistant to surgery.

[0008] In even more specific aspects, the cancer is selected from the group consisting of cervicopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, pharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, gastrointestinal cancer, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymus cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0009] In some embodiments, the method further comprises preventing recurrence in a cancer patient in remission.

[0010] In a further embodiment, the method further comprises administering a therapeutically effective amount of a compound of Formula (I) in combination with immunotherapy. In some embodiments, the immunotherapy comprises checkpoint inhibition therapy. In certain embodiments, the method comprises administering a compound of Formula (I) in combination with one or more checkpoint inhibitors. In more particular embodiments, the one or more checkpoint inhibitors are selected from anti-PD-1 Ab, anti-PD-L1 Ab, anti-CTLA-4 Ab, anti-TIGIT Ab, and combinations thereof.

[0011] Certain aspects of the presently disclosed subject matter have been described hereinabove and are addressed in whole or in part by the presently disclosed subject matter; other aspects will become apparent as the description proceeds in connection with the accompanying examples and figures best described herein below.

[0012] The patent or application contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] Having thus generally described the subject matter of the present disclosure, reference is now made to the accompanying drawings, which are not necessarily drawn to scale. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows a single time point pharmacokinetic screen in mice showing DON exposure (at 30 or 60 minutes) in EL4 tumors and plasma of azotomycin prodrugs P1-P19. [Figure 2] Figure 2A shows the pharmacokinetics of P3 in mice. The full pk of DON exposure for P3 in plasma, tumor, and jejunum is shown. Figure 2B shows the pharmacokinetics of P3 in mice. The full pk of unchanged prodrug exposure for P3 in plasma, tumor, and jejunum is shown. Figure 2C shows the pharmacokinetics of P4 in mice. The full pk of DON exposure for P4 in plasma, tumor, and jejunum is shown. Figure 2D shows the pharmacokinetics of P4 in mice. The full pk of unchanged prodrug exposure for P4 in plasma, tumor, and jejunum is shown. Figure 2E shows the pharmacokinetics of P10 in mice. The full pk of DON exposure for P10 in plasma, tumor, and jejunum is shown. DETAILED DESCRIPTION OF THE INVENTION

[0015] The presently disclosed subject matter is described more fully below with reference to the accompanying drawings, which illustrate some, but not all, embodiments of the invention. Like numerals refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter described herein will be readily apparent to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it should be understood that the presently disclosed subject matter is not limited to the particular embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0016] I. Compounds of Formula (I) In some embodiments, the subject of the present disclosure is a compound of formula (I): [ka] R1 and R2 each independently represent -OR4, -NR5R6, -O - M + , and -O-(CH2CH2O) n -R7 is selected from n is an integer selected from 1, 2, 3, and 4; R4 is selected from H and C1-C4 substituted or unsubstituted branched or unbranched alkyl; R5 and R6 are each independently H, C1-C4 substituted or unsubstituted branched or unbranched alkyl, -AA-COOR7, where R7 is C1-C4 substituted or unsubstituted branched or unbranched alkyl and AA is an amino acid; M + Na + , K. + , Li + and in some embodiments, NH4 +metal cations, including but not limited to: R3 is H, -(C=O)-R8, -C(=O)-CH2-(NH-C(=O)-CH2-NR9R 10 )-CH2-R 11 , -C(=O)-CH-(NH-C(=O)-CH(NH2)-CH-(CH3)2)-((CH2)3NH-C(=O)NH2, -C(=O)-CH((CH2)4-NH-C(=O)-CH3)(NH-C(=O)-adamantane), -C(=O)-AA-R 12 , acetyl (Ac), dimethylglycine, dimethylglycyl (DMG), L-Lys(Ac), D-Val-L-Leu-L-Lys(Ac), D-Val-L-Leu-L-Lys(H), D-Val-L-Leu-L-Lys(DMG), L-Val-L-Cit, para-aminobenzoic acid (PABA), L-Cit-PABA, L-Val-L-Cit-PABA, Val-Cit-OH, dimethylglycyl-L-tryptophan, acetoxymethyl pivalate, 1-acetoxyethyl pivalate, acetoxy(phenyl)methyl pivalate, and 2-acetoxypropan-2-yl pivalate, wherein AA is an amino acid, and the amino acids of R3, R5, and R6 may be selected from the amino acids or amino acid-related substituents listed in Table 2 or Table 3, and combinations thereof; R8 is a C1-C4 substituted or unsubstituted branched or unbranched alkyl and -CH2-NR 14 R 15 is selected from R9, R 10 , R 14 , and R 15 are each independently selected from H and C1-C4 substituted or unsubstituted branched or unbranched alkyl; R 11 is selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; R 12 is H, -(C=O)-R 13 and -NH-dimethylglycyl; R 13is a C1-C4 substituted or unsubstituted branched or unbranched alkyl; provided that when R3 is H, R1 and R2 cannot both be -OH; stereoisomers and pharmaceutically acceptable salts thereof, A compound of formula (I) is provided.

[0017] Those skilled in the art will recognize that azotomycin is expressly excluded from the compounds of formula (I).

[0018] In certain embodiments, the compound of Formula (I) is a compound of Formula (Ia): [ka] R1 and R2 are each independently -OR4, -NR5R6, and -O-(CH2CH2O) n -R7 is selected from n is an integer selected from 1, 2, 3, and 4; R4 is a C1-C4 substituted or unsubstituted branched or unbranched alkyl; R5 and R6 are each independently H, C1-C4 substituted or unsubstituted branched or unbranched alkyl, -AA-COOR7, where R7 is C1-C4 substituted or unsubstituted branched or unbranched alkyl and AA is an amino acid; R3 is H, -(C=O)-R8, -C(=O)-CH(NR9R 10 )-R7, -C(=O)-CH2-(NH-C(=O)-CH2-NR9R 10 )-CH2-R 11 , -C(=O)-AA-R 12, acetyl (Ac), dimethylglycine, dimethylglycyl (DMG), L-Lys(Ac), D-Val-L-Leu-L-Lys(Ac), D-Val-L-Leu-L-Lys(H), D-Val-L-Leu-L-Lys(DMG), L-Val-L-Cit, para-aminobenzoic acid (PABA), L-Cit-PABA, L-Val-L-Cit-PABA, dimethylglycyl-L-tryptophan, acetoxymethyl pivalate, 1-acetoxyethyl pivalate, acetoxy(phenyl)methyl pivalate, and 2-acetoxypropan-2-yl pivalate, wherein AA is an amino acid, and the amino acids of R3, R5, and R6 may be selected from the amino acids or amino acid-related substituents listed in Table 2 or Table 3, and combinations thereof; R8 is a C1-C4 substituted or unsubstituted branched or unbranched alkyl and -CH2-NR 14 R 15 is selected from R9, R 10 , R 14 , and R 15 are each independently selected from H and C1-C4 substituted or unsubstituted branched or unbranched alkyl; R 11 is selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; R 12 is H, -(C=O)-R 13 and -NH-dimethylglycyl; R 13 is a C1-C4 substituted or unsubstituted branched or unbranched alkyl; stereoisomers and their pharmaceutically acceptable salts.

[0019] In some embodiments, R and R are each independently selected from the group consisting of -OCHCH, -O-CH(CH), -OC(CH), -NH, -NHCH, -NH-AA-COO-CH(CH), -NH-AA-COO-C(CH), -O-(CHCHO) n -H, -O-(CH2CH2O) n-AA-COO-CH(CH3)2, and -O-(CH2CH2O) n -AA-COO-C(CH3)3.

[0020] In some embodiments, R3 is selected from H, acetyl (Ac), dimethylglycine, dimethylglycyl (DMG), L-Lys(Ac), D-Val-L-Leu-L-Lys(Ac), D-Val-L-Leu-L-Lys(H), D-Val-L-Leu-L-Lys(DMG), L-Val-L-Cit, para-aminobenzoic acid (PABA), L-Cit-PABA, L-Val-L-Cit-PABA, dimethylglycyl-L-tryptophan, acetoxymethyl pivalate, 1-acetoxyethyl pivalate, acetoxy(phenyl)methyl pivalate, and 2-acetoxypropan-2-ylpivalate, —C(═O)—AA-H, —C(═O)—AA-Ac, —C(═O)—AA-NH-dimethylglycyl.

[0021] In some embodiments, R1 and R2 are each -OR4. In certain embodiments, one or both of R1 and R2 are each selected from -OCH2CH3, -OCH(CH3)2, and -OC(CH3)3.

[0022] In some embodiments, R1 and R2 are each -NR5R6. In certain embodiments, R1 and R2 are each -NH2.

[0023] In some embodiments, R is selected from the group consisting of H, —C(═O)—R, and —C(═O)—CH—(NH—C(═O)—CH—NR 10 )-CH2-R 11 In certain embodiments, R3 is selected from H, -C(=O)-CH3, -C(=O)-CH2-N(CH3)2, and -C(=O)-CH2-(NH-C(=O)-CH2-N(CH3)2)-CH2-(1H)indole.

[0024] In certain embodiments, the compound is [ka] is selected from.

[0025] In a more specific embodiment, the compound of formula (I) is [ka] is selected from.

[0026] In an even more particular embodiment, the compound of formula (I) is [ka] is.

[0027] In certain embodiments, the compound is [ka] is selected from.

[0028] In one embodiment, at least one of R1 and R2 is -O - M + In certain embodiments, M + Na + and the compound is [ka] is selected from.

[0029] In certain embodiments, the compound is [ka] and Ad is adamantane.

[0030] Representative azotomycin prodrugs of formula (I) are shown in Table 1.

[0031] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0032] In some embodiments, the prodrug moiety may comprise an amino acid (AA). As used herein, the term "amino acid" includes a moiety having a carboxylic acid group and an amino group. The term "amino acid" includes both natural amino acids (including proteinogenic amino acids) and unnatural amino acids. The term "natural amino acids" also includes other amino acids (including pyrrolysine and selenocysteine) that are incorporated into proteins during translation. Furthermore, the term "natural amino acids" also includes other amino acids produced during intermediary metabolism, such as ornithine, which is produced from arginine in the urea cycle. A non-limiting list of representative amino acids is provided in Table 2. Representative amino acid-related substituents are provided in Table 3.

[0033] In some embodiments, the amino acid is selected from the proteinogenic amino acids, which include glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, serine, threonine, glutamine, asparagine, arginine, lysine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, and histidine.

[0034] The term "amino acid" includes, but is not limited to, alpha amino acids and beta amino acids, such as beta alanine and 2-methyl beta alanine. The term "amino acid" also includes, but is not limited to, certain lactam analogs of natural amino acids, such as pyroglutamine. The term "amino acid" also includes, but is not limited to, amino acid homologs, such as homocitrulline, homoarginine, homoserine, homotyrosine, homoproline, and homophenylalanine.

[0035] Examples of non-proteinogenic amino acids include citrulline, hydroxyproline, 4-hydroxyproline, β-hydroxyvaline, ornithine, β-aminoalanine, albidine, 4-aminophenylalanine, biphenylalanine, 4-nitrophenylalanine, 4-fluorophenylalanine, 2,3,4,5,6-pentafluorophenylalanine, norleucine, cyclohexylalanine, α-aminoisobutyric acid, α-aminobutyric acid, α-aminoisobutyric acid, 2-aminoisobutyric acid, 2-aminoindan-2-carboxylic acid, selenite, cetearyl alcohol ... These include, but are not limited to, thiamin, thiamin, thiamin, thiamin-2-carboxylic acid, thiamin-3-carboxylic acid, thiamin-4-carboxylic acid, thiamin-5-carboxylic acid, thiamin-6-carboxylic acid, thiamin-7-carboxylic acid, thiamin-8-carboxylic acid, thiamin-9-carboxylic acid, thiamin-10-carboxylic acid, thiamin-11-carboxylic acid, thiamin-12-carboxylic acid, thiamin-13-carboxylic acid, thiamin-14-carboxylic acid, thiamin-15-carboxylic acid, thiamin-16-carboxylic acid, thiamin-17-carboxylic acid, thiamin-18-carboxylic acid, thiamin-19-carboxylic acid, thiamin-20-carboxylic acid, thiamin-21-carboxylic acid, thiamin-22-carboxylic acid, thiamin-23-carboxylic acid, thiamin-24-carboxylic acid, thiamin-25-carboxylic acid, thiamin-26-carboxylic acid, thiamin-27-carboxylic acid, thiamin-28-carboxylic acid, thiamin-29-carboxylic acid, thiamin-30-carboxylic acid, thiamin-31-carboxylic acid, thiamin-32-carboxylic acid, thiamin-33-carboxylic acid, thiamin-34-carboxylic acid, thiamin-35-carboxylic acid, thiamin-36-carboxylic acid, thiamin-37-carboxylic acid, thiamin-38-carboxylic acid, thiamin-40-carboxylic acid, thiamin-41-carboxylic acid, thiamin-42-carboxylic acid, thiamin-43-carboxylic acid, thiamin-44-carboxylic acid, thiamin-45-carboxylic acid, thiamin-45-carboxylic acid, thiamin-46-carboxylic acid,

[0036] The terminal portion of an amino acid residue or peptide may be in the form of a free acid, i.e., terminated with a -COOH group, or in a masked (protected) form, such as a carboxylic acid ester or carboxamide. In certain embodiments, the amino acid or peptide residue terminates with an amino group. In one embodiment, the residue terminates with a carboxylic acid group -COOH or an amino group -NH. In another embodiment, the residue terminates with a carboxamide group. In yet another embodiment, the residue terminates with a carboxylic acid ester.

[0037] As noted above, the term "amino acid" includes compounds having a -COOH group and a -NH group. Substituted amino acids include amino acids with mono- or di-substituted amino groups. In certain embodiments, the amino group may be mono-substituted. (A proteinogenic amino acid may be substituted at another site from its amino group to form an amino acid that is a substituted proteinogenic amino acid.) Thus, the term substituted amino acid includes N-substituted metabolites of natural amino acids, including, but not limited to, N-acetylcysteine, N-acetylserine, N-acetylthreonine, and the like.

[0038] For example, the term "N-substituted amino acid" refers to an N-alkyl amino acid (e.g., C sarcosine, N-methylalanine, N-methylglutamic acid, N-tert-butylglycine, etc.). 1-6 N-alkylamino acids), including C 1-6 N-substituted alkyl amino acids (e.g., N-(carboxyalkyl) amino acids (e.g., N-(carboxymethyl) amino acids) and N-methylcycloalkyl amino acids (e.g., N-methylcyclopropyl amino acids)) can be included; N,N-dialkyl amino acids (e.g., N,N-di-C 1-6 Alkyl amino acids (e.g., N,N-dimethyl amino acids); N,N,N-trialkyl amino acids (e.g., N,N,N-tri-C 1-6 Alkyl amino acids (e.g., N,N,N-trimethyl amino acids); N-acyl amino acids (e.g., C 1-6 N-acyl amino acids), N-aryl amino acids (e.g., N-phenyl amino acids such as N-phenylglycine), and N-amidinyl amino acids (e.g., N-amidine amino acids, i.e., amino acids in which the amine group is replaced by a guanidino group).

[0039] The term "amino acid" includes amino acid alkyl esters (e.g., amino acid C 1-6In the case of amino acids having a hydroxy group in the side chain, the term "amino acid" also includes O-alkylamino acids (e.g., C 1-6 Also included are S-alkylamino acids (e.g., C-alkylamino acid ethers), O-aryl amino acids (e.g., O-phenyl amino acid ethers), O-acyl amino acid esters, and O-carbamoyl amino acids. In the case of amino acids having a thiol group in the side chain, the term "amino acid" includes S-alkylamino acids (e.g., C- 1-6 S-methylmethionine and S-methylcycloalkylamino acids (such as S-methylcyclopropylamino acids) which may contain S-alkylamino acids. 1-6 S-substituted alkyl amino acids), S-acyl amino acids (e.g., C 1-6 Also included are S-acyl amino acids, S-aryl amino acids (e.g., S-phenyl amino acids), sulfoxide analogs of sulfur-containing amino acids (e.g., methionine sulfoxide) or sulfoxide analogs of S-alkyl amino acids (e.g., S-methylcysteine ​​sulfoxide) or S-aryl amino acids.

[0040] The subject matter of the present disclosure also contemplates derivatives of such natural amino acids that have been functionalized by straightforward synthetic transformations known in the art (e.g., those described in "Protective Groups in Organic Synthesis" by T.W. Greene and P.G.M. Wuts, John Wiley & Sons Inc. (1999)) and references therein.

[0041] In one embodiment, the amino acid side chain is attached to another amino acid, hi yet another embodiment, the side chain is attached to the amino acid through the N-terminus, C-terminus, or side chain of the amino acid. Examples of side chains of natural amino acids include hydrogen (glycine), methyl (alanine), isopropyl (valine), sec-butyl (isoleucine), -CHCH(CH) (leucine), benzyl (phenylalanine), p-hydroxybenzyl (tyrosine), -CHOH (serine), -CH(OH)CH (threonine), -CH-3-indoyl (tryptophan), -CHCOOH (aspartic acid), -CHCHCOOH (glutamic acid), -CHC(O)NH (asparagine), -CHCHC(O)NH (glutamine), -CHSH (cysteine), -CHCHSCH (methionine), -(CH)NH (lysine), -(CH)NHC(=NH)NH (arginine), and -CH-3-imidazoyl (histidine).

[0042] In some embodiments, the amino acid may be substituted with a monocyclic ring. Exemplary monocyclic and bicyclic rings include, but are not limited to, benzene, pyrimidine, purine, and the like, and more generally, aryl and heteroaryl rings. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, furanyl, thienyl, pyrazolyl, oxazolyl, thiazolyl, isothiazolyl, isoxazolyl, pyrrolyl, imidazolyl, indolyl, indolinolyl, imidazopyridazinyl, and the like. Aryl groups include phenyl (C6), benzyl, naphthyl (C7), and the like. 10 ), biphenyl group (C 12 Examples of pyrimidines include, but are not limited to, cytosine, thymine, uracil, etc. Examples of purines include, but are not limited to, purine, adenine, N-substituted adenines, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, isoguanine, etc. Examples of purine nucleosides include, but are not limited to, adenine and guanine, etc.

[0043] The term "peptide" refers to an amino acid chain of 2 to 9 amino acids, unless otherwise specified. In a preferred embodiment, peptides used in the present invention are 2 or 3 amino acids in length. In one embodiment, the peptide may be a branched peptide. In this embodiment, at least one amino acid side chain in the peptide is attached to another amino acid (either terminally or via a side chain). The term "N-substituted peptide" refers to an amino acid chain of 2 to 9 amino acids in which one or more NH groups have been replaced, for example, by a substituent described elsewhere herein for substituted amino groups. Optionally, N-substituted peptides have a substituted N-terminal amino group; in one embodiment, the amide bond is not substituted.

[0044] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0045] [Table 3-1] [Table 3-2] [Table 3-3]

[0046] (II. Treatment method) In some aspects, the presently disclosed subject matter provides methods for treating a disease, disorder, or condition associated with excessive and / or abnormal glutamine utilization, the methods comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of formula (I) or a pharmaceutical formulation thereof.

[0047] In certain embodiments, the disease, disorder or condition associated with excessive and / or abnormal glutamine utilization is selected from an infectious disease, a cancer, an autoimmune disease, an inflammatory disease, and a neurodegenerative or neurological disease.

[0048] In certain embodiments, the cancer is selected from a newly diagnosed cancer, a recurrent cancer, a refractory cancer, and a combination thereof.

[0049] In more particular embodiments, the cancer is selected from (i) a cancer of the central nervous system, (ii) a cancer associated with transplantation and / or immunosuppression, (iii) a cancer resistant to chemotherapy, (iv) a cancer resistant to photodynamic therapy, (v) a cancer resistant to proton therapy, (vi) a cancer resistant to radiation therapy, and (vii) a cancer resistant to surgery.

[0050] In even more specific embodiments, the cancer is cervicopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, pharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, gastrointestinal cancer, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, esophageal cancer, esophageal cancer, esophageal carcinoma, esophageal cancer ... cancer of the esophagus, gallbladder, head, eye, neck, kidney, Wilms' tumor, liver, Kaposi's sarcoma, prostate, lung, testicular, Hodgkin's disease, non-Hodgkin's lymphoma, oral cavity, skin, mesothelioma, multiple myeloma, ovarian, endocrine pancreatic, glucagonoma, pancreatic, parathyroid, penile, pituitary, soft tissue sarcoma, retinoblastoma, small intestine, stomach, thymus, thyroid, choriocarcinoma, hydatidiform mole, uterine, endometrial, vaginal, vulvar, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum, heart, lip, meningeal, oral, nerve, palate, parotid, peritoneal, pharyngeal, pleural, salivary, tongue, and tonsillar cancer.In even more specific aspects, the cancer is selected from the group consisting of cervicopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, pharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, gastrointestinal cancer, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymus cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0051] In some embodiments, the method further comprises preventing recurrence in a cancer patient in remission.

[0052] In other embodiments, the method further comprises administering a therapeutically effective amount of a compound of Formula (I) in combination with immunotherapy. In some embodiments, the immunotherapy comprises checkpoint inhibitor therapy. In certain embodiments, the compound of Formula (I) is administered in combination with one or more checkpoint inhibitors. In more specific embodiments, the one or more checkpoint inhibitors are selected from anti-PD-1 Ab, anti-PD-L1 Ab, anti-CTLA-4 Ab, anti-TIGIT Ab, and combinations thereof. See, for example, Yokoyama, Y. et al., 2022; Rais, R. et al., 2022; Leone RD et al., 2019.

[0053] As used herein, the term "treatment" can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of a disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Prevention refers to preventing a disease, disorder, condition, or its symptoms or signs, or worsening of its severity, from occurring. Thus, the compositions disclosed herein can be administered prophylactically to prevent or reduce the occurrence or recurrence of a disease, disorder, or condition.

[0054] As used herein, the term "inhibit" means to decrease, suppress, attenuate, reduce, halt, or stabilize the activity of a factor, e.g., an enzyme, associated with a disease or disease-related pathway, or the development or progression of a disease, disorder, or condition, e.g., brain tumor, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% compared to an untreated control subject, cell, biological pathway, or biological activity.

[0055] While the "subject" treated by many embodiments of the disclosed methods is desirably a human subject, it should be understood that the methods described herein are effective on all vertebrate species intended to be encompassed by the term "subject." Accordingly, a "subject" can include a human subject for medical purposes, such as treatment of an existing condition or disease or prophylactic treatment to prevent the onset of a condition or disease, or an animal subject for medical, veterinary, or developmental purposes. Suitable animal subjects include mammals, including, but not limited to, primates such as humans, monkeys, and apes; bovines, such as cows and oxen; ovines, such as goats; caprines, such as pigs and domestic pigs; equines, such as horses, donkeys, and zebras; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; rodents, including mice, rats, and the like. The animal may be a transgenic animal. In some embodiments, the subject is a human, including, but not limited to, a fetal, neonatal, infant, juvenile, or adult subject. Additionally, a "subject" can also include a patient suffering from or suspected of suffering from a condition or disease. Thus, the terms "subject" and "patient" are used interchangeably herein. The term "subject" also refers to an organism, tissue, cell, or collection of cells from a subject.

[0056] Generally, an "effective amount" of an active agent refers to the amount necessary to elicit a desired biological response. As one skilled in the art would understand, the effective amount of an agent may vary depending on factors such as the desired biological endpoint, the agent being delivered, the composition of the pharmaceutical composition, and the target tissue.

[0057] The term "combination" is used in the broadest sense and means that a subject is administered at least two agents, more specifically, a compound of Formula (I) described herein and at least one other therapeutic agent, such as a chemotherapeutic or immunotherapeutic agent. More specifically, the term "combination" refers to the simultaneous administration of two (or more) active agents, for example, to treat a single medical condition. As used herein, the active agents can be administered in combination in a single dosage form, simultaneously in separate dosage forms, or in separate dosage forms that are administered alternately or sequentially on the same or different days. In one embodiment of the presently disclosed subject matter, the active agents are administered in combination in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., when it is desired to vary the amount of one agent while maintaining the amount of the other). The single dosage form may include additional active agents for the treatment of a medical condition.

[0058] Additionally, the compositions described herein may be used alone or in combination with one or more therapeutic agents to enhance the stability of the composition, in certain embodiments facilitate administration of pharmaceutical compositions containing them, increase dissolution or dispersion, increase inhibitory activity, provide adjunctive therapy, or the like, including other active ingredients, and may be administered alone or in combination with other adjunctive agents. Advantageously, such combination therapy may require lower doses of conventional therapeutic agents, thereby avoiding potential toxicity and side effects that may occur when those agents are used as monotherapies.

[0059] The timing of administration of the compound of formula (I) described herein and at least one additional therapeutic agent can be changed, as long as the beneficial effect of the combination of these drugs is achieved.Therefore, the term "in combination" refers to administering the compound of formula (I) described herein and at least one additional therapeutic agent simultaneously, sequentially, or in combination.Therefore, the subject who is administered the combination of the compound of formula (I) described herein and at least one additional therapeutic agent can receive the at least one additional therapeutic agent simultaneously (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or different days), as long as the combined effect of both drugs is achieved in the subject.

[0060] When administered sequentially, the agents may be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes, or more of each other. In other embodiments, sequentially administered agents may be administered within 1, 5, 10, 15, 20 days, or more of each other. When at least one additional therapeutic agent described herein is administered simultaneously, they may be administered to the subject as separate pharmaceutical compositions, each comprising either a compound of Formula (I) or at least one additional therapeutic agent, or they may be administered to the subject as a single pharmaceutical composition comprising both agents.

[0061] When administered in combination, the effective concentration of each agent that induces a particular biological response may be lower than the effective concentration when each agent is administered alone, thereby reducing the dosage of one or more agents compared to the dosage required when the agent is administered as a single agent. The effects of multiple agents are not necessarily additive or synergistic, but may be additive. Agents may be administered multiple times.

[0062] In some embodiments, administering two or more agents in combination can result in a synergistic effect. As used herein, the terms "synergy," "synergistic," "synergistically," and derivatives thereof, such as "synergistic effect" or "synergistic combination" or "synergistic composition," refer to a situation in which the biological activity of a combination of a miR described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of each agent when administered individually.

[0063] Synergy can be expressed in terms of the "synergy index (SI)," which can generally be determined from the ratio determined by the method described in F.C. Kull et al., Applied Microbiology 9, 538 (1961), according to the following formula: Q a / Q A +Q b / Q B = Synergy Index (SI) Q A is the concentration of component A acting alone to produce the endpoint relative to component A, Q a is the concentration of component A in the mixture that produced the endpoint, Q B is the concentration of component B acting alone to produce the endpoint relative to component B, Q b is the concentration of component B in the mixture that produced the endpoint.

[0064] Generally, Q a / Q A and Q b / Q BA sum greater than 1 indicates antagonism, a sum equal to 1 indicates additive action, and a sum less than 1 demonstrates synergy. The lower the SI, the greater the synergistic effect exhibited by that particular mixture. Thus, a "synergistic combination" exhibits greater activity than would be expected based on the observed activity of the individual components when used alone. Furthermore, a "synergistically effective amount" of a component refers to the amount of that component needed to elicit a synergistic effect, for example, with another therapeutic agent present in the composition.

[0065] III. Pharmaceutical Compositions and Administration In some embodiments, the present disclosure provides pharmaceutical compositions comprising one compound of Formula (I) of the present disclosure, alone or in combination with one or more additional therapeutic agents in a pharmaceutically acceptable excipient. Those skilled in the art will recognize that pharmaceutical compositions include pharmaceutically acceptable salts of the compounds. Pharmaceutically acceptable salts are generally well known to those skilled in the art and include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituent moieties found on the compounds described herein. When a compound of the present disclosure contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent, or by ion exchange, replacing one base counterion (base) in the ionic complex with another. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts.

[0066] When a compound of the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent, or by ion exchange, whereby one acidic counterion (acid) in the ionic complex is replaced by another acid. Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphate acid, diphosphoric acid, sulfuric acid, monohydrogensulfate acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, trifluoroacetic acid (TFA), and the like. Also included are salts of amino acids, such as arginic acid, and salts of organic acids, such as glucuronic acid or galacturonic acid (see, e.g., Berge et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the present disclosure contain both basic and acidic functional groups that allow the compounds to be converted into either base or acid addition salts.

[0067] Thus, pharmaceutically acceptable salts suitable for use in the presently disclosed subject matter include, for example, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolic acid arsanilate, hexylresorcinate, hydrabamine, hydrobromide, Pharmaceutically acceptable salts include, but are not limited to, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, acetate, succinate, sulfate, tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts are described, for example, in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000). For therapeutic and / or diagnostic applications, the compounds of the present disclosure can be formulated for various modes of administration, including systemic, topical, or local administration. Techniques and formulations are generally described in Remington: The Science and Practice of Pharmacy (20th ed.). th (ed.) Lippincott, Williams & Wilkins (2000).

[0068] Such agents may be formulated into liquid or solid dosage forms and administered systemically or locally, depending on the particular condition being treated. The agents may be delivered, for example, in time-release or sustained-release forms, as known to those skilled in the art. Techniques for formulation and administration are described in Remington: The Science and Practice of Pharmacy (2004). th(ed.) Lippincott, Williams & Wilkins (2000). Suitable routes of administration include oral, buccal, inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration, parenteral administration including intramuscular, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal or intraocular injection, or other modes of administration.

[0069] For injection, the agent of the present invention can be prepared and diluted in an aqueous solution such as Hank's solution, Ringer's solution, or physiologically compatible buffer such as physiological saline buffer. For such transmucosal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art.

[0070] It is within the scope of the present disclosure to use a pharmaceutically acceptable inert carrier to formulate the compounds disclosed herein into dosages suitable for systemic administration for the practice of the present disclosure. By appropriately selecting the carrier and employing an appropriate manufacturing method, the compositions of the present disclosure, particularly those formulated as solutions, can be administered parenterally, such as by intravenous injection. The compounds can be easily formulated into dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. With such carriers, the compounds of the present disclosure can be formulated into tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by a treatment subject (e.g., a patient).

[0071] For nasal or inhalation delivery, the agents of the present disclosure may also be formulated by methods known to those skilled in the art, and may include, for example, but not limited to, solubilizing, diluting, or dispersing agents such as saline, preservatives such as benzyl alcohol, absorption enhancers, and fluorocarbons.

[0072] In certain embodiments, the compounds of formula (I) of the present disclosure are administered intranasally in a form selected from the group consisting of nasal spray, nasal drops, powder, granules, sachets, tablets, aerosols, pastes, creams, gels, ointments, salves, foams, pastes, lotions, creams, oil suspensions, emulsions, solutions, patches, and sticks.

[0073] Pharmaceutical compositions suitable for use in the present disclosure include compositions containing the active ingredient in an amount effective to achieve its intended purpose. Determination of an effective amount is within the capabilities of one of ordinary skill in the art, especially in light of the detailed disclosure provided herein. Generally, compounds according to the present disclosure are effective over a wide dosage range. For example, for adult treatment, dosages of 0.01-1000 mg, 0.5-100 mg, 1-50 mg per day, 5-40 mg per day, etc. are examples of dosages that may be used. Dosages are not limited to 10-30 mg per day. The exact dosage will depend on the route of administration, the dosage form of the compound, the subject being treated, the subject's body weight, the bioavailability of the compound, the adsorption, distribution, metabolism, and excretion (ADME) toxicity of the compound, and the preference and experience of the attending physician.

[0074] These pharmaceutical compositions may contain, in addition to the active ingredient, a suitable pharmaceutically acceptable carrier, including excipients and adjuvants that facilitate the processing of the active compound into a preparation that can be used as a medicine. Preparations formulated for oral administration may be in the form of tablets, sugar-coated tablets, capsules, or solutions.

[0075] Oral pharmaceutical preparations can be obtained by combining active compounds with solid excipients, grinding the resulting mixture as needed, adding suitable excipients as needed, and then processing the granulated mixture to obtain tablets or sugar-coated tablet cores.Suitable excipients are, in particular, fillers such as sugars including lactose, sucrose, mannitol, or sorbitol, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose (CMC), and / or cellulose preparations such as polyvinylpyrrolidone (PVP: povidone).If necessary, disintegrants can be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate.

[0076] The core of the sugar-coated tablet is provided with a suitable coating.For this purpose, concentrated sugar solutions are used, which may contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures as needed.For identification or to characterize different combinations of dosages of active compounds, dyes or pigments may be added to the tablet or sugar-coated tablet coating.

[0077] Pharmaceuticals that can be used for oral administration include push-fit capsules made of gelatin and sealed soft capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally, a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol (PEG). Additionally, stabilizers may be added.

[0078] (IV. Definition) Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this disclosure belongs.

[0079] While the following terms relating to compounds of formula (I-III) are believed to be well understood by those of ordinary skill in the art, the following definitions are provided to facilitate explanation of the subject matter of the present disclosure. These definitions are intended to supplement and explain, not preclude, definitions that would be apparent to one of ordinary skill in the art upon review of the present disclosure.

[0080] As used herein, the terms "substituted" and "substituent," whether preceded by the term "optionally" or not, refer to the ability to replace one functional group on a molecule with another functional group, provided that the valences of all atoms are maintained, as understood by those skilled in the art. When multiple positions in a structure can be substituted with multiple substituents selected from a particular group, the substituents can be either the same or different at each position. Substituents can also be further substituted (e.g., an aryl group substituent can have another substituent, such as another aryl group, which can be further substituted at one or more positions).

[0081] When substituents or linking groups are designated by conventional chemical formulas written from left to right, they equally encompass the chemically identical substituents that would occur if the structure were written from right to left. For example, -CHO- is equivalent to -OCH-, -C(=O)O- is equivalent to -OC(=O)-, -OC(=O)NR- is equivalent to NRC(=O)O-, etc.

[0082] When the term "independently selected" is used, the referenced substituents (e.g., R groups such as R, R groups, or variables such as "m" and "n") can be the same or different. For example, both R and R can be substituted alkyl, R can be hydrogen and R can be substituted alkyl, etc.

[0083] The terms "a," "an," or "a(n)," as used herein with respect to a group of substituents, mean at least one. For example, if a compound is substituted with "an" alkyl or aryl, the compound is optionally substituted with at least one alkyl and / or at least one aryl. Furthermore, if a moiety is substituted with R substituents, the group may be referred to as "R-substituted." If a moiety is R-substituted, the moiety is substituted with at least one R substituent, and each R substituent can optionally be different.

[0084] Unless otherwise indicated herein, a designated "R" or group generally has the structure recognized in the art as corresponding to the group having that name. For illustrative purposes, representative "R" groups shown above are defined below.

[0085] The description of the compounds of the present disclosure is limited by the principles of chemical bonding known to those skilled in the art.Therefore, the substitution of one or more substituents that group can be substituted is selected to give a compound that conforms to the principles of chemical bonding and is not inherently unstable and / or is known to those skilled in the art to be likely to be unstable under ambient conditions, such as aqueous, neutral and some known physiological conditions.For example, heterocycloalkyl or heteroaryl is attached to the rest of the molecule through ring heteroatom according to the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0086] Unless expressly defined otherwise, a "substituent" as used herein includes a functional group selected from one or more of the following moieties, as defined herein:

[0087] As used herein, the term "hydrocarbon" refers to any chemical group comprising hydrogen and carbon. Hydrocarbons can be substituted or unsubstituted. As known to those skilled in the art, all valences must be satisfied when substitutions are made. Hydrocarbons can be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic. Specific hydrocarbons are further defined herein below and include, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, and the like.

[0088] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, the monovalent radical —C derived by removing a hydrogen atom from any carbon atom of an alkane. n H 2n+1 An alkyl group derived by removing a hydrogen atom from the terminal carbon atom of an unbranched alkane has the formula H(CH2) n RCH2, R2CH (R≠H), and R3C (R≠H) groups are primary, secondary, and tertiary alkyl groups, respectively. Alkyl can be a straight-chain (i.e., unbranched) or branched acyclic hydrocarbon having the specified number of carbon atoms (i.e., C 1-10 means 1 to 10 carbons, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbons). In certain embodiments, the term "alkyl" refers to the inclusive C groups, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons. 1-20 Refers to...

[0089] Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, etc. Representative C1-C4 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, etc.

[0090] "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl, or propyl, is attached to a linear alkyl chain. A "lower alkyl" refers to a group having from 1 to about 8 carbon atoms (i.e., C), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. 1-8 "Higher alkyl" refers to an alkyl group having from about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In one embodiment, "alkyl" refers specifically to C 1-8 In another embodiment, "alkyl" refers to a straight chain alkyl, specifically C 1-8 Refers to branched chain alkyl.

[0091] An alkyl group can be optionally substituted with one or more alkyl group substituents ("substituted alkyl"), which can be the same or different. "Alkyl group substituents" include, but are not limited to, alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can be optionally inserted along the alkyl chain, where the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl"), or aryl.

[0092] Thus, the term "substituted alkyl," as used herein, includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group have been replaced with another atom or functional group, including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, cyano, and mercapto.

[0093] The term "heteroalkyl," by itself or in combination with other terms, means, unless otherwise specified, a stable linear or branched chain or cyclic hydrocarbon group having 1 to 20 carbon atoms or heteroatoms, or 3 to 10 carbon atoms or heteroatoms, or combinations thereof, composed of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen, phosphorus, and sulfur atoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized. The heteroatom(s) O, N, P, S, and Si can be placed at any position within the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0094] As noted above, heteroalkyl groups as used herein include groups attached to the remainder of the molecule via a heteroatom, such as -C(O)NR', ​​-NR'R'', -OR', -SR, -S(O)R, and / or -S(O2)R'. When "heteroalkyl" is followed by a specific heteroalkyl group, such as -NR'R, it is understood that the terms "heteroalkyl" and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, as used herein, the term "heteroalkyl" should not be construed to exclude specific heteroalkyl groups, such as -NR'R''.

[0095] The terms "cyclic" and "cycloalkyl" refer to a monovalent group derived from a cycloalkane by removing a hydrogen atom from a ring carbon atom. Cycloalkanes are saturated monocyclic hydrocarbons (with or without side chains), such as cyclobutane. Unsaturated monocyclic hydrocarbons with a single double or triple bond within the ring are called cycloalkenes and cycloalkynes, respectively. Compounds with multiple multiple bonds include cycloalkadienes and cycloalkatrienes. Collective terms for cyclic hydrocarbons with any number of multiple bonds include cyclic olefins and cyclic acetylenes. As used herein, cycloalkyl can be a non-aromatic monocyclic or polycyclic ring structure having from about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Cycloalkyl groups can optionally be partially unsaturated. Cycloalkyl groups can also be optionally substituted with alkyl group substituents, oxo groups, and / or alkylene groups, as defined herein. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can be optionally inserted along the cyclic alkyl chain, where the nitrogen substituent is hydrogen, unsubstituted alkyl, substituted alkyl, aryl, or substituted aryl, thereby providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like. Polycyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, noradamantyl, and fused ring systems such as dihydronaphthalene and tetrahydronaphthalene.

[0096] As used herein, the term "cycloalkylalkyl" refers to a cycloalkyl group as defined above which is optionally substituted with an alkylene group as defined above (e.g., C 1-20 An alkylene group is attached to the parent molecular moiety through an alkylene group. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

[0097] The term "cycloheteroalkyl" or "heterocycloalkyl" refers to an unsaturated or partially unsaturated non-aromatic ring system, such as a 3- to 10-membered substituted or unsubstituted cycloalkyl ring system, containing one or more heteroatoms, which may be the same or different, selected from the group consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si), and which may optionally contain one or more double bonds.

[0098] Cycloheteroalkyl rings may optionally be fused to or attached to other cycloheteroalkyl rings and / or non-aromatic hydrocarbon rings. Heterocyclic rings include rings having 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, where the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the term heterocyclic refers to a non-aromatic 5-, 6-, or 7-membered ring or polycyclic group in which at least one ring atom is a heteroatom selected from O, S, and N (the nitrogen and sulfur heteroatoms can optionally be oxidized), including, but not limited to, bicyclic or tricyclic groups having fused 6-membered rings with 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, where (i) each 5-membered ring has 0-2 double bonds, each 6-membered ring has 0-2 double bonds, and each 7-membered ring has 0-3 double bonds, (ii) the nitrogen and sulfur heteroatoms can be optionally oxidized, (iii) the nitrogen heteroatom can be optionally quaternized, and (iv) any of the above heterocycles can be fused to an aryl or heteroaryl ring. Representative cycloheteroalkyl ring systems include, but are not limited to, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.

[0099] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl," respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The terms "cycloalkylene" and "heterocycloalkylene" refer to the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.

[0100] As used herein, the terms "bicycloalkyl" and "bicycloheteroalkyl" refer to two cycloalkyl or cycloheteroalkyl groups bonded together. Non-limiting examples include bicyclohexane and bipiperidine.

[0101] Unsaturated hydrocarbons have one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and their higher homologs and isomers. Alkyl groups limited to hydrocarbon groups are referred to as "homoalkyl."

[0102] More specifically, the term "alkenyl" as used herein refers to a C alkyl group having at least one carbon-carbon double bond. 2-20Alkenyl refers to a monovalent group derived by removing one hydrogen molecule from a straight-chain or branched-chain hydrocarbon moiety containing: Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, 1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, allenyl, and butadienyl.

[0103] The term "cycloalkenyl" as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

[0104] As used herein, the term "alkynyl" refers to a straight or branched chain C alkyl group of a specified number of carbon atoms containing at least one carbon-carbon triple bond. 2-20 It refers to a monovalent group derived from a hydrocarbon. Examples of "alkynyl" include ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, heptynyl, and the like.

[0105] The term "alkylene," by itself or as part of another substituent, refers to a straight- or branched-chain divalent aliphatic hydrocarbon group derived from an alkyl group having 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Alkylene groups can be straight-chain, branched, or cyclic. Additionally, alkylene groups can be optionally unsaturated and / or substituted with one or more "alkyl group substituents." One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl") can be optionally inserted along the alkylene group, where the nitrogen substituents are alkyl as previously described. Examples of alkylene groups include methylene (-CH-), ethylene (-CH-CH-), propylene (-(CH-), cyclohexylene (-CH-), and cyclohexylene (-CH-).10 -), -CH=CH-CH=CH-, -CH=CH-CH2-, -CH2CH2CH2CH2-, -CH2CH=CHCH2-, -CH2CsCCH2-, -CH2CH2CH(CH2CH2CH3)CH2-, -(CH2) q -N(R)-(CH2) r where q and r are each independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or a lower alkyl, such as methylenedioxyl (-O-CH2-O-), and ethylenedioxyl (-O-(CH2)2-O-). Alkylene groups have from about 2 to about 3 carbon atoms and can further have 6 to 20 carbon atoms. Typically, alkyl (or alkylene) groups have 1 to 24 carbon atoms, with some embodiments of the present disclosure being groups having 10 or fewer carbon atoms. A "lower alkyl group" or "lower alkylene group" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms.

[0106] The term "heteroalkylene," alone or as part of another substituent, means a divalent radical derived from heteroalkyl, including, but not limited to, -CH-CH-S-CH-CH, CH-S-CH-CH-NH-CH-, and the like. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Further, for alkylene and heteroalkylene groups, the direction of the written formula of the linking group does not dictate orientation of the linking group. For example, the formula -C(O)OR'- represents both -C(O)OR'- and -R'OC(O)-.

[0107] The term "aryl," unless otherwise specified, refers to a substituent derived from an arene, i.e., a monocyclic or polycyclic aromatic hydrocarbon, by removing a hydrogen atom from a ring carbon atom. Aryl groups can include monocyclic or polycyclic (e.g., 1 to 3 rings) aromatic hydrocarbon substituents, which are fused or covalently linked to each other. The term "heteroaryl" refers to an aryl group (or ring) containing 1 to 4 heteroatoms (in each ring if multiple rings) selected from N, O, and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Heteroaryl groups can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-phenyl-4-ox ...

[0023] Examples of aryl and heteroaryl ring systems include 1-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms "arylene" and "heteroarylene" refer to the divalent forms of aryl and heteroaryl, respectively.

[0108] For brevity, the term "aryl," when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl), includes both aryl and heteroaryl rings as defined above. Thus, the terms "arylalkyl" and "heteroarylalkyl" are intended to include groups in which an aryl or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, etc.), including alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced with, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.). However, the term "haloaryl," as used herein, is intended to encompass only aryls substituted with one or more halogens.

[0109] When a heteroalkyl, heterocycloalkyl, or heteroaryl contains a specific number of members (eg, "3 to 7 members"), the term "member" refers to a carbon or heteroatom.

[0110] Furthermore, in this specification, the formula [ka] refers to aliphatic and / or aromatic ring compounds having ring structures, such as, but not limited to, 3-carbon, 4-carbon, 5-carbon, 6-carbon, 7-carbon, etc., including saturated, partially saturated, and unsaturated ring structures, with substituent R groups, which may or may not be present, and if present, one or more R groups may each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of an R group and the number of R groups is determined by the value of the variable "n," which is typically an integer ranging from 0 to the number of carbon atoms on the ring available for substitution. Each R group, when present in multiples, can substitute on an available carbon atom on the ring structure rather than on another R group. For example, when n is 0 to 2, the above structure comprises composite groups such as, but not limited to, the following formulae: [ka]

[0111] A dashed line representing a bond in a cyclic ring structure indicates that the bond may or may not be present in the ring, i.e., that the ring structure is selected from the group consisting of saturated, partially saturated, and unsaturated ring structures.

[0112] symbol( JPEG2025540593000028.jpg825) denotes the point of attachment of the moiety to the rest of the molecule.

[0113] When a named atom of an aromatic ring or heteroaromatic ring is defined as "absent," the named atom is replaced by a direct bond.

[0114] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl," "heterocycloalkyl," "aryl," "heteroaryl," "phosphonate," and "sulfonate," as well as their divalent derivatives) are intended to include both substituted and unsubstituted forms of the indicated radical. Optional substituents for each radical are described below.

[0115] Substituents for monovalent and divalent derivative groups of alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halo gen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R',-C(O)NR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR' 'R''', -NR''C(O)OR', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -CN, CF3, Fluorinated C 1-4and —NO2, where m′ is the total number of carbon atoms in such group, and a number ranging from 0 to (2m′+1). R′, R″, R′′, and R′″ can each independently represent hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 to 3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or arylalkyl. As used herein, an “alkoxy” group is an alkyl attached to the remainder of the molecule through a divalent oxygen. When compounds of the present disclosure contain multiple R groups, for example, each R group is independently selected, as are multiple R′, R″, R′′, and R′″ groups. When R′ and R″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl groups. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is intended to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.).

[0116] Similar to the substituents described above for alkyl groups, exemplary substituents for aryl and heteroaryl groups (and their divalent derivatives) are varied and include, for example, halogen, —OR′, —NR′R″, —SR′, SiR′R″R′″, —OC(O)R′, —C(O)R′, —COR′, —C(O)NR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′, —NR—C(NR′R″R′″)═NR′″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)R′, —S(O)NR′R″, —NRRS02R′, —CN and —NO2, —R′, —N3, —CH(Ph)2, fluoro(C 1-4 ) alkoxo, and fluoro(C 1-4 ) alkyl, a number ranging from 0 to the total number of open valences on the aromatic ring system, and R', R", R'", R'", and R"" can be independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. For example, when a compound of the present disclosure includes multiple R groups, each R group is independently selected, and when multiple of these groups are present, each R', R", R'", and R"" group is also independently selected.

[0117] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -TC(O)-(CRR')qU-, where T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may form a ring of the formula -A-(CH2) r A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 4.

[0118] One of the single bonds in the new ring thus formed can optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring can optionally be replaced with a group of the formula -(CRR') s -X'-(C''R'') d -, where s and d are each an integer from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituents R, R', R" and R'" can be independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0119] As used herein, the term "acyl" refers to an organic acid group in which the -OH of the carboxyl group is replaced with another substituent and has the general formula RC(=O)-, where R is an alkyl, alkenyl, alkynyl, aryl, carbocyclic, heterocyclic, or aromatic heterocyclic group as defined herein. Thus, the term "acyl" specifically includes arylacyl groups such as 2-(furan-2-yl)acetyl and 2-phenylacetyl. Specific examples of acyl groups include acetyl and benzoyl groups. Acyl groups also include amides, -RC(=O)NR', ​​esters, -RC(=O)OR', ketones, -RC(=O)R', and aldehydes, -RC(=O)H.

[0120] The terms “alkoxyl” or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl-O—) or unsaturated (i.e., alkenyl-O— and alkynyl-O—) group attached to the parent molecular moiety through an oxygen atom; the terms “alkyl,” “alkenyl,” and “alkynyl” are as defined above and include, for example, C alkoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the like. 1-20The oxo- or oxo-hydrocarbon chain may be linear, branched, or cyclic, saturated or unsaturated.

[0121] The term "alkoxyalkyl" as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.

[0122] "Aryloxyl" refers to an aryl-O- group in which the aryl group is as previously described, including substituted aryl. As used herein, the term "aryloxyl" can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl-substituted phenyloxyl or hexyloxyl.

[0123] "Aralkyl" refers to an arylalkyl group in which the aryl and alkyl groups are as previously described, and includes substituted aryl and alkyl groups. Examples of aralkyl groups include benzyl, phenylethyl, naphthylmethyl, and the like.

[0124] "Aralkyloxyl" refers to an aralkyl-O- group in which the aralkyl group is as previously described. Examples of aralkyloxyl groups include benzyloxyl groups, i.e., C6H5-CH2-O-. Aralkyloxyl groups can be optionally substituted.

[0125] "Alkoxycarbonyl" refers to an alkyl-OC(=O)- group. Examples of alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, tert-butyloxycarbonyl, and the like.

[0126] "Aryloxycarbonyl" refers to an aryl-OC(=O)- group. Examples of aryloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl groups.

[0127] "Aralkoxycarbonyl" refers to an aralkyl-OC(=O)- group. Examples of aralkoxycarbonyl groups include benzyloxycarbonyl.

[0128] "Carbamoyl" refers to an amide group represented by the formula -C(=O)NH2. "Alkylcarbamoyl" refers to the group R'RN-C(=O)- where one of R and R' is hydrogen and the other of R and R' is alkyl and / or substituted alkyl as defined above. "Dialkylcarbamoyl" refers to the group R'RN-C(=O)- where R and R' are each independently alkyl and / or substituted alkyl as defined above.

[0129] The term "carbonyldioxyl," as used herein, refers to a carbonate group of formula -OC(=O)-OR.

[0130] "Acyloxyl" refers to an acyl-O- group in which acyl is as previously described.

[0131] The term "amino" refers to the -NH group and, as known in the art, also refers to nitrogen-containing groups derived from ammonia by replacing one or more hydrogen radicals with an organic radical. For example, the terms "acylamino" and "alkylamino" refer to specific N-substituted organic radicals having acyl and alkyl substituents, respectively.

[0132] As used herein, "aminoalkyl" refers to an amino group covalently bonded to an alkylene linker. More specifically, as used herein, the terms alkylamino, dialkylamino, and trialkylamino refer to one, two, or three alkyl groups, as defined above, attached to the parent molecular moiety via a nitrogen atom, respectively. The term alkylamino refers to a group having the structure -NHR', where R' is an alkyl group as defined above. Meanwhile, the term dialkylamino refers to a group having the structure -NR'R''', where R' and R'' are each independently selected from the group consisting of alkyl groups. Trialkylamino refers to a group having the structure -NR'R''R''', where R', R'', and R''' are each independently selected from the group consisting of alkyl groups. Furthermore, R', R'', and / or R''' together may optionally be -(CH2) k -, where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, isopropylamino, piperidino, trimethylamino, and propylamino.

[0133] An amino group is -NR'R'', where R' and R'' are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0134] The terms alkylthioether and thioalkoxyl refer to saturated (i.e., alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) groups attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxyl groups include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0135] "Acylamino" means an acyl-NH- group in which the acyl group is as previously described. "Aroylamino" means an aroyl-NH- group in which the aroyl group is as previously described.

[0136] The term "carbonyl" refers to the group -C(=O)- and can include aldehyde groups represented by the general formula RC(=O)H.

[0137] The term "carboxyl" refers to a -COOH group. Such a group is also referred to herein as a "carboxylic acid" moiety.

[0138] The term "cyano" refers to the group --C.ident.N.

[0139] As used herein, the terms "halo," "halide," or "halogen" refer to fluoro, chloro, bromo, and iodo groups. Additionally, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, "halo(C 1-4 The term "alkyl" is meant to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0140] The term "hydroxyl" refers to an --OH group.

[0141] The term "hydroxyalkyl" refers to an alkyl group substituted with an --OH group.

[0142] The term "mercapto" refers to the group --SH.

[0143] The term "oxo" as used herein means an oxygen atom that is double bonded to a carbon atom or other element.

[0144] The term "nitro" refers to the group --NO.sub.2.

[0145] The term "thio" refers to a compound previously described herein in which a carbon or oxygen atom has been replaced by a sulfur atom.

[0146] The term "sulfate" refers to the -SO4 group.

[0147] The term thiohydroxyl or thiol, as used herein, refers to a group of formula -SH.

[0148] More specifically, the term "sulfide" refers to a compound having a group of formula -SR.

[0149] The term "sulfone" refers to a compound having a sulfonyl group -S(O2)R.

[0150] The term "sulfoxide" refers to a compound with a sulfinyl group -S(O)R.

[0151] The term ureido refers to a urea group of the chemical formula -NH-CO-NH2.

[0152] Throughout the specification and claims, a particular chemical formula or name is intended to encompass all tautomers, homologs, optical isomers and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

[0153] Certain compounds of the present disclosure may have asymmetric carbon atoms (optical or chiral centers) or double bonds, and enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms, and individual isomers, which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or D- or L- of amino acids, are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include compounds known in the art to be too unstable for synthesis and / or isolation. The present disclosure is intended to include compounds in racemic, scale, and optically pure form. Optically active (R)- and (S)-, or D- and L-, isomers may be prepared using chiral synthons or chiral reagents or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, unless otherwise specified, both E- and Z-geometric isomers are intended to be included in the compounds.

[0154] Unless otherwise indicated, structures depicted herein are intended to include all stereochemical forms of the structure, i.e., both R and S configurations of each asymmetric center. Accordingly, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

[0155] It will be apparent to one of ordinary skill in the art that certain compounds of the present disclosure may exist as tautomers, and all such tautomers of the compounds are within the scope of the present disclosure. As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another.

[0156] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds in which a hydrogen atom is replaced by deuterium or tritium, or a carbon atom is replaced by 13 C- or I4 Compounds substituted at C-enriched carbon atoms are also included within the scope of this disclosure.

[0157] The compounds of the present disclosure may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds. For example, the compounds of the present disclosure may contain tritium ( 3 H), iodine-125( 125 I), or carbon-14 ( 14 C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0158] The compounds of the present disclosure may exist as salts. The present disclosure also includes such salts. Examples of applicable salt forms include hydrochloride, hydrobromide, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate (e.g., (+)-tartrate, (-)-tartrate, or a mixture thereof, including a racemic mixture), succinate, benzoate, and salts with amino acids such as glutamic acid. These salts can be prepared by methods known to those skilled in the art. Also included are base addition salts of sodium, potassium, calcium, ammonium, organic amino, magnesium, and similar salts. When a compound of the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent, or by ion exchange. Examples of acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonate, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfate, hydroiodic acid, or phosphorous acid, as well as salts derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. Also included are salts of amino acids such as alginic acid, and salts of organic acids such as glucuronic acid and galacturonic acid. Some compounds of the present disclosure contain both basic and acidic functional groups, allowing these compounds to be converted into either base or acid addition salts.

[0159] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0160] Some compounds of the present disclosure can exist in unsolvated form as well as solvated form, including hydrated form.In general, solvated form is equivalent to unsolvated form and is included in the scope of the present disclosure.Some compounds of the present disclosure can exist in multiple crystalline forms or amorphous forms.In general, all physical forms are equivalent in the applications envisioned by the present disclosure and are intended to be included in the scope of the present disclosure.

[0161] Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, a reference to "a subject" includes a plurality of subjects unless the context clearly dictates otherwise (e.g., a plurality of subjects).

[0162] Throughout this specification and claims, the terms "comprises," "comprises," and "comprising" are used in a non-exclusive sense unless the context otherwise requires. Similarly, the term "comprises" and its grammatical variations are intended to be non-limiting, and the recitation of items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0163] For purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, properties, and other numerical values ​​used in the specification and claims are to be understood as being modified in all instances by the term "about," even if the value, amount, or range does not explicitly state otherwise. Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are not, and need not be, exact, but may be approximate and / or may be made larger or smaller as necessary to reflect tolerances, conversion factors, rounding, measurement error, and the like, as well as other factors known to those of ordinary skill in the art, depending upon the desired properties sought to be obtained by the subject matter of the present disclosure. For example, the term "about" when referring to a value may be intended to encompass variations in some embodiments of ±100%, in some embodiments of ±50%, in some embodiments of ±20%, in some embodiments of ±10%, in some embodiments of ±5%, in some embodiments of ±1%, in some embodiments of ±0.5%, and in some embodiments of ±0.1% from the specified amount, as appropriate for practicing the disclosed methods or using the disclosed compositions.

[0164] Furthermore, the term "about," when used in connection with one or more numerical values ​​or numerical ranges, is understood to refer to all such numerical values, including all numerical values ​​within the range, and modifies that range by extending the boundaries above and below the numerical values ​​set forth. The recitation of numerical ranges by endpoints includes all numbers contained within that range, including, for example, integers and fractions thereof (e.g., recitation of 1 to 5 includes 1, 2, 3, 4, 5, as well as fractions thereof such as 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range. [Example]

[0165] The following examples are included to provide guidance to those skilled in the art for practicing representative embodiments of the presently disclosed subject matter. Given this disclosure and the general level of skill in the art, those skilled in the art will appreciate that the following examples are intended to be merely illustrative and that numerous changes, modifications, and variations may be adopted without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples below are for illustrative purposes only and should not be construed in any way as limiting the ability to prepare the disclosed compositions by other methods. [Example]

[0166] Azotomycin prodrugs with preferential tumor delivery

[0167] (1.1 Overview) The subject matter of the present disclosure provides for the development of novel tumor cell-targeted azotomycin prodrugs intended to circulate unchanged in plasma as inactive prodrugs and to be preferentially biotransformed within tumor cells to the active glutamine antagonist metabolite "DON." Multiple azotomycin prodrugs were synthesized and subsequently characterized using a well-defined screening paradigm. Through this screening process, two prodrugs, P3 and P4, were identified that exhibited selective delivery to tumors. P3 and P4 were stable in vitro in mouse and human plasma and liver microsomes. C57BL / 6 CES1 mice bearing flank EL4 tumors were used for this study. - / - When administered subcutaneously to mice, P3 exhibited excellent pharmacokinetics, resulting in approximately 3.5-fold higher DON exposure in tumors compared with plasma administration.

[0168] Most importantly, P3 demonstrated approximately 8-fold higher tumor exposure (site of efficacy; AUC = 2.3 nmol / mL*h) versus GI tissue (site of toxicity; AUC = 0.27 nmol / mL*h). P4 also demonstrated tumor targeting in mice, but its selective delivery was less pronounced than P3. P3 demonstrated a 3-fold increase in exposure versus GI tissue (site of toxicity; AUC = 0.97 nmol / mL*h) versus tumor (site of efficacy; AUC = 3.0 nmol / mL*h). In summary, the subject matter of the present disclosure presents the rationale for the design and discovery of tumor-targeted azotomic singletamine antagonist prodrugs, demonstrating significantly lower GI exposure to tumors. While the development of this effective class of therapeutics has been hindered to date by GI toxicity, it is believed that these prodrugs could be developed for future clinical trials.

[0169] (1.2 Experiment) [ka]

[0170] Scheme 1: Azotomycin prodrugs bearing tBu esters

[0171] tert-Butyl (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoamido)-6-diazo-5-oxohexanoato (3) [ka] Fmoc-L-DON-OH (1.65 g, 4.18 mmol, 1 equiv.) and HATU (1.67 g, 4.39 mmol, 1.05 equiv.) were dissolved in anhydrous DCM (40 mL). The mixture was cooled to 0 °C, and DIPEA (1.62 g, 2.18 mL, 12.5 mmol, 3 equiv.) was added. The mixture was stirred for 5 min, and a solution of HL-DON-OtBu (950 mg, 4.18 mmol, 1 equiv.) in anhydrous DCM (12 mL) was added. The resulting mixture was stirred at 0 °C for 30 min and at room temperature for 1.5 h. The DCM was evaporated, and the residue was dissolved in EtOAc (400 mL), washed with saturated NaHCO (200 mL), 10% KHSO (200 mL), HO (200 mL), brine (200 mL), dried over MgSO, and the DCM was evaporated. The crude product was purified by silica LC (DCM / EtOAc, 1:1) to give the product 3 as a yellow solid (1.60 g) in 63% yield.

[0172] 1 H NMR(CDCl3):1.48(s,9H),1.96-2.07(m,2H),2.14-2.28(m,2H),2.31-2.46(m,2H),2.48-2.65( m,2H),4.24(q,J=7.6,7.1Hz,2H),4.38(d,J=7.3Hz,2H),4.45(td,J=8.1,4.6Hz,1H),5.26(bs, 1H),5.35(bs,1H),5.95(d,J=7.5Hz,1H),7.27-7.31(m,1H),7.33(tt,J=7.4,1.0Hz,2H),7.36- 7.45(m,2H),7.60(dd,J=7.8,3.0Hz,2H),7.75(dq,J=7.6,1.0Hz,2H).ESI-MS:625.400([M+Na] + ).

[0173] tert-Butyl (S)-2-((S)-2-amino-6-diazo-5-oxohexanamide)-6-diazo-5-oxohexanoate (4) [ka] Compound 3 (1.29 g, 2.14 mmol, 1 equiv.) was dissolved in anhydrous DCM (15 mL), diethylamine (2.20 mL, 21.4 mmol, 10 equiv.) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 2.5 h. Volatiles were removed in vacuo, and the crude product 4 was used immediately in the next step without purification.

[0174] tert-Butyl(5S,10S,13S)-5-(tert-butoxycarbonyl)-10,13-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-3,8,11-trioxo-2-oxa-4,9,12-triazatetradecane-14-oate (5) [ka] Fmoc-L-Glu(H)-OtBu (765 mg, 1.80 mmol, 1 equiv.) and HATU (752 mg, 1.98 mmol, 1.1 equiv.) were dissolved in anhydrous DCM (15 mL). The mixture was cooled to 0 °C, and DIPEA (0.94 mL, 5.39 mmol, 3.0 equiv.) was added. After 5 min, a solution of compound 4 (684 mg, 1.80 mmol, 1.0 equiv.) in anhydrous DCM (5 mL) was added, and the mixture was stirred at 0 °C for 30 min and at room temperature overnight (16 h). The DCM was evaporated, and the residue was dissolved in EtOAc (250 mL) and washed with saturated NaHCO (100 mL), HO (100 mL), 10% KHSO (100 mL), and brine (100 mL). The organic layer was dried over MgSO, and the EtOAc was evaporated. The crude product was purified by silica LC (DCM / MeOH, 30:1) to give the product 5 as a yellow solid (734 mg, 54% yield over two steps).

[0175] 1H NMR(CDCl3):1.45(s,9H),1.47(s,9H),1.87-2.01(m,3H),2.11-2.26(m,3H),2.2 8-2.42(m,4H),2.46-2.66(m,2H),4.20-4.27(m,2H),4.35-4.45(m,4H),5.30(bs, 1H),5.34(bs,1H),5.64(d,J=8.1Hz,1H),6.81(d,J=7.0Hz,1H),7.24(m,1H),7.2 8-7.45(m,4H),7.61(d,J=7.4Hz,2H),7.73-7.80(m,2H).ESI-MS:810.647([M+Na] + ).

[0176] tert-Butyl (S)-2-((S)-2-((S)-4-amino-5-(tert-butoxy)-5-oxopentanamide)-6-diazo-5-oxohexamide)-6-diazo-5-oxohexanoate (P1) [ka] Compound 5 (734 mg, 0.932 mmol, 1 equiv.) was dissolved in anhydrous DCM (7.5 mL), diethylamine (1.93 mL, 18.6 mmol, 20 equiv.) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 2 h. The volatiles were removed in vacuo, and the residue was purified by silica LC (DCM / MeOH, 15:1). The product P1 was isolated as a yellow solid (350 mg) in 66% yield.

[0177] 1 H NMR(CDCl3):1.45(s,9H),1.46(s,9H),1.74-1.90(m,3H),1.93-2.09(m,3H),2.09-2.25(m,2H),2.29-2.45(m,4H),2.44-2.65(m,2H),3 .34(dd,J=8.7,4.8Hz,1H),4.36-4.46(m,2H),5.36(2xCH,bs,2H),7.04(d,J=7.1Hz,1H),7.42(d,J=7.9Hz,1H).ESI-MS:566.416([M+H] + ).

[0178] tert-Butyl (S)-2-((S)-2-((S)-4-acetamido-5-(tert-butoxy)-5-oxopentanamido)-6-diazo-5-oxohexanoate)-6-diazo-5-oxohexanoate (P2) [ka] Compound P1 (200 g, 0.354 mmol, 1 equiv.) was dissolved in anhydrous DCM (2 mL), and pyridine (57 μL, 0.707 mmol, 2.0 equiv.) was added, followed by acetone (40 μL, 0.424 mmol, 1.2 equiv.). The reaction mixture was stirred at room temperature under an inert nitrogen atmosphere for 1 h. The volatiles were removed in vacuo, and the residue was purified by silica LC (DCM / MeOH, 30:1 to 10:1). The product P2 was isolated as a yellow solid (143 mg) in 67% yield.

[0179] 1 H NMR(CDCl3):1.46(s,9H),1.47(s,9H),1.90(ddd,J=14.1,7.1,2.1Hz,1H),1.94 -2.01(m,2H),2.02(s,3H),2.10-2.26(m,3H),2.28-2.34(m,2H),2.33-2.46(m, 2H),2.47-2.65(m,2H),4.36-4.50(m,3H),5.36(bs,1H),5.38(bs,1H),6.43(d, J=7.7Hz,1H),7.01(d,J=7.0Hz,1H),7.26-7.29(m,1H).ESI-MS:630.490([M+Na] + ).

[0180] tert-Butyl (6S,11S,14S)-6-(tert-butoxycarbonyl)-11,14-bis(4-diazo-3-oxobutyl)-2-methyl-4,9,12-trioxo-2,5,10,13-tetraazapentadecan-15-oate (P3) [ka] Compound P1 (158 mg, 0.279 mmol, 1 equiv.) was dissolved in anhydrous DCM (6 mL), 2,5-dioxopyrrolidin-1-yldimethylglycinate (67 mg, 0.335 mmol, 1.2 equiv.) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. DCM (50 mL) was added, and the organic layer was washed with saturated NaHCO (2 × 30 mL) and brine (30 mL), dried over anhydrous MgSO, and the solvent was evaporated. The crude product was purified by silica LC (DCM / MeOH, 10:1), and product P3 was isolated as a yellow solid (178 mg, 98%).

[0181] 1 H NMR(CDCl3):1.45(s,9H),1.47(s,9H),1.65-1.81(m,1H),1.82-1.93(m,1H),1.93-2.05(m ,2H),2.11-2.25(m,4H),2.33(s,6H),2.35-2.70(m,4H),2.92(d,J=16.3Hz,1H),3.04(d,J =16.3Hz,1H),4.37-4.45(m,2H),4.49(td,J=9.1,3.9Hz,1H),5.36(bs,1H),5.39(bs,1H), 7.10(d,J=6.6Hz,1H),7.24(d,J=8.0Hz,1H),7.71(d,J=8.7Hz,1H).ESI-MS:651.513([M+H] + ).

[0182] tert-Butyl(5S,8S,13S,16S)-5-((1H-indol-3-yl)methyl)-8-(tert-butoxycarbonyl)-13,16-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,11,14-tetraoxo-2-oxa-4,7,12,15-tetraazaheptadecan-17-oate (6) [ka] Fmoc-L-Trp-OH (249 mg, 0.583 mmol, 1.1 equiv) and HATU (232 mg, 0.610 mmol, 1.15 equiv) were dissolved in anhydrous DMF (10 mL). The mixture was cooled to 0 °C, and DIPEA (277 μL, 1.59 mmol, 3.0 equiv) was added. After 5 min, a solution of compound P1 (300 mg, 0.530 mmol, 1.0 equiv) in anhydrous DMF (6 mL) was added, and the mixture was stirred at 0 °C for 30 min and at room temperature for 2 h. The DCM was evaporated, and the residue was purified by reverse-phase HPLC (AcN / HO) to give product 6 as a yellow solid (448 mg, 88% yield).

[0183] 1 H NMR(CDCl3):1.41(s,9H),1.46(s,9H),1.86-2.07(m,4H),2.10-2.28(m,4H),2.30-2.63(m,4H),3. 11-3.22(m,1H),3.47(dd,J=14.9,4.8Hz,1H),4.22(t,J=7.1Hz,1H),4.26-4.50(m,5H),4.60-4.71 (m,1H),5.30(bs,1H),5.53(bs,1H),6.81-6.93(m,2H),7.11-7.24(m,4H),7.28-7.46(m,6H),7.54 -7.60(m,2H),7.60-7.67(m,1H),7.77(d,J=7.6Hz,2H),9.00-9.10(m,1H).ESI-MS:996.725([M+Na] + ).

[0184] tert-Butyl (S)-2-((S)-2-((S)-4-((S)-2-amino-3-(1H-indol-3-yl)propanamido)-5-(tert-butoxy)-5-oxopentanamido)-6-diazo-5-oxohexanoate (7) [ka] Compound 6 (445 mg, 0.457 mmol, 1 equiv.) was dissolved in anhydrous DCM (5 mL), diethylamine (946 μL, 9.14 mmol, 20 equiv.) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 2 h. The volatiles were removed in vacuo, and the residue was purified by silica LC (DCM / MeOH, 15:1 to 10:1). The product 7 was isolated as a yellow solid (27 mg) in 79% yield.

[0185] 1 H NMR(CDCl3):1.44(s,9H),1.47(s,9H),1.57-1.78(m,2H),1.81-2.07(m,4H),2.08-2.30(m,4H),2. 56(d,J=17.7Hz,4H),3.26(dd,J=14.5,5.3Hz,2H),3.80(dd,J=6.1,4.5Hz,1H),4.33-4.50(m,3H),5 .33(bs,1H),5.36(bs,1H),6.83(d,J=7.0Hz,1H),7.07-7.22(m,3H),7.32(d,J=7.6Hz,1H),7.39(d t,J=8.2,1.0Hz,1H),7.63-7.70(m,1H),7.92(d,J=8.5Hz,1H),9.08(s,1H).ESI-MS:752.312([M+H] + ).

[0186] tert-Butyl (6S,9S,14S,17S)-6-((1H-indol-3-yl)methyl)-9-(tert-butoxycarbonyl)-14,17-bis(4-diazo-3-oxobutyl)-2-methyl-4,7,12,15-tetraoxo-2,5,8,13,16-pentaazaoctadecane-18-oate (P4) [ka] Compound 7 (270 mg, 0.359 mmol, 1 equiv.) was dissolved in anhydrous DCM (5 mL), 2,5-dioxopyrrolidin-1-yldimethylglycinate (144 mg, 0.718 mmol, 2.0 equiv.) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. DCM (300 mL) was added, and the organic layer was washed with saturated NaHCO (2 × 150 mL) and brine (150 mL), dried over anhydrous MgSO, and the solvent was evaporated. The crude product was purified by silica LC (DCM / MeOH, 15:1 to 10:1), and product P4 was isolated as a yellow solid (262 mg, 87%).

[0187] 1 H NMR(CDCl3):1.44(s,9H),1.49(s,9H),1.81-2.13(m,5H),2.17(s,6H),2.20-2.32(m,3H),2.32-2.62(m,4H),2.89(d,J=16.3Hz, 1H),3.01(d,J=16.3Hz,1H),3.23-3.41(m,2H),4.36(q,J=7.2Hz,1H),4.42(dt,J=8.0,3.9Hz,1H),4.49(td,J=8.1,4.5Hz,1H),4. 79(q,J=6.8Hz,1H),5.35(bs,1H),5.37(bs,1H),7.07(d,J=7.7Hz,1H),7.13(ddd,J=8.1,7.0,1.1Hz,1H),7.17-7.23(m,2H),7.26 (bs,1H),7.30-7.34(m,1H),7.36-7.40(m,1H),7.68(d,J=7.9Hz,1H),7.74(d,J=7.2Hz,1H),8.67(s,1H).ESI-MS:837.716([M+H] + ).

[0188] [ka]

[0189] Scheme 2: Amide-bearing azotomycin prodrugs

[0190] (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-amino-6-diazo-1,5-dioxohexan-2-yl)amino)-6-diazo-1,5-dioxohexan-2-yl)carbamate (9) [ka] Fmoc-L-DON-OH (1.95 g, 4.96 mmol, 1.1 equiv.) and HATU (1.97 g, 5.18 mmol, 1.15 equiv.) were dissolved in anhydrous DCM (30 mL). The mixture was cooled to 0 °C, and DIPEA (2.40 mL, 3.5 mmol, 3 equiv.) was added. The mixture was stirred for 5 min, and a solution of HL-DON-CONH2 (746 mg, 4.38 mmol, 1 equiv.) in anhydrous DCM (17 mL) was added. The resulting mixture was stirred at 0 °C for 30 min and at room temperature for 2 h. DMF was evaporated, and the crude product was purified by silica LC (DCM / MeOH, 20:1 to 15:1) to give product 9 as a yellow solid (1.46 g) in 61% yield.

[0191] 1 H NMR(DMSO-d6):1.73-1.80(m,2H),1.85-1.96(m,2H),2.23-2.42(m,4H),3.95-4.02(m,1H),4.16-4.30(m,4H),6.01(bs,2H) ),7.05-7.08(m,1H),7.30-7.43(m,5H),7.54-7.58(m,1H),7.71-7.74(m,2H),7.86-7.95(m,3H).ESI-MS:568.320([M+Na] + ).

[0192] (S)-2-Amino-N-((S)-1-amino-6-diazo-1,5-dioxohexan-2-yl)-6-diazo-5-oxohexanamide (10) [ka] Compound 9 (1.46 mg, 2.68 mmol, 1 equiv.) was dissolved in a mixture of anhydrous solvents DCM / DMF (20 + 8 mL), diethylamine (2.80 mL, 27.0 mmol, 10 equiv.) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. Volatiles were removed in vacuo, and the crude product 10 was used immediately in the next step without purification.

[0193] (9H-Fluoren-9-yl)methyl ((S)-1-amino-5-(((S)-1-(((S)-1-amino-6-diazo-1,5-dioxohexan-2-yl)amino)-6-diazo-1,5-dioxohexan-2-yl)amino)-1,5-dioxopentan-2-yl)carbamate (11) [ka] Fmoc-L-Glu(H)-CONH2 (1.08 g, 2.94 mmol, 1.1 equiv.) and HATU (1.17 g, 3.08 mmol, 1.15 equiv.) were dissolved in anhydrous DMF (35 mL). The mixture was cooled to 0 °C, and DIPEA (1.40 mL, 8.03 mmol, 3.0 equiv.) was added. After 5 min, a solution of compound 10 (865 mg, 2.68 mmol, 1.0 equiv.) in anhydrous DMF (15 mL) was added, and the mixture was stirred at 0 °C for 30 min and at room temperature overnight (16 h). The DMF was evaporated, and the crude product was purified by reverse-phase HPLC (AcN / HO) to give product 11 as a yellow solid (1.12 g, 62% yield over two steps).

[0194] 1 H NMR(DMSO-d6):1.68-1.96(m,6H),2.18-2.22(m,2H),2.29-2.36(m,4H),3.92(d,J=6.6Hz,1H),4.12-4.27(m,5H),6.01(bs,2H) ,7.04-7.07(m,2H),7.26-7.52(m,7H),7.73(d,J=7.5Hz,2H),7.88-7.95(m,3H),8.03(d,J=7.5Hz,1H).ESI-MS:696.397([M+Na] + ).

[0195] (S)-4-amino-N1-((S)-1-(((S)-1-amino-6-diazo-1,5-dioxohexan-2-yl)amino)-6-diazo-1,5-dioxohexan-2-yl)pentanediamide (P5) [ka] Compound 11 (1.12 mg, 1.66 mmol, 1 equiv.) was dissolved in anhydrous DCM (25 mL), diethylamine (5 mL) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. The volatiles were removed in vacuo, and the residue was purified by silica LC (DCM / MeOH, 2:1 + 1% EtN). The product P5 was isolated as a yellow solid (590 mg) in 79% yield.

[0196] 1 H NMR(DMSO-d6):1.62(dq,J=13.3,7.8Hz,1H),1.69-1.82(m,3H),1.83-2.00(m, 3H),2.16-2.25(m,2H),2.26-2.42(m,4H),3.10(dd,J=7.8,5.3Hz,1H),4.11-4 .24(m,2H),6.04(2xCH,bs,2H),6.97(bs,1H),7.06(bs,1H),7.28(bs,1H),7.3 2(bs,1H),7.94(d,J=8.1Hz,1H),8.08(d,J=7.4Hz,1H).ESI-MS:452.302([M+H] + ).

[0197] (S)-4-Acetamido-N1-((S)-1-(((S)-1-amino-6-diazo-1,5-dioxohexan-2-yl)amino)-6-diazo-1,5-dioxohexan-2-yl)pentanediamide (P6) [ka] Compound P5 (175 g, 0.388 mmol, 1 equiv.) was dissolved in anhydrous DCM (3 mL) and pyridine (94 μL, 1.16 mmol, 3.0 equiv.) was added, followed by acetone (73 μL, 0.775 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. The volatiles were removed in vacuo, and the residue was purified by silica LC (DCM / MeOH, 5:1 + 1% EtN). The product P6 was isolated as a yellow solid (114 mg) in 60% yield.

[0198] 1 H NMR(DMSO-d6):1.72(tq,J=13.5,7.4Hz,3H),1.85(s,3H),1.86-1.98(m,3H),2.16(t,J=8.0Hz,2H),2.24-2.40(m,4H),4.07-4.22(m,3H),6. 03(2xCH,bs,2H),7.01(bs,1H),7.06(bs,1H),7.29(bs,1H),7.33(bs,1H),7.88-7.96(m,2H),8.04(d,J=7.4Hz,1H).ESI-MS:516.251([M+Na] + ).

[0199] (S)-N1-((S)-1-(((S)-1-amino-6-diazo-1,5-dioxohexan-2-yl)amino)-6-diazo-1,5-dioxohexan-2-yl)-4-(2-(dimethylamino)acetamido)pentanediamide (P7) [ka] Compound P5 (140 mg, 0.310 mmol, 1 equiv.) was dissolved in anhydrous DCM (5 mL), 2,5-dioxopyrrolidin-1-yldimethylglycinate (75 mg, 0.372 mmol, 1.2 equiv.) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. DMF was evaporated, and the crude product was purified by silica LC (DCM / MeOH, 5:1 + 1% EtN) to isolate product P7 as a yellow solid (140 mg, 84%).

[0200] 1H NMR(DMSO-d6):1.67-1.83(m,3H),1.83-1.98(m,3H),2.14(t,J=7.9Hz,2H),2.22(s,6) H),2.26-2.41(m,4H),2.85-2.94(m,2H),4.08-4.22(m,2H),4.25(td,J=8.0,5.2Hz,1 H),6.02(2xCH,bs,2H),7.05(bs,1H),7.14(bs,1H),7.28(bs,1H),7.48(bs,1H),7.70 (d,J=8.3Hz,1H),7.91(d,J=8.0Hz,1H),8.06(d,J=7.5Hz,1H).ESI-MS:537.345([M+H] + ).

[0201] (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-amino-5-(((S)-1-(((S)-1-amino-6-diazo-1,5-dioxohexan-2-yl)amino)-6-diazo-1,5-dioxohexan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate (12) [ka] Fmoc-L-Trp-OH (208 mg, 0.488 mmol, 1.1 equiv) and HATU (194 mg, 0.510 mmol, 1.15 equiv) were dissolved in anhydrous DMF (6 mL). The mixture was cooled to 0 °C, and DIPEA (232 μL, 1.33 mmol, 3.0 equiv) was added. After 5 min, a solution of compound P5 (200 mg, 0.444 mmol, 1.0 equiv) in anhydrous DMF (4 mL) was added, and the mixture was stirred at 0 °C for 30 min and at room temperature for 1.5 h. The DMF was evaporated, and the residue was purified by reverse-phase HPLC (AcN / HO) to give product 12 as a yellow solid (356 mg, 94% yield).

[0202] 1H NMR(DMSO-d6):1.66-2.03(m,4H),2.20(t,J=8.0Hz,2H),2.26-2.37(m,4H),2.41-2.46(m,2H),2.94 -3.21(m,2H),4.11-4.25(m,6H),4.27-4.36(m,1H),6.00(bs,2H),6.97(t,J=7.4Hz,1H),7.01-7.11 (m,3H),7.15-7.20(m,1H),7.21-7.35(m,4H),7.39(q,J=7.0Hz,2H),7.52(d,J=8.2Hz,1H),7.58-7. 71(m,3H),7.86(d,J=7.6Hz,2H),7.92-8.02(m,4H),8.07(d,J=8.0Hz,1H).ESI-MS:882.621([M+Na] + ).

[0203] (S)-4-((S)-2-amino-3-(1H-indol-3-yl)propanamide)-N1-((S)-1-(((S)-1-amino-6-diazo-1,5-dioxohexan-2-yl)amino)-6-diazo-1,5-dioxohexan-2-yl)pentanediamide (13) [ka] Compound 12 (356 g, 0.414 mmol, 1 equiv.) was dissolved in anhydrous DCM (8 mL), diethylamine (856 μL, 8.28 mmol, 20 equiv.) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. The volatiles were removed in vacuo, and the residue was purified by silica LC (DCM / MeOH, 2:1 + 1% EtN). Product 13 was isolated as a yellow solid (222 mg) in 84% yield.

[0204] 1H NMR(DMSO-d6):1.64-1.83(m,3H),1.87-2.01(m,3H),2.13-2.28(m,4H),2.28-2.43(m,4H),2.71(d,J=15 .4Hz,1H),2.86(d,J=15.4Hz,1H),4.07-4.30(m,2H),4.50-4.65(m,2H),6.00(bs,1H),6.05(bs,1H),6.9 8(t,J=7.3Hz,1H),7.00-7.24(m,3H),7.22-7.30(m,3H),7.58(d,J=7.6Hz,1H),7.70(d,J=7.2Hz,1H),7. 92(d,J=7.6Hz,1H),8.04(d,J=7.1Hz,1H),8.19(d,J=7.6Hz,1H),10.42(bs,1H).ESI-MS:638.462([M+H] + ).

[0205] (S)-N1-((S)-1-(((S)-1-amino-6-diazo-1,5-dioxohexan-2-yl)amino)-6-diazo-1,5-dioxohexan-2-yl)-4-((S)-2-(2-(dimethylamino)acetamido)-3-(1H-indol-3-yl)propanamido)pentanediamide (P8) [ka] Compound 13 (222 mg, 0.348 mmol, 1 equiv.) was dissolved in anhydrous DMF (2.5 mL), 2,5-dioxopyrrolidin-1-yldimethylglycinate (140 mg, 0.696 mmol, 2.0 equiv.) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. DMF was evaporated, and the crude product was purified by reverse-phase HPLC (AcN / HO) to isolate product P8 as a yellow solid (133 mg, 53% yield).

[0206] 1H NMR(DMSO-d6):1.66-1.84(m,3H),1.84-1.99(m,3H),2.01(s,6H),2.13-2.25(m,2H),2.26-2.41(m,4H),2.70(d,J= 15.5Hz,1H),2.83(d,J=15.5Hz,1H),2.98-3.09(m,1H),3.14-3.24(m,1H),4.05-4.28(m,3H),4.55-4.69(m,1H),6. 03(2xCH,bs,2H),6.96(t,J=7.2Hz,1H),7.01-7.19(m,4H),7.24-7.36(m,3H),7.57(d,J=7.6Hz,1H),7.69(d,J=7.2 Hz,1H),7.94(d,J=7.7Hz,1H),8.01(d,J=7.0Hz,1H),8.17(d,J=7.7Hz,1H),10.80(bs,1H).ESI-MS:723.491([M+H] + ).

[0207] [ka]

[0208] Scheme 3: Azotomycin prodrugs with various esters

[0209] 1.2.1 General Procedure for Compounds 15a-c Fmoc-L-DON-OH (1.50 g, 3.81 mmol, 1.1 equiv.) and HATU (1.52 g, 3.98 mmol, 1.15 equiv.) were suspended in anhydrous DCM (40 mL). The mixture was cooled to 0° C., and DIPEA (1.81 mL, 10.4 mmol, 3 equiv.) was added. The mixture was stirred for 5 min, and the resulting mixture was mixed with HL-DON-OH in anhydrous DCM (10 mL). 1 A solution of (3.47 mmol, 1 equiv) was added dropwise. The resulting mixture was stirred at 0 °C for 30 min and at room temperature for 16 h. The volatiles were evaporated and the residue was dissolved in EtOAc (200 mL) and washed with saturated NaHCO (150 mL), HO (80 mL), and brine (80 mL). The organic layer was dried over anhydrous MgSO and the DCM was evaporated. The crude product was purified by LC on silica (DCM / EtOAc, 30:1).

[0210] Allyl(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoamido)-6-diazo-5-oxohexanoate (15a) [ka] HL-DON-OAll (14a) (732 mg) was purified by silica LC (DCM / EtOAc, 3:5) to give product 15a (1.55 g, 76%) as a yellow solid.

[0211] 1 H NMR(DMSO-d6):1.71-1.93(m,3H),1.96-2.08(m,2H),2.26-2.46(m,4H),3.97-4. 08(m,1H),4.17-4.33(m,4H),4.53-4.62(m,2H),5.18-5.23(m,1H),5.26-5.34(m, 1H),5.87-5.97(m,1H),5.98-6.09(m,1H),7.29-7.44(m,4H),7.53-7.60(m,1H),7 .70-7.78(m,2H),7.90(d,J=7.9Hz,2H),8.35-8.45(m,1H).ESI-MS:609.3([M+Na] + ).

[0212] Ethyl (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanamido)-6-diazo-5-oxohexanoate (15b) (124-189) [ka] HL-DON-Oet (14b) (690 mg) was purified by silica LC (DCM / EtOAc, 1:2) to give product 15b (677 mg, 34%) as a yellow oil. ESI-MS: 597.3 ([M+Na] + ).

[0213] Isopropyl(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoamido)-6-diazo-5-oxohexanoate (15c) [ka] HL-DON-OiPr (14c) (739 mg) was purified by silica LC (DCM / EtOAc, 1:1) to give the product 15c (1.22 g, 60%) as a yellow solid.

[0214] 1 H NMR(CDCl3):1.20-1.26(m,6H),1.90-2.00(m,2H),2.08-2.23(m,2H),2.30-2.41(m,2H ),2.42-2.52(m,2H),4.20(t,J=7.0Hz,2H),4.31-4.40(m,2H),4.44(dd,J=8.9,4.6Hz, 1H),4.95-5.05(m,1H),5.26-5.31(m,1H),5.32-5.41(m,1H),6.01-6.12(m,1H),7.34( dt,J=42.4,7.5Hz,4H),7.55-7.62(m,3H),7.74(d,J=7.5Hz,2H).ESI-MS:611.3([M+Na] + ).

[0215] General procedure for compounds 16a-c: Compound 15c (1.70 mmol, 1 equiv.) was dissolved in anhydrous DCM (7 mL) and diethylamine (17.0 mmol, 10 equiv.) was added. The mixture was stirred at room temperature for 2 h. Volatiles were evaporated in vacuo, and the crude product was used in the next reaction without further purification due to the extreme instability of the product.

[0216] Allyl(S)-2-((S)-2-amino-6-diazo-5-oxohexanamide)-6-diazo-5-oxohexanoate (16a) [ka]

[0217] ESI-MS: 387.2 ([M+Na] + ).

[0218] Ethyl (S)-2-((S)-2-amino-6-diazo-5-oxohexanamido)-6-diazo-5-oxohexanoate (16b) [ka]

[0219] ESI-MS: 353.2 ([M+H] + ).

[0220] Isopropyl (S)-2-((S)-2-amino-6-diazo-5-oxohexanamide)-6-diazo-5-oxohexanoate (16c) ESI-MS: 367.2 ([M+H] + ). [ka]

[0221] 5-(tert-butyl)1-isopropyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamic acid (18) [ka] Fmoc-L-Glu(t-Bu)-OH (17) (5.0 g, 11.8 mmol, 1 equiv.) was dissolved in anhydrous DCM (50 mL) and i-PrOH (4.50 mL, 58.7 mmol, 5 equiv.) was added. CDI (3.81 g, 23.5 mmol, 2 equiv.) was added in one portion, and the resulting mixture was stirred at room temperature for 24 h. The volatiles were evaporated in vacuo, and the crude mixture was diluted with DCM (100 mL) and washed with 10% KHSO (100 mL) and brine (50 mL). The organic phase was dried over MgSO and concentrated in vacuo, then purified by liquid silica chromatography (hexane / EtOAc, gradient 7:1 to 4:1) to give product 18 (5.16 g, 94%) as a colorless oil.

[0222] 1 H NMR(CDCl3):1.22-1.29(m,6H),1.45(s,9H),1.90-1.99(m,1H),2.11-2.19(m ,1H),2.29-2.39(m,2H),4.12(q,J=7.1Hz,1H,),4.23(t,J=7.1Hz,1H),4.32- 4.45(m,2H),5.07(hept,J=6.1Hz,1H),5.45(d,J=8.1Hz,1H),7.36(dt,J=42. 1,7.4Hz,4H),7.54-7.66(m,2H),7.77(d,J=7.6Hz,2H).ESI-MS:468.2([M+H] + ).

[0223] (S)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-isopropoxy-5-oxopentanoic acid (19) [ka] Compound 18 (5.16 g, 11.04 mmol, 1 equiv.) was dissolved in anhydrous DCM (25 mL), cooled to 0 °C, and TFA (25 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The crude mixture was triturated with hexane, filtered, and dried in vacuo. Product 19 (4.40 g, 97%) was obtained as a colorless solid.

[0224] 1 H NMR(CDCl3):1.19-1.39(m,6H),1.82-2.60(m,4H),4.00-4.57(m,4H),5.00-5.19(m,1H),5.51-5.62 (m,1H),7.18-7.48(m,4H),7.55-7.62(m,2H),7.72-7.89(m,2H),9.50(s,1H).ESI-MS:412.2([M+H] + ).

[0225] General procedure for compounds 20a-e: Fmoc-Glu(OH)-OR 2(1.1 equiv.) and HATU (1.2 equiv.) were suspended in anhydrous DCM, and the mixture was cooled to 0°C. DIPEA (3 equiv.) was added, and the reaction mixture was stirred at the same temperature for 10 min. A solution of compound 16a-c (1 equiv.) in anhydrous DCM was added dropwise. The resulting mixture was stirred at 0°C for 30 min and at room temperature for 16 h, then concentrated in vacuo. Workup (if necessary) and purification were performed according to the individual reactions.

[0226] Allyl(5S,10S,13S)-10,13-bis(4-diazo-3-oxobutyl)-5-(ethoxycarbonyl)-1-(9H-fluoren-9-yl)-3,8,11-trioxo-2-oxa-4,9,12-triazatetradecane-14-oate (20a) [ka] A solution of Fmoc-L-Glu(OH)-OEt (745 mg, 1.87 mmol) and HATU (778 mg, 2.05 mmol) in DCM (15 mL), DIPEA (891 μL, 5.11 mmol), and amine 16a (621 mg, 1.71 mmol) in DCM (7 mL) was purified by silica chromatography (DCM / MeOH, 30:1) to give product 20a (1.02 g, 80%) as a yellow solid. ESI-MS: 766.3 ([M+Na]+).

[0227] Allyl(5S,10S,13S)-5-(tert-butoxycarbonyl)-10,13-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-3,8,11-trioxo-2-oxa-4,9,12-triazatetradecane-14-oate (20b) [ka] A solution of Fmoc-L-Glu(OH)-OtBu (374 mg, 0.879 mmol) and HATU (349 mg, 0.918 mmol), DIPEA (417 μL, 2.40 mmol), and amine 16a (291 mg, 0.800 mmol) in DCM (6 mL) was purified by silica chromatography (DCM / MeOH, 30:1) to give product 20b (425 mg, 69%) as a yellow oil.

[0228] 1 H NMR(DMSO-d6)1.39(s,9H),1.67-2.06(m,6H),2.17-2.25(m,2H),2.27-2.46(m,4H),3.85- 3.95(m,1H),4.18-4.35(m,6H),4.56(dt,J=5.5,1.4Hz,2H),5.20(dq,J=10.5,1.4Hz,1H), 5.30(dq,J=17.2,1.6Hz,1H),5.95-6.10(m,2H),7.30-7.45(m,4H),7.71(t,J=8.6Hz,3H), 7.90(d,J=7.5Hz,2H),8.03(d,J=7.9Hz,1H),8.40(d,J=7.4Hz,1H).ESI-MS:794.3([M+Na] + ).

[0229] Ethyl (5S,10S,13S)-5-((allyloxy)carbonyl)-10,13-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-3,8,11-trioxo-2-oxa-4,9,12-triazatetradecane-14-oate (20c) [ka] A solution of Fmoc-L-Glu(OH)-OAll (689 mg, 1.68 mmol) and HATU (698 mg, 1.84 mmol), DIPEA (799 μL, 4.59 mmol), and amine 16b (539 mg, 1.53 mmol) in DCM (6 mL) was purified by silica chromatography (DCM / MeOH, 30:1) to give product 20c (1.06 g, 93%) as a yellow oil.

[0230] 1 H NMR(CDCl3):1.26(t,J=7.1Hz,3H),1.92-2.05(m,4H),2.08-2.31(m,3H),2.32-2.45(m,3H),2.45-2.6 2(m,4H),4.17(q,J=7.1Hz,2H),4.18-4.24(m,1H),4.33-4.44(m,3H),4.48(td,J=8.4,4.9Hz,1H),4.6 5(d,J=5.6Hz,1H),5.26(d,J=10.4Hz,1H),5.31-5.42(m,2H),5.81(d,J=7.9Hz,1H),5.85-6.05(m,3H) ,6.82(d,J=6.3Hz,1H),7.30-7.42(m,4H),7.58-7.64(m,2H),7.75-7.78(m,2H).ESI-MS:766.4([M+Na] + ).

[0231] tert-Butyl (5S,10S,13S)-5-((allyloxy)carbonyl)-10,13-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-3,8,11-trioxo-2-oxa-4,9,12-triazatetradecane-14-oate (20d) [ka] A mixture of Fmoc-L-Glu(OH)-OAll (485 mg, 1.18 mmol) and HATU (491 mg, 129 mmol), DIPEA (563 μL, 3.23 mmol), and amine 4 (410 mg, 1.08 mmol) in DCM (10 mL) was purified by silica chromatography (DCM / MeOH, 30:1) to give product 20d (640 mg, 59%) as a yellow oil.

[0232] 1H NMR(DMSO-d6)1.38(s,9H),1.66-1.83(m,3H),1.85-2.08(m,3H),2.20-2.44(m,7H),4.01-4.13( m,2H),4.20-4.36(m,4H),4.55-4.62(m,2H),5.15-5.22(m,1H),5.30(dq,J=17.3,1.6Hz,1H),5. 99(bs,1H),6.06(bs,1H),7.38(dt,J=42.4,7.4Hz,4H),7.71(d,J=7.4Hz,2H),7.85(d,J=8.0Hz, 1H),7.90(d,J=7.5Hz,2H),8.02(d,J=7.9Hz,1H),8.25(d,J=7.2Hz,1H).ESI-MS:744.3([M-N2+H] + ).

[0233] Isopropyl(5S,10S,13S)-10,13-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-5-(isopropoxycarbonyl)-3,8,11-trioxo-2-oxa-4,9,12-triazatetradecane-14-oate (20e) [ka] Fmoc-L-Glu(OH)-OiPr (19) (1.46 g, 3.55 mmol) and HATU (1.47 g, 3.87 mmol), DIPEA (1.69 mL, 9.68 mmol) in DCM (30 mL), and amine 16c (1.18 g, 3.23 mmol) in DCM (15 mL) were diluted with EtOAc (250 mL) and washed with saturated NaHCO (130 mL), 50% aqueous NaCl (120 mL), and brine (90 mL). The organic phase was dried over MgSO and concentrated in vacuo, then purified by silica chromatography (DCM / MeOH, 30:1) to give product 20e (370 mg, 14%) as a yellow oil.

[0234] 1H NMR(CDCl3):1.22-1.30(m,12H),1.90-2.05(m,3H),2.07-2.28(m,3H),2.31(t,J=7.1Hz,2H),2.34 -2.47(m,2H),2.47-2.68(m,2H),4.23(t,J=7.1Hz,1H),4.28-4.35(m,1H),4.35-4.43(m,3H),4.46( td,J=8.1,4.7Hz,1H),4.98-5.12(m,2H),5.29(bs,1H),5.34(bs,1H),5.70(d,J=7.8Hz,1H),6.77( d,J=6.4Hz,1H),7.29-7.44(m,5H),7.57-7.64(m,2H),7.77(d,J=7.5Hz,2H).ESI-MS:782.3([M+Na] + ).

[0235] General procedure for compounds 21a-d and P9: Compounds 20a-e (1 equivalent) were dissolved in anhydrous DCM and diethylamine (10 equivalents) was added. The reaction mixture was stirred at room temperature for 4 hours and then concentrated in vacuo. The crude mixture was purified by silica chromatography (DCM / MeOH, 10:1).

[0236] Allyl(S)-2-((S)-2-((S)-4-amino-5-ethoxy-5-oxopentanamide)-6-diazo-5-oxohexanoamide)-6-diazo-5-oxohexanoate (21a) [ka] Compound 20a (1.0 g, 1.34 mmol) in DCM (7 mL), EtNH (1.4 mL, 13.4 mmol) afforded product 21a (500 mg, 71%) as a yellow amorphous compound.

[0237] 1H NMR(CDCl3):1.28(t,J=7.1Hz,3H),1.90-2.03(m,3H),2.12-2.30(m,4H),2.37-2.52 (m,6H),3.07(q,J=7.2Hz,2H),4.17-4.26(m,2H),4.32(t,J=6.7Hz,1H),4.44-4.49( m,1H),4.54-4.65(m,2H),5.18-5.27(m,1H),5.26-5.35(m,1H),5.44-5.50(m,2H),5 .82-5.93(m,1H),7.40(d,J=6.7Hz,1H),7.68(d,J=7.8Hz,1H).ESI-MS:522.3([M+H] + ).

[0238] Allyl(S)-2-((S)-2-((S)-4-amino-5-(tert-butoxy)-5-oxopentanamide)-6-diazo-5-oxohexanoamide)-6-diazo-5-oxohexanoate (21b) [ka] Compound 20b (423 mg, 0.548 mmol) in DCM (3 mL), EtNH (567 μL, 5.98 mmol) afforded product 21b (180 mg, 60%) as a yellow amorphous compound.

[0239] 1 H NMR(CDCl3):1.49(s,9H),2.00-2.15(m,4H),2.16-2.28(m,3H),2.39-2.64(m,6H),3.68-3.79(m,2H),4.32-4.41(m,1H),4.48-4.55(m,1H) ,4.56-4.68(m,2H),5.23-5.38(m,2H),5.41-5.50(m,2H),5.80-5.91(m,1H),7.31-7.37(m,1H),7.52(d,J=8.8Hz,1H).ESI-MS:550.3([M+H] + ).

[0240] Ethyl (S)-2-((S)-2-((S)-5-(allyloxy)-4-amino-5-oxopentanamido)-6-diazo-5-oxohexamido)-6-diazo-5-oxohexanoate (21c) [ka] Compound 20c (1.09 g, 1.46 mmol) in DCM (8 mL), EtNH (1.52 mL, 14.6 mmol) afforded product 21c (523 mg, 68%) as a yellow amorphous compound.

[0241] 1 H NMR(CDCl3):1.26(t,J=7.1Hz,3H),1.97-2.11(m,4H),2.13-2.32(m,3H),2.32-2.39(m,1H ),2.40-2.47(m,2H),2.50-2.63(m,3H),2.70-2.79(m,1H),4.12-4.20(m,2H),4.22(dd,J= 8.2,3.1Hz,1H),4.34-4.41(m,1H),4.46(td,J=8.4,4.5Hz,1H),4.70-4.76(m,2H),5.28-5 .40(m,2H),5.43-5.51(m,1H),5.86-5.97(m,2H),7.42-7.49(m,2H).ESI-MS:522.3([M+H] + ).

[0242] tert-Butyl (S)-2-((S)-2-((S)-5-(allyloxy)-4-amino-5-oxopentanamide)-6-diazo-5-oxohexanoate (21d) [ka] Compound 20d (634 mg, 0.821 mmol) in DCM (4 mL), EtNH (850 μL, 8.21 mmol) afforded product 21d (360 mg, 80%) as a yellow amorphous compound.

[0243] 1H NMR(CDCl3):1.39(s,9H),1.81-1.98(m,3H),2.00-2.20(m,3H),2.27-2.47(m,6H),3.23-3.30(m,2H),3.55-3.69(m,1H),4.21-4.32(m,2H) ,4.60(d,J=5.9Hz,2H),5.18-5.33(m,2H),5.42-5.48(m,2H),5.82-5.91(m,1H),7.29-7.33(m,1H),7.45-7.52(m,1H).ESI-MS:550.3([M+H] + ).

[0244] Isopropyl (S)-2-((S)-2-((S)-4-amino-5-isopropoxy-5-oxopentanamide)-6-diazo-5-oxohexanoate (P9) [ka] Compound 20e (365 mg, 0.480 mmol) in DCM (10 mL), EtNH (497 μL, 4.80 mmol) afforded product P9 (220 mg, 85%) as a yellow amorphous compound.

[0245] 1 H NMR(CDCl3):1.21-1.26(m,12H),1.62-1.70(m,2H),1.81-1.90(m,1H),1.95 -2.27(m,5H),2.30-2.45(m,4H),2.47-2.55(m,1H),2.55-2.66(m,1H),3.42( dd,J=8.8,4.9Hz,1H),4.34-4.57(m,2H),5.03(hept,J=6.6Hz,2H),5.26-5. 42(m,2H),6.99(d,J=6.8Hz,1H),7.51(d,J=7.8Hz,1H).ESI-MS:538.3([M+H] + ).

[0246] [ka]

[0247] Scheme 4: Azotomycin prodrugs with free carboxylates

[0248] Allyl(8S,13S,16S)-13,16-bis(4-diazo-3-oxobutyl)-8-(ethoxycarbonyl)-1-(9H-fluoren-9-yl)-3,6,11,14-tetraoxo-2-oxa-4,7,12,15-tetraazaheptadecan-17-oate (22) [ka] Fmoc-Gly-OH (94 mg, 0.316 mmol, 1.1 equiv) and HATU (126 mg, 0.330 mmol, 1.15 equiv) were suspended in anhydrous DCM (8 mL) and cooled to 0 °C. DIPEA (150 μL, 0.863 mmol, 3 equiv) was added, and the mixture was stirred at the same temperature for 5 min. A solution of compound 21a (150 mg, 0.288 mmol, 1 equiv) in anhydrous DCM (6 mL) was added dropwise, and the solution was stirred at 0 °C for 30 min and at room temperature for 4 h. The reaction mixture was concentrated in vacuo and purified by silica chromatography (DCM / MeOH, gradient 20:1) to give product 22 (159 mg, 69%) as a yellow solid. ESI-MS: 823.4 ([M+Na] + ).

[0249] Allyl(S)-2-((S)-2-((S)-4-(2-aminoacetamido)-5-ethoxy-5-oxopentanamido)-6-diazo-5-oxohexanoate)-6-diazo-5-oxohexanoate (23) [ka] Compound 22 (157 mg, 0.196 mmol, 1 equiv.) was dissolved in anhydrous DCM (2 mL) and diethylamine (193 μL, 10 equiv.) was added. The reaction mixture was stirred at room temperature for 3 h and then concentrated in vacuo. The residue was purified by silica chromatography (DCM / MeOH, gradient 5:1 to 1:1) to give product 23 (54 mg, 48%).

[0250] 1 H NMR(CDCl3):1.28(t,J=7.1Hz,3H),1.92-2.08(m,4H),2.10-2.19(m,1H),2.19-2.30(m,2H),2.33(t,J=7 .2Hz,2H),2.38-2.61(m,5H),3.45-3.52(m,2H),4.20(q,J=7.1Hz,2H),4.44(q,J=6.9Hz,1H),4.51-4.60( m,2H),4.62(dq,J=4.2,1.4Hz,2H),5.23-5.28(m,1H),5.33(dq,J=17.2,1.4Hz,1H),5.39-5.44(m,2H),5. 84-5.97(m,1H),7.23(d,J=6.0Hz,1H),7.55(d,J=7.5Hz,1H),7.97(d,J=8.1Hz,1H).ESI-MS:579.3([M+H] + ).

[0251] (S)-2-((S)-2-((S)-4-(2-aminoacetamido)-4-carboxylatobutanamido)-6-diazo-5-oxohexamido)-6-diazo-5-oxohexanoate sodium salt (P10) [ka] Compound 23 (23 mg, 0.040 mmol, 1 equiv.) was dissolved in a mixture of THF (250 μL) and MeOH (150 μL), and 1 M NaOH (240 μL, 0.239 mmol, 6 equiv.) was added. The reaction mixture was stirred at room temperature for 45 min and then concentrated in vacuo. The residue was purified by preparative HPLC (gradient 0–20% MeCN / HO over 50 min) to give product P10 (6 mg, 30%) as a pale orange solid.

[0252] 1H NMR(D2O):1.85-1.98(m,3H),2.08-2.19(m,3H),2.31-2.42(m,4H),2.47(t,J=7.4Hz,2H),3.78-3.83(m,2H),4.13(dd,J =8.6,4.8Hz,1H),4.17(dd,J=9.0,4.7Hz,1H),4.28(dd,J=8.5,5.9Hz,1H),5.83(s,1H),5.88(s,1H).ESI-MS:509.2([MH] + ).

[0253] 1.22. General Procedure for Compounds 24a-d Compounds 21a-d (1 equiv.) were dissolved in anhydrous DCM and 2,5-dioxopyrrolidin-1-yldimethylglycinate (1.1 equiv.) was added. The mixture was stirred at room temperature for 1.5 h, then diluted with DCM (70 mL) and washed with saturated NaHCO (35 mL) and brine (35 mL). The organic phase was dried over MgSO and concentrated in vacuo. The residue was purified by silica chromatography (DCM / MeOH, 10:1).

[0254] Allyl(6S,11S,14S)-11,14-bis(4-diazo-3-oxobutyl)-6-(ethoxycarbonyl)-2-methyl-4,9,12-trioxo-2,5,10,13-tetraazapentadecan-15-oate (24a) [ka] Compound 21a (75 mg, 0.144 mmol), 2,5-dioxopyrrolidin-1-yldimethylglycinate (32 mg, 0.158 mmol) in DCM (4 mL), product 24a (35 mg, 40%) in the form of a yellow solid.

[0255] 1H NMR(CDCl3):1.28(t,J=7.1Hz,3H),1.87-2.09(m,3H),2.11-2.21(m,1H),2.22-2.31(m,4H),2.33(s,6H) ),2.36-2.65(m,4H),2.83-3.11(m,2H),4.20(qd,J=7.1,1.0Hz,2H),4.42(q,J=7.0Hz,1H),4.54(td,J=8 .2,4.7Hz,1H),4.57-4.66(m,3H),5.26(dq,J=10.4,1.2Hz,1H),5.29-5.44(m,3H),5.90(ddt,J=17.1,10 .4,5.8Hz,1H),7.05(d,J=6.7Hz,1H),7.45(d,J=7.6Hz,1H),7.73(d,J=8.8Hz,1H).ESI-MS:607.3([M+H] + ).

[0256] Allyl(6S,11S,14S)-6-(tert-butoxycarbonyl)-11,14-bis(4-diazo-3-oxobutyl)-2-methyl-4,9,12-trioxo-2,5,10,13-tetraazapentadecan-15-oate (24b) [ka] Compound 21b (74 mg, 0.135 mmol), 2,5-dioxopyrrolidin-1-yldimethylglycinate (30 mg, 0.148 mmol) in DCM (3 mL), product 24b (40 mg, 47%) in the form of a yellow solid.

[0257] 1H NMR(CDCl3):1.47(s,9H),1.82-1.92(m,1H),1.95-2.08(m,2H),2.11-2.20(m,1H),2.21-2.31(m,4H),2.33( s,6H),2.36-2.65(m,4H),2.87-3.08(m,2H),4.42(q,J=6.9Hz,1H),4.49(td,J=9.2,3.9Hz,1H),4.55(td,J=8 .1,4.6Hz,1H),4.62(dq,J=5.8,1.5Hz,2H),5.26(dq,J=10.4,1.2Hz,1H),5.29-5.44(m,3H),5.90(ddt,J=17. 1,10.4,5.8Hz,1H),7.13(d,J=6.9Hz,1H),7.44(d,J=7.8Hz,1H),7.72(d,J=8.7Hz,1H).ESI-MS:635.4([M+H] + ).

[0258] Ethyl (6S,11S,14S)-6-((allyloxy)carbonyl)-11,14-bis(4-diazo-3-oxobutyl)-2-methyl-4,9,12-trioxo-2,5,10,13-tetraazapentadecan-15-oate (24c) [ka] Compound 21c (150 mg, 0.288 mmol), 2,5-dioxopyrrolidin-1-yldimethylglycinate (63 mg, 0.317 mmol) in DCM (4 mL), product 24c (58 mg, 64%) in the form of a yellow solid.

[0259] 1H NMR(CDCl3):1.27(t,J=7.1Hz,3H),1.89-2.07(m,3H),2.10-2.26(m,3H),2.27-2.31(m,2H), 2.33(s,6H),2.37-2.64(m,4H),2.90-3.09(m,2H),4.19(q,J=7.2Hz,2H),4.42(q,J=7.1Hz,1H ),4.49-4.53(m,1H),4.59-4.69(m,3H),5.24-5.37(m,2H),5.36-5.45(m,1H),5.85-5.96(m, 2H),7.02(d,J=7.3Hz,1H),7.38(d,J=7.8Hz,1H),7.74(d,J=9.0Hz,1H).ESI-MS:607.3([M+H] + ).

[0260] tert-Butyl (6S,11S,14S)-6-((allyloxy)carbonyl)-11,14-bis(4-diazo-3-oxobutyl)-2-methyl-4,9,12-trioxo-2,5,10,13-tetraazapentadecan-15-oate (24d) [ka] Compound 21d (115 mg, 0.209 mmol), 2,5-dioxopyrrolidin-1-yldimethylglycinate (46 mg, 0.230 mmol) in DCM (5 mL), product 24c (46 mg, 35%) as a yellow amorphous compound.

[0261] 1H NMR(CDCl3):1.46(s,9H),1.89-2.05(m,3H),2.11-2.29(m,5H),2.33(s,6H),2.35-2. 47(m,2H),2.48-2.68(m,2H),2.89-3.10(m,2H),4.35-4.43(m,2H),4.60-4.69(m,3H) ,5.27(dq,J=10.4,1.2Hz,1H),5.31-5.44(m,3H),5.91(ddt,J=16.9,10.4,5.9Hz,1H) ,7.00(d,J=8.1Hz,1H),7.27(d,J=2.5Hz,1H),7.70-7.78(m,1H).ESI-MS:635.4([M+H] + ).

[0262] Sodium (6S,11S,14S)-11,14-bis(4-diazo-3-oxobutyl)-6-(ethoxycarbonyl)-2-methyl-4,9,12-trioxo-2,5,10,13-tetraazapentadecan-15-oate (P11) [ka] Compound 24a (27 mg, 0.045 mmol, 1 equiv.) was dissolved in anhydrous DCM (1 mL), and phenylsilane (11 μL, 0.089 mmol, 2 equiv.) and Pd(PPh3)4 (2.6 mg, 2.25 μmol, 0.05 equiv.) were added. The reaction mixture was stirred at room temperature for 50 min and then concentrated in vacuo. The residue was purified by preparative HPLC (gradient 0–50% MeCN / HO over 50 min) to give product P11 (19 mg, 73%) as a yellow solid.

[0263] 1H NMR(D2O):1.23(t,J=7.3Hz,3H),1.89-2.04(m,3H),2.08-2.13(m,2H), 2.14-2.22(m,1H),2.33-2.47(m,6H),2.48(s,6H),3.38-3.45(m,2H),4. 12(dd,J=8.3,4.7Hz,1H),4.19(q,J=7.1Hz,2H),4.24-4.32(m,1H),4.38(dd,J=9.1,5.1Hz,1H),5.82(s,1H),5.86(s,1H).ESI-MS:567.3([M+H] + ).

[0264] Sodium (6S,11S,14S)-6-carboxylato-11,14-bis(4-diazo-3-oxobutyl)-2-methyl-4,9,12-trioxo-2,5,10,13-tetraazapentadecan-15-oate (P12) [ka] Compound 24c (23 mg, 0.041 mmol, 1 equiv.) was dissolved in MeOH (250 μL) and 1 M NaOH (165 μL, 0.165 mmol, 4 equiv.) was added. The reaction mixture was stirred at room temperature for 30 min and then concentrated in vacuo. The residue was purified by preparative HPLC (gradient 0 to 40% MeCN / HO over 50 min) to give product P12 (16 mg, 73%) as a yellow solid.

[0265] 1 H NMR(D2O):1.85-1.99(m,3H),2.05-2.15(m,3H),2.30-2.41(m,4H),2.44-2.49(m,2H),2.9 0(s,6H),3.98(s,2H),4.10-4.15(m,2H),4.27(dd,J=8.5,5.8Hz,1H).ESI-MS:539.3([M+H] + ).

[0266] N 5-((S)-6-diazo-1-(((S)-6-diazo-1-ethoxy-1,5-dioxohexan-2-yl)amino)-1,5-dioxohexan-2-yl)-N 2 -(Dimethylglycyl)-L-glutamine (P13) [ka] Compound 24c (30 mg, 0.049 mmol, 1 equiv.) was dissolved in anhydrous DCM (1 mL), followed by the addition of phenylsilane (12 μL, 0.099 mmol, 2 equiv.) and Pd(PPh3)4 (2.9 mg, 2.47 μmol, 0.05 equiv.). The reaction mixture was stirred at room temperature for 1 h and then concentrated in vacuo. The residue was purified by preparative HPLC (gradient 0–60% MeCN / HO over 50 min) to give product P13 (19 mg, 68%) as a colorless solid.

[0267] 1 H NMR(D2O):1.22(t,J=7.1Hz,3H),1.86-2.01(m,3H),2.03-2.12(m,2H),2.14-2.24(m,2H),2.30-2.36(m,2H),2.46(q, J=7.0Hz,3H),2.69(s,6H),3.69(s,2H),4.12-4.20(m,3H),4.22-4.30(m,1H),4.31-4.38(m,1H).ESI-MS:567.3([M+H] + ).

[0268] Sodium (6S,11S,14S)-6-(tert-butoxycarbonyl)-11,14-bis(4-diazo-3-oxobutyl)-2-methyl-4,9,12-trioxo-2,5,10,13-tetraazapentadecan-15-oate (P14) [ka] Compound 24b (36 mg, 0.057 mmol, 1 equiv.) was dissolved in MeOH (400 μL) and 1 M NaOH (170 μL, 0.170 mmol, 3 equiv.) was added. The solution was stirred at room temperature for 30 min. The volatiles were evaporated, and the residue was purified by preparative HPLC (gradient 0 to 60% MeCN / HO over 50 min) to give product P14 (21 mg, 62%) as a colorless solid.

[0269] 1 H NMR(D2O):1.43(s,9H),1.84-2.01(m,3H),2.03-2.20(m,3H),2.34-2.39(m,4H),2.40(s,6H),2.43-2.48(m,2H),3 .25-3.37(m,2H),4.12(dd,J=8.8,4.9Hz,1H),4.24(dd,J=9.1,5.2Hz,1H),4.26-4.31(m,1H).ESI-MS:595.3([M+H] + ).

[0270] Sodium N 5 -((S)-1-(((S)-1-(tert-butoxy)-6-diazo-1,5-dioxohexan-2-yl)amino)-6-diazo-1,5-dioxohexan-2-yl)-N 2 -(Dimethylglycyl)-L-glutamic acid (P15) [ka] Compound 24d (42 mg, 0.066 mmol, 1 equiv.) was dissolved in MeOH (300 μL) and 1 M NaOH (200 μL, 0.199 mmol, 3 equiv.) was added. The solution was stirred at room temperature for 30 min. The volatiles were evaporated, and the residue was purified by preparative HPLC (gradient 0–50% MeCN / HO over 50 min) to give product P15 (18 mg, 46%) as a colorless solid.

[0271] 1H NMR(D2O):1.42(s,9H),1.84-2.01(m,3H),2.02-2.21(m,3H),2.28-2.37(m,2H),2.38-2.51(m, 4H),2.61(s,6H),4.09-4.18(m,1H),4.19-4.23(m,1H),4.23-4.32(m,1H).ESI-MS:595.3([M+H] + ).

[0272] Allyl(5S,8S,13S,16S)-13,16-bis(4-diazo-3-oxobutyl)-8-(ethoxycarbonyl)-1-(9H-fluoren-9-yl)-5-isobutyl-3,6,11,14-tetraoxo-2-oxa-4,7,12,15-tetraazaheptadecan-17-oate (25) [ka] Compound 21a (185 mg, 0.355 mmol, 1 equiv.) and HATU (155 mg, 0.408 mmol, 1.15 equiv.) were suspended in anhydrous DCM (9 mL), cooled to 0 °C, and DIPEA (185 μL, 1.06 mmol, 3 equiv.) was added. The mixture was stirred at 0 °C for 30 min and at room temperature for 2 h. The volatiles were evaporated in vacuo, and the residue was purified by silica chromatography (DCM / MeOH, 20:1) to give product 25 (300 mg, 99%) as a yellow amorphous compound.

[0273] 1H NMR(DMSO-d6):0.88(dd,J=15.9,6.6Hz,6H),1.16(t,J=7.1Hz,3H),1.40-1.50(m,2H),1.60-1.74(m,2H),1.79-1.89(m,3H),1.93 -2.06(m,2H),2.15-2.44(m,6H),4.07(tq,J=11.5,6.0Hz,3H),4.16-4.35(m,6H),4.50-4.59(m,2H),5.16-5.23(m,1H),5.29(dq, J=17.2,1.6Hz,1H),5.87(ddt,J=17.0,10.7,5.4Hz,1H),5.99-6.09(m,2H),7.31(td,J=7.4,2.9Hz,2H),7.41(t,J=7.4Hz,2H),7. 49(d,J=8.2Hz,1H),7.72(t,J=7.9Hz,2H),7.86-7.92(m,3H),8.31(d,J=7.5Hz,1H),8.40(d,J=7.4Hz,1H).ESI-MS:879.4([M+Na] + ).

[0274] Allyl(S)-2-((S)-2-((S)-4-((S)-2-amino-4-methylpentanamide)-5-ethoxy-5-oxopentanamide)-6-diazo-5-oxohexanoate (26) [ka] Compound 25 (300 mg, 0.350 mmol, 1 equiv.) was dissolved in anhydrous DCM (3 mL), and DMF (0.5 mL) and diethylamine (724 μL, 7.00 mmol, 20 equiv.) were added. The mixture was stirred at room temperature for 1 h. The volatiles were evaporated in vacuo. The residue was purified by silica chromatography (DCM / MeOH, 10:1) and repurified by preparative HPLC (gradient 0–50% MeCN / HO over 50 min) to give product 26 (129 mg, 57%) as a yellow solid.

[0275] 1H NMR(CDCl3):0.97(dd,J=15.8,5.9Hz,6H),1.28(t,J=7.1Hz,3H),1.65-1.87(m,3H),1.88-2.15(m,4H),2.16-2.30 (m,2H),2.35(t,J=6.7Hz,2H),2.40-2.69(m,6H),4.09-4.16(m,1H),4.17-4.25(m,2H),4.40(q,J=6.7Hz,1H),4.5 2(q,J=7.9Hz,1H),4.56-4.67(m,3H),5.23-5.28(m,1H),5.31-5.37(m,1H),5.47(s,1H),5.51(s,1H),5.90(ddt,J =16.3,10.6,5.8Hz,1H),7.17(d,J=8.1Hz,1H),7.39(d,J=6.0Hz,1H),7.72(d,J=7.2Hz,1H).ESI-MS:635.4([M+H] + ).

[0276] (S)-2-((S)-2-((S)-4-((S)-2-amino-4-methylpentanamide)-4-carboxylatobutanamide)-6-diazo-5-oxohexanoamide)-6-diazo-5-oxohexanoate sodium (P16) [ka] Compound 26 (22.5 mg, 0.035 mmol, 1 equiv.) was dissolved in MeOH (250 μL) and 1 M NaOH (215 μL, 0.213 mmol, 6 equiv.) was added. The mixture was stirred at room temperature for 45 min. The volatiles were evaporated in vacuo, and the residue was purified by preparative HPLC (gradient 0–50% MeCN / HO over 50 min) to give product P16 (12 mg, 55%) as a yellow solid.

[0277] 1H NMR(D2O):0.93(dd,J=11.0,6.1Hz,6H),1.63-1.76(m,3H),1.83-2.00(m,3H),2.04-2.14(m,3H),2.30-2.41(m,4H),2.46(t,J=7 .4Hz,2H),3.95(t,J=6.8Hz,1H),4.09-4.16(m,2H),4.27(dd,J=8.6,5.7Hz,1H),5.82(s,1H),5.86(s,1H).ESI-MS:567.3([M+H] + ).

[0278] [ka]

[0279] Scheme 5: Synthesis of azotomycin prodrug P17

[0280] Isopropyl(8S,13S,16S)-13,16-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-8-(isopropoxycarbonyl)-3,6,11,14-tetraoxo-2-oxa-4,7,12,15-tetraazaheptadecan-17-oate (27) [ka] Fmoc-Gly-OH (24 mg, 0.082 mmol, 1.1 equiv.) and HATU (33 mg, 0.086 mmol, 1.15 equiv.) were dissolved in anhydrous DCM (3 mL), and DIPEA (39 μL, 0.223 mmol, 3 equiv.) was added, followed by a solution of compound P9 (40 mg, 0.074 mmol, 1 equiv.) in DCM (2 mL). The mixture was stirred at room temperature for 2 h, diluted with DCM (50 mL), and washed with saturated NaHCO (30 mL) and brine (30 mL). The organic phase was dried over anhydrous MgSO and concentrated in vacuo. The residue was purified by silica chromatography (DCM / MeOH, 20:1) to give product 27 (52 mg, 82%) as a yellow solid.

[0281] 1H NMR(CDCl3):1.21-1.29(m,12H),1.93-2.08(m,3H),2.08-2.35(m,4H),2.35-2.62(m,5H),3.8 5-4.01(m,2H),4.22-4.27(m,1H),4.38-4.44(m,3H),4.43-4.49(m,1H),4.50-4.56(m,1H),5.0 0-5.09(m,2H),5.25-5.39(m,2H),5.70(t,J=5.1Hz,1H),6.92(d,J=6.3Hz,1H),7.16(d,J=6.8 Hz,1H),7.29-7.43(m,5H),7.62(d,J=7.5Hz,2H),7.76(d,J=7.6Hz,2H).ESI-MS:839.3([M+Na] + ).

[0282] Isopropyl (S)-2-((S)-2-((S)-4-(2-aminoacetamido)-5-isopropoxy-5-oxopentanamido)-6-diazo-5-oxohexanoate (28) [ka] Compound 27 (52 mg, 0.064 mmol, 1 equiv.) was dissolved in anhydrous DCM (1.1 mL) and diethylamine (132 μL, 1.27 mmol, 20 equiv.) was added. The mixture was stirred at room temperature for 3 h, and then the volatiles were evaporated in vacuo. The residue was purified by silica chromatography (DCM / MeOH, 5:1) to give product 28 (24 mg, 63%) as a yellow solid.

[0283] 1H NMR(CDCl3):1.14-1.30(m,12H),1.92-2.06(m,5H),2.11-2.30(m,3H),2.30-2.35(m,2 H),2.36-2.49(m,2H),2.49-2.64(m,2H),3.43(s,2H),4.42(q,J=6.6Hz,1H),4.48(td, J=7.7,4.2Hz,1H),4.56(td,J=9.0,4.2Hz,1H),4.98-5.09(m,2H),5.32-5.42(m,2H),7 .11(d,J=6.6Hz,1H),7.37(d,J=9.3Hz,1H),7.90(d,J=8.5Hz,1H).ESI-MS:595.3([M+H] + ).

[0284] Isopropyl (9S,14S,17S)-14,17-bis(4-diazo-3-oxobutyl)-9-(isopropoxycarbonyl)-2-methyl-4,7,12,15-tetraoxo-2,5,8,13,16-pentaazaoctadecane-18-oate (P17) [ka] Compound 28 (23 mg, 0.039 mmol, 1 equiv.) and 2,5-dioxopyrrolidin-1-yldimethylglycinate (9.3 mg, 0.046 mmol, 1.2 equiv.) were dissolved in anhydrous DMF (1 mL), and the solution was stirred at room temperature for 3 h. The mixture was diluted with DCM (30 mL) and washed with saturated NaHCO (15 mL) and brine (15 mL). The organic phase was dried over MgSO and concentrated in vacuo. The residue was purified by silica chromatography (DCM / MeOH, 5:1) to give product P17 (17 mg, 65% yield) as a yellow solid.

[0285] 1H NMR(CDCl3):1.24(d,J=6.1Hz,12H),1.92-2.16(m,4H),2.17-2.31(m,4H),2. 33(s,6H),2.36-2.60(m,4H),2.94-3.11(m,2H),3.91-4.06(m,2H),4.37-4.5 4(m,3H),4.97-5.06(m,2H),5.37-5.46(m,2H),7.09(d,J=6.1Hz,1H),7.30(d ,J=7.3Hz,1H),7.51(d,J=7.6Hz,1H),7.76-7.83(m,1H).ESI-MS:680.3([M+H] + ).

[0286] [ka]

[0287] Scheme 6: Synthesis of azotomycin prodrug P18

[0288] Isopropyl(5S,8S,11S,16S,19S)-16,19-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-11-(isopropoxycarbonyl)-5-isopropyl-3,6,9,14,17-pentaoxo-8-(3-ureidopropyl)-2-oxa-4,7,10,15,18-pentaazaicosan-20-oate (29) [ka] Fmoc-Val-Cit-OH (41 mg, 0.818 mmol, 1.1 equiv) and HATU (33 mg, 0.086 mmol, 1.15 equiv) were dissolved in anhydrous DCM (2 mL), and DIPEA (39 μL, 0.223 mmol, 3 equiv) was added. The mixture was cooled to 0 °C, and a solution of compound P9 (40 mg, 0.074 mmol, 1 equiv) was added dropwise. The mixture was stirred at room temperature for 19 h and then concentrated in vacuo. The crude product 29 was used for further reactions without purification or characterization.

[0289] Isopropyl(6S,9S,14S,17S)-1-amino-6-((S)-2-amino-3-methylbutanamido)-14,17-bis(4-diazo-3-oxobutyl)-9-(isopropoxycarbonyl)-1,7,12,15-tetraoxo-2,8,13,16-tetraazaoctadecane-18-oate (P18) [ka] The crude product 29 was dissolved in anhydrous DCM (2 mL) and diethylamine (600 μL, 5.80 mmol, 78 equiv.) was added. The mixture was stirred at room temperature for 1 h and concentrated in vacuo. The residue was purified by silica chromatography (DCM / MeOH, gradient 7:1 to 2:1) to give product 18 (41 mg, 54%) as a yellow solid. ESI-MS: 794.5 ([M+H] + ).

[0290] [ka]

[0291] Scheme 7: Synthesis of azotomycin prodrug P19

[0292] (E)-Prop-1-en-1-ylN2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-acetyl-L-lysine acid (31) [ka] Fmoc-Lys(Ac)-OH (1.0 g, 2.44 mmol, 1 equiv.) was dissolved in anhydrous DMF (15 mL), and KCO (505 mg, 3.65 mmol, 1.5 equiv.) was added, followed by allyl bromide (274 μL, 3.17 mmol, 1.3 equiv.). The resulting mixture was stirred at room temperature for 4 h. The volatiles were evaporated, and the residue was diluted with EtOAc (100 mL) and washed with distilled HO (100 mL) and brine (100 mL). The organic layer was dried over anhydrous MgSO and concentrated in vacuo. Product 31 (1.05 g, 95%) was obtained as a colorless solid. ESI-MS: 451.3 ([M+H] + ).

[0293] (E)-Prop-1-en-1-yl N6-acetyl-L-lysinate (32) [ka] Compound 31 (1.05 g, 2.33 mmol, 1 equiv.) was dissolved in anhydrous DCM (20 mL) and DMF (1 mL), and diethylamine (4.8 mL, 46.6 mmol, 20 equiv.) was added. The mixture was stirred at room temperature for 2 hours and concentrated in vacuo. The crude product 32 was used in further reactions without purification or characterization.

[0294] (E)-Prop-1-en-1-yl N6-acetyl-N2-((3S,5S,7S)-adamantane-1-carbonyl)-L-lysine acid (33) [ka] Adamantanecarboxylic acid (420 mg, 2.33 mmol, 1 equiv.) and HATU (975 mg, 2.56 mmol, 1.1 equiv.) were dissolved in anhydrous DMF (15 mL), and DIPEA (1.2 mL, 6.99 mmol, 3 equiv.) was added, followed by a solution of compound 32 (532 mg, 2.33 mmol, 1 equiv.) in anhydrous DMF (10 mL). The reaction mixture was stirred at room temperature for 1.5 h and concentrated in vacuo. The residue was diluted with EtOAc (100 mL) and washed with saturated NaHCO (100 mL), distilled HO (100 mL), 10% KHSO (100 mL), and brine (100 mL). The organic layer was dried over anhydrous MgSO and concentrated in vacuo. The residue was purified by silica chromatography (DCM / MeOH, 20:1) to give product 33 (700 mg, 77%) as a yellow solid.

[0295] 1 H NMR(CDCl3):1.35(p,J=7.6Hz,2H),1.49-1.62(m,2H),1.63-1.79(m,8H) ,1.82-1.90(m,6H),1.98(s,3H),2.02-2.07(m,3H),3.14-3.32(m,J=6.9 Hz,2H),4.54-4.69(m,3H),5.22-5.38(m,2H),5.79-5.85(m,1H),5.90(d dt,J=16.6,11.4,5.8Hz,1H),6.22(d,J=7.7Hz,1H).ESI-MS:391.3([M+H] + ).

[0296] N6-Acetyl-N2-((3S,5S,7S)-adamantane-1-carbonyl)-L-lysine (34) [ka] Compound 33 (668 mg, 1.71 mmol, 1 equiv.) was dissolved in anhydrous DCM (18 mL), and PhSiH (422 μL, 3.42 mmol, 2 equiv.) was added, followed by Pd(PPh) (40 mg, 0.034 mmol, 0.02 equiv.). The reaction mixture was stirred at room temperature for 1.5 h. The volatiles were evaporated, and the residue was purified by silica chromatography (DCM / MeOH, gradient 10:1 to 5:1) to give product 34 (480 mg, 80%) as a colorless solid.

[0297] 1 H NMR(DMSO-d6):1.16-1.28(m,2H),1.30-1.39(m,2H),1.58-1.72(m,8H),1.74-1.83(m,9H),1.96(s,3H) ,2.92-3.02(m,2H),4.05-4.11(m,1H),7.29(d,J=6.8Hz,1H),7.79(t,J=5.0Hz,1H).ESI-MS:349.2([MH] - ).

[0298] Isopropyl(2S,5S,10S,13S)-13-((3S,5S,7S)-adamantane-1-carboxamido)-2,5-bis(4-diazo-3-oxobutyl)-10-(isopropoxycarbonyl)-4,7,12,19-tetraoxo-3,6,11,18-tetraazaicosanoic acid (P19) [ka] Compound 34 (34 mg, 0.063 mmol, 1 equiv.) and HATU (28 mg, 0.073 mmol, 1.15 equiv.) were dissolved in anhydrous DMF (1.5 mL) and cooled to 0 °C. DIPEA (33 μL, 0.190 mmol, 3 equiv.) was added, followed by a solution of compound P9 (34 mg, 0.063 mmol, 1 equiv.) in anhydrous DMF (1.5 mL). The reaction mixture was stirred at room temperature for 1.5 h and concentrated in vacuo. The residue was purified by silica chromatography (DCM / MeOH, 15:1) to give product P19 (32 mg, 58%) as a yellow solid.

[0299] 1 H NMR(CDCl3):1.20-1.28(m,12H),1.58-1.64(m,5H),1.66-1.78(m,6H),1.84-1.90(m,6H),1.98(s ,3H),2.00-2.07(m,3H),2.10-2.62(m,9H),3.18(ddq,J=11.5,7.4,4.0Hz,2H),3.22-3.28(m,2H), 3.68-3.77(m,2H),4.31-4.58(m,4H),4.96-5.09(m,2H),5.33-5.53(m,2H),6.30(d,J=6.4Hz,1H), 7.12-7.21(m,1H),7.47(d,J=6.7Hz,1H),7.70(d,J=7.8Hz,1H),8.02(s,1H).ESI-MS:870.5([M+H] + ).

[0300] 1.3. Metabolic stability 1.3.1 Metabolic stability assessment method

[0301] In vitro stability studies were performed using CES1KO mouse plasma, human plasma, CES1-KO mouse liver, and human liver microsomes as previously described [1]. For tissue homogenate stability studies, washed tissues were diluted 10-fold with 0.1 M potassium phosphate buffer and homogenized using probe sonication. The crude homogenate was then aliquoted to 1 mL, and a 10 mM stock of the analyte in DMSO was added to achieve a final concentration of 10 μM. Plasma stability of the prodrug was measured by adding the analyte to 1 mL of plasma. Plasma and tissues were incubated at 37°C in an orbital shaker. Stability in human liver microsomes was evaluated at a protein concentration of 0.2 mg / mL per microsome and a final prodrug concentration of 1 μM. All stability tests were performed at predetermined times (0 and 60 min). Triplicate 50 μL aliquots of the mixture were removed and the reaction was stopped by adding a 5-fold volume of ice-cold methanol spiked with an internal standard (losartan: 0.5 μM). Samples were vortexed for 30 seconds and centrifuged at 10,000 g for 10 minutes at 4°C. A 50 μL aliquot of the supernatant was diluted with 50 μL of water and transferred to a 250 μL polypropylene vial sealed with a Teflon cap. The disappearance of the prodrug was monitored over time using liquid chromatography-mass spectrometry (LC-MS).

[0302] 1.3.2 Bioanalysis - Metabolic Stability Analysis was performed using a Dionex Ultra High Performance LC system coupled to a Q Exactive Focus Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). Separation was performed using an Agilent Eclipse Plus column (100 x 2.1 mm i.d., maintained at 35 °C) packed with a 1.8 μm C18 stationary phase. The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The pump was operated at a flow rate of 0.3 mL / min for 7 min using gradient elution. The mass spectrometer was controlled with Xcalibur software 4.0.27.13 (Thermo Scientific) and operated in positive ionization mode using a HESI ion source. Metabolites were compared with the t = 0 sample using full scan mode from m / z 50 to 1600.

[0303] [Table 4]

[0304] CES1KO = carboxylesterase 1 knockout

[0305] 1.4 Pharmacokinetic screening of prodrugs (1.4.1 Method)C57BL / 6 CES1 - / - Pharmacokinetic studies in mice were conducted under protocols reviewed and approved by the Johns Hopkins University Animal Care and Use Committee, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International (AACS) and the Public Health Service Policy for the Humane Care and Use of Laboratory Animals (PHS Policy). Naïve male and female C57BL / 6 CES1 mice, 6-8 weeks old, were used in this study. - / - Mice (weight 25-30 g) were used. The animals were housed under a 12-hour light-dark cycle and had free access to food and water. Tumors were established by inoculating EL4 cells (1 × 10 cells in 0.2 mL of phosphate-buffered saline) into one site on the flank of each mouse. 6 The tumors were generated by subcutaneous injection of 1000 mg / kg of IgG4. The average tumor volume was approximately 400 mm. 3Before administration, the interscapular area was wiped with alcohol gauze.

[0306] Compounds were dissolved in ethanol / Tween 80 / saline (5:10:85 v / v / v) and administered subcutaneously to mice at a single dose of 1 mg / kg azotomycin equivalent. Mice were euthanized with carbon dioxide at the designated time points for pK analysis. Blood samples (approximately 0.8 mL) were collected into heparinized microtubes by cardiac puncture, and the jejunum and tumor were excised and flash-frozen on dry ice. Blood samples were centrifuged at 3000 g for 10 minutes at 4°C. All samples were kept refrigerated throughout processing. Plasma samples (approximately 300 μL) were collected into polypropylene tubes and stored at -80°C until bioanalysis. Flash-frozen jejunum and tumor samples were also stored at -80°C until bioanalysis.

[0307] To quantify the unchanged analyte in the pharmacokinetic samples, plasma samples (25 μL) were processed using a single-step protein precipitation method by adding 125 μL of methanol containing the internal standard (losartan: 0.5 μM), followed by vortex mixing for 30 seconds and centrifugation at 16,000 g for 5 minutes at 4°C. Jejunum and tumor tissues were diluted 1:5 w / v with methanol containing losartan (0.5 μM), homogenized, vortex mixed, and centrifuged at 16,000 g for 5 minutes at 4°C. The supernatant was transferred to an autosampler vial. 2 μL of sample was injected into the LCMS system.

[0308] Samples were analyzed by LC-MS / MS after dabsyl chloride derivatization as described in Tenora et al. (2019). DON was extracted from plasma samples by protein precipitation with methanol. Briefly, standards, QCs, and samples (50 μL) were mixed with 250 μL of methanol containing 10 μM glutamic acid-d5 (internal standard) in low-retention microcentrifuge tubes. Jejunum and tumor samples were weighed. Five μL of methanol containing 10 μM glutamic acid-d5 was added per mg of tissue sample and mechanically homogenized. For plasma, jejunum, and tumor samples, the mixture was vortexed and centrifuged at 16,000 g for 5 min at 4°C. The supernatant (100 μL) was transferred to a new tube and vacuum-dried at 45°C for 1 h. Samples were analyzed by LC-MS / MS after dabsyl chloride derivatization as described in Tenora et al. (2019).

[0309] All publications, patent applications, patents, and other references mentioned herein are indicative of the level of ordinary skill in the art to which the subject matter of this disclosure pertains. All publications, patent applications, patents, and other references are incorporated herein by reference to the same extent as if each individual publication, patent application, patent, and other reference were specifically and individually indicated to be incorporated by reference. Although numerous patent applications, patents, and other references are referenced herein, it should be understood that such reference does not constitute an admission that any of these documents form part of the general knowledge in the art. [Prior art documents] [Non-patent literature]

[0310] [Non-Patent Document 1] Tenora, L. et al., Tumor-Targeted Delivery of 6-Diazo-5-oxo-l-norleucine(DON)Using Substituted Acetylated Lysine Prodrugs, J.Med.Chem.2019,62,3524-3538. [Non-patent document 2] Yokoyama, Y. et al., Sirpiglenastat (DRP-104) Induces Antitumor Efficacy through Direct, Broad Antagonism of Glutamine Metabolism and Stimulation of the Innate and Adaptive Immune Systems, Mol. Cancer Ther., 2022, 21(10), 1561-1572. [Non-patent document 3] Rais, R. et al., Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug, Sci. Adv., 2022, 8, eabq5925. [Non-patent document 4] Leone RD, et al., Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion, Science, 2019, 366, 1013-1021.

[0311] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood that changes and modifications may be practiced by those skilled in the art that are within the scope of the appended claims.

Claims

1. A compound of formula (I) 【Chemistry 1】 R １ and R ２ are each independently -OR ４ , -NR ５ R ６ , -O-M ＋ , and —O—(CH ２ CH ２ O) ｎ -R ７ is selected from n is an integer selected from 1, 2, 3, and 4; R ４ is H and C １ -C ４ and is selected from substituted or unsubstituted branched or unbranched alkyl of the formula: R ５ and R ６ are each independently H, C １ -C ４ substituted or unsubstituted branched or unbranched alkyl, -AA-COOR ７ where R ７ is C １ -C ４ substituted or unsubstituted branched or unbranched alkyl, and AA is an amino acid; M ＋ is a metal cation, R ３ is H, -(C=O)-R ８ , -C(=O)-CH ２ -(NH-C(=O)-CH ２ -NR ９ R １０ )-CH ２ -R １１ , -C(=O)-CH-(NH-C(=O)-CH(NH ２ )-CH-(CH ３ ) ２ )-((CH ２ ) ３ NH-C(=O)NH ２ , -C(=O)-CH((CH ２ ) ４ -NH-C(=O)-CH ３ ) (NH—C(═O)-adamantane), —C(═O)-AA-R １２ , acetyl (Ac), dimethylglycine, dimethylglycyl (DMG), L-Lys(Ac), D-Val-L-Leu-L-Lys(Ac), D-Val-L-Leu-L-Lys(H), D-Val-L-Leu-L-Lys(DMG), L-Val-L-Cit, para-aminobenzoic acid (PABA), L-Cit-PABA, L-Val-L-Cit-PABA, Val-Cit-OH, dimethylglycyl-L-tryptophan, acetoxymethyl pivalate, 1-acetoxyethyl pivalate, acetoxy(phenyl)methyl pivalate, and 2-acetoxypropan-2-yl pivalate, wherein AA is an amino acid and R ３ , R ５ , and R ６ may be selected from the amino acids or amino acid related substituents listed in Table 2 or Table 3, and combinations thereof; R ８ is C １ -C ４ and -CH ２ -NR １４ R １５ is selected from R ９ , R １０ , R １４ , and R １５ are each independently H and C １ -C ４ and is selected from substituted or unsubstituted branched or unbranched alkyl of the formula: R １１ is selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; R １２ is H, -(C=O)-R １３ and —NH-dimethylglycyl; R １３ is C １ -C ４ is a substituted or unsubstituted branched or unbranched alkyl of R ３ If H, then R １ and R ２ provided that both cannot be -OH, stereoisomers and pharmaceutically acceptable salts thereof, A compound of formula (I).

2. The compound of formula (I) is a compound of formula (Ia), 【Chemistry 2】 R １ and R ２ are each independently -OR ４ , -NR ５ R ６ , and —O—(CH ２ CH ２ O) ｎ -R ７ is selected from n is an integer selected from 1, 2, 3, and 4; R ４ is C １ -C ４ is a substituted or unsubstituted branched or unbranched alkyl of R ５ and R ６ are each independently H, C １ -C ４ substituted or unsubstituted branched or unbranched alkyl, -AA-COOR ７ where R ７ is C １ -C ４ substituted or unsubstituted branched or unbranched alkyl, and AA is an amino acid; R ３ is H, -(C=O)-R ８ , -C(=O)-CH(NR ９ R １０ )-R ７ , -C(=O)-CH ２ -(NH-C(=O)-CH ２ -NR ９ R １０ )-CH ２ -R １１ , -C(=O)-AA-R １２ , acetyl (Ac), dimethylglycine, dimethylglycyl (DMG), L-Lys(Ac), D-Val-L-Leu-L-Lys(Ac), D-Val-L-Leu-L-Lys(H), D-Val-L-Leu-L-Lys(DMG), L-Val-L-Cit, para-aminobenzoic acid (PABA), L-Cit-PABA, L-Val-L-Cit-PABA, dimethylglycyl-L-tryptophan, acetoxymethyl pivalate, 1-acetoxyethyl pivalate, acetoxy(phenyl)methyl pivalate, and 2-acetoxypropan-2-yl pivalate, wherein AA is an amino acid and R ３ , R ５ , and R ６ may be selected from the amino acids or amino acid related substituents listed in Table 2 or Table 3, and combinations thereof; R ８ is C １ -C ４ and -CH ２ -NR １４ R １５ is selected from R ９ , R １０ , R １４ , and R １５ are each independently H and C １ -C ４ and is selected from substituted or unsubstituted branched or unbranched alkyl of the formula: R １１ is selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; R １２ is H, -(C=O)-R １３ and —NH-dimethylglycyl; R １３ is C １ -C ４ is a substituted or unsubstituted branched or unbranched alkyl of stereoisomers and pharmaceutically acceptable salts thereof, The compound of claim 1.

3. R １ and R ２ are each independently —OCH ２ CH ３ , —O—CH(CH ３ ) ２ , —O—C(CH ３ ) ３ , -NH ２ , -NHCH ３ , -NH-AA-COO-CH(CH ３ ) ２ , -NH-AA-COO-C(CH ３ ) ３ , —O—(CH ２ CH ２ O) ｎ -H, -O-(CH ２ CH ２ O) ｎ -AA-COO-CH(CH ３ ) ２ , and —O—(CH ２ CH ２ O) ｎ -AA-COO-C(CH ３ ) ３ 3. The compound of claim 1 or 2, selected from:

4. R ３ is selected from H, acetyl (Ac), dimethylglycine, dimethylglycyl (DMG), L-Lys(Ac), D-Val-L-Leu-L-Lys(Ac), D-Val-L-Leu-L-Lys(H), D-Val-L-Leu-L-Lys(DMG), L-Val-L-Cit, para-aminobenzoic acid (PABA), L-Cit-PABA, L-Val-L-Cit-PABA, dimethylglycyl-L-tryptophan, acetoxymethyl pivalate, 1-acetoxyethyl pivalate, acetoxy(phenyl)methyl pivalate, and 2-acetoxypropan-2-yl pivalate, —C(═O)-AA-H, —C(═O)-AA-Ac, —C(═O)-AA-NH-dimethylglycyl.

5. R １ and R ２ are respectively -OR ４ 5. The compound of claim 1, wherein

6. R １ and R ２ One or both of ２ CH ３ , -OCH(CH ３ ) ２ , and —OC(CH ３ ) ３ 6. The compound of claim 5, selected from:

7. R １ and R ２ are respectively -NR ５ R ６ 5. The compound of claim 1, wherein

8. R １ and R ２ are each -NH ２ 8. The compound of claim 7, wherein:

9. R ３ is H, -C(=O)-R ８ and —C(═O)—CH ２ -(NH-C(=O)-CH ２ -NR ９ R １０ )-CH ２ -R １１ 9. The compound according to any one of claims 1 to 8, selected from:

10. R ３ is H, —C(═O)—CH ３ , -C(=O)-CH ２ -N(CH ３ ) ２ and —C(═O)—CH ２ -(NH-C(=O)-CH ２ -N(CH ３ ) ２ )-CH ２ 10. The compound of claim 9, wherein the compound is selected from -(1H)indoles.

11. The compound is 【Transformation 3】 7. The compound of claim 6, selected from:

12. The compound of formula (I) is 【Chemistry 4】 12. The compound of claim 11 selected from:

13. The compound of formula (I) is 【Transformation 5】 13. The compound of claim 12, selected from:

14. The compound is 【Transformation 6】 9. The compound of claim 8 selected from:

15. R １ and R ２ At least one of the groups is —O － M ＋ 2. The compound of claim 1, wherein:

16. The compound is 【Transformation 7】 16. The compound of claim 15, selected from:

17. The compound is 【Transformation 8】 2. The compound of claim 1, wherein Ad is selected from the group consisting of:

18. 18. A formulation comprising a compound of any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

19. A method for treating a disease, disorder, or condition associated with excessive and / or abnormal glutamine utilization, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 17 or a formulation of claim 18.

20. 20. The method of claim 19, wherein the disease, disorder or condition associated with excessive and / or abnormal glutamine utilization is selected from infectious diseases, cancer, autoimmune diseases, inflammatory diseases, and neurodegenerative or neurological diseases.

21. 21. The method of claim 20, wherein the cancer is selected from a newly diagnosed cancer, a recurrent cancer, a refractory cancer, and a combination thereof.

22. 22. The method of claim 20 or 21, wherein the cancer is selected from (i) cancer of the central nervous system, (ii) cancer associated with transplantation and / or immunosuppression, (iii) cancer resistant to chemotherapy, (iv) cancer resistant to photodynamic therapy, (v) cancer resistant to proton beam therapy, (vi) cancer resistant to radiation therapy, and (vii) cancer resistant to surgery.

23. The cancers include cervicopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, pharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, gastrointestinal cancer, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, kidney cancer, 22. The method of any one of claims 19 to 21, wherein the cancer is selected from pancreatic cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymus cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

24. 22. The method of claim 21, further comprising preventing recurrence in a cancer patient in remission.

25. 20. The method of claim 19, further comprising administering a therapeutically effective amount of a compound of formula (I) in combination with immunotherapy.

26. 26. The method of claim 25, wherein the immunotherapy comprises checkpoint inhibition therapy.

27. 27. The method of claim 26, comprising administering a compound of formula (I) in combination with one or more checkpoint inhibitors.

28. 28. The method of claim 27, wherein the one or more checkpoint inhibitors are selected from anti-PD-1 Ab, anti-PD-L1 Ab, anti-CTLA-4 Ab, anti-TIGIT Ab, and combinations thereof.

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