Drug delivery conjugates, and methods for treating diseases caused by PSMA expressing cells

Abstract

To provide compounds capable of targeting PSMA expressing cells, for use in the diagnosis, imaging, and / or treatment of diseases caused by PSMA expressing cells, such as prostate cancer cells.SOLUTION: The present invention provides a conjugate represented by the formula B-L-(D)n or a pharmaceutically acceptable salt thereof [B comprises urea or thiourea from lysine and an amino acid, or one or more carboxylic acid derivatives thereof, including, but not limited to urea or thiourea from lysine and aspartic acid, or glutamic acid, or homoglutamic acid (where the urea or thiourea is capable of binding to PSMA), L is a polyvalent linker, D is a radical of a drug, and n is an integer selected from 1, 2, 3, and 4. A specific example is a conjugate represented by the following structural formula or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61 / 726,991, filed November 16, 2011; U.S. Provisional Application No. 61 / 790,234, filed March 15, 2013; U.S. Provisional Application No. 61 / 788,382, filed March 15, 2013; and U.S. Provisional Application No. 61 / 875,971, filed September 10, 2013. This application also claims priority under 35 U.S.C. § 365(c) to U.S. Patent Application No. 13 / 837,539, filed March 15, 2013. The entire disclosures of each of the above applications are incorporated herein by reference. Technical Field The invention described herein relates to the diagnosis, imaging, and / or treatment of pathogenic cell populations. More particularly, the invention described herein relates to the diagnosis, imaging, and / or treatment of diseases caused by PSMA - expressing cells, such as prostate cancer cells, using compounds capable of targeting PSMA - expressing cells.

Background Art

[0002] The prostate is a male reproductive organ that produces and stores semen, which provides nutrients and fluids for the survival of sperm introduced into the vagina during reproduction. Like other tissues, the prostate has the potential to develop either malignant (cancerous) or benign (non - cancerous) tumors. In fact, prostate cancer is one of the most common cancers in Western societies and is the second leading form of malignancy in American men. Current treatment methods for prostate cancer include hormonal therapy, radiation therapy, surgery, chemotherapy, photodynamic therapy, and combination therapies. However, many of these treatments affect the quality of life of patients, especially men diagnosed with prostate cancer over the age of 50. For example, the use of hormonal drugs often involves side effects such as osteoporosis and liver damage. Such side effects may be mitigated by the use of treatments that are more selective or specific to the tissues involved in the disease state and avoid non - target tissues such as bone or liver. [Overview of the project] [Problems that the invention aims to solve]

[0003] Prostate-specific membrane antigen (PSMA) is a biomarker overexpressed in prostate cancer. PSMA is overexpressed in malignant prostate tissue compared to other organs of the human body, such as the kidneys, proximal small intestine, and salivary glands. PSMA is also expressed in neovascularization within many non-prostate solid tumors, such as lung, colon, breast, kidney, liver, and pancreatic cancers, but not in normal vascular systems. PSMA is also minimally expressed in the brain. PSMA is a type II cell surface membrane-bound glycoprotein with a molecular weight of ~110 kD, containing an intracellular segment (1-18 amino acids), a transmembrane domain (19-43 amino acids), and a large extracellular domain (44-750 amino acids). The functions of the intracellular segment and transmembrane domain are currently considered insignificant, but the extracellular domain is involved in several distinct activities. For example, PSMA plays a role in the central nervous system, where it metabolizes N-acetyl-aspartylglutamate (NAAG) to glutamate and aspartate. PSMA also plays a role in the proximal small intestine, where it removes γ-linked glutamate from poly-γ-glutamated folate and α-linked glutamate from peptides and small molecules. However, the specific function of PSMA in prostate cancer cells remains unresolved. [Means for solving the problem]

[0004] Unlike many other membrane-bound proteins, PSMA undergoes rapid internalization into the cell in a manner similar to cell surface-bound receptors such as vitamin receptors. PSMA is internalized via clathrin-coated pits and can subsequently be recycled to the cell surface or travel to lysosomes. Therefore, diagnostic agents, contrast agents, and therapeutic agents can target PSMA for delivery into PSMA-expressing cells, such as prostate cancer cells.

[0005] This specification describes compounds that can bind to PSMA. It also describes compounds that can target PSMA for the delivery of diagnostic agents, contrast agents, and therapeutic agents. Furthermore, this specification describes compounds and compositions, as well as methods and uses thereof, for diagnosing, contrast-enhancing, and treating diseases caused by pathogenic cell populations expressing or overexpressing PSMA.

[0006] Unexpectedly, the conjugates described herein were found to exhibit high affinity for PSMA. The compounds described in this specification were also found to be effective in treating diseases caused by pathogenic cells expressing PSMA, such as prostate cancer cells.

[0007] In one exemplary embodiment of the present invention, formula: BL-(D)n [In the formula, B comprises lysine and an amino acid or thiourea, or one or more carboxylic acid derivatives thereof (where urea or thiourea can bind to PSMA), L is a polyvalent linker, D is a drug radical, and n is an integer selected from 1, 2, 3, and 4.] The present invention describes PSMA-conjugated drug delivery conjugates or pharmaceutically acceptable salts thereof. It should be understood that as used in the present invention, such drugs and the term “drug” include therapeutic agents, diagnostic agents, contrast agents, and other compounds that are preferably delivered to or target PSMA and / or PSMA-expressing cells.

[0008] In another exemplary embodiment, the formula is: BL-(D)n [In the formula, B is a PSMA-binding or targeting ligand, L is a polyvalent linker containing an aminomethylphenylacetic acid diradical or both, D is a drug radical, and n is an integer selected from 1, 2, 3, and 4.] The PSMA-conjugated drug delivery conjugate or a pharmaceutically acceptable salt thereof is described.

[0009] All combinations of the various embodiments of B, L, D, and n described herein form exemplary embodiments of the compound of the present invention, where it should be understood that the various embodiments of B, L, and D are species, subgenera, or genera. It should be further understood that each of these further exemplary embodiments of the compound may be used in any composition, unit dose, method and / or use described herein.

[0010] Another embodiment also describes a pharmaceutical composition containing one or more compounds. In one embodiment, the composition is in bulk form and is suitable for producing unit doses, unit dosage forms, etc., which may be included in the uses and / or methods described herein. In another embodiment, the composition comprises a therapeutically effective amount of one or more compounds for the diagnosis, contrast enhancement, and / or treatment of a disease caused by PSMA-expressing cells in a patient. Exemplary compositions include unit doses, unit dosage forms, etc. It should be understood that the composition may, but is not limited to, other therapeutically effective compounds, and / or one or more carriers, and / or one or more diluents, and / or one or more excipients, and other components and / or ingredients. In another embodiment, a method for using compounds and pharmaceutical compositions for the diagnosis, contrast enhancement, and / or treatment of a disease caused by PSMA-expressing cells in a patient is also described herein. In one embodiment, the method comprises the step of administering one or more compounds and / or compositions described herein to a patient. In another embodiment, the use of compounds and compositions in the manufacture of pharmaceuticals for the diagnosis, contrast imaging, and / or therapeutic use of diseases caused by PSMA-expressing cells in patients is also described herein. In one embodiment, the pharmaceutical comprises one or more of the compounds and / or compositions described herein in a therapeutically effective amount.

[0011] It is understood herein that the compounds described herein may be used alone or in combination with other compounds useful for the diagnosis, contrast enhancement, and / or treatment of diseases caused by PSMA-expressing cells in patients, such as compounds that may be therapeutically effective by the same or different mechanisms of action. Furthermore, it is understood herein that the compounds described herein may be used in combination with other compounds administered to treat other symptoms of the disease, such as compounds administered to reduce pain. [Brief explanation of the drawing]

[0012] [Figure 1] In 10% serum / FDRPMI, the relative affinity of PMPA (black square), 1.0 (normalized); DUPA (black circle), 0.05 (1 / 19); EC1067 (white circle), 30X; EC1069 (white square), 22X; and EC1080 (black inverted triangle), 6X to PSMA is shown. [Figure 2] The relative affinity of PMPA, 1.0 (normalized) (black square); EC1100, 20X (black circle); EC1168, 17X (black inverted triangle); EC1169, 7X (black triangle); and EC1170, 7X (white square) to PSMA is shown in 10% serum / FDRPMI. [Figure 3] This paper shows the dose-response and IC50 (2 hours to 72 hours) of EC1169 to LNCaP cells, as determined in vitro by 3H-thymidine-uptake cells. [Figure 4] The dose-response and IC50 (2-72 hours) of (black inverted triangle) EC1718, (black diamond) EC1677, (black triangle) EC1719, (black circle) EC1720, and (black square) EC1721 in LNCaP cells, as determined in vitro by 3H-thymidine-uptake cells, are shown. [Figure 5]This shows the in vivo efficacy of EC1169 (black triangle), EC1550 (black circle), and EC1551 (black square) at 2 μmol / kg, TIW (3 times weekly), and 2 weeks, compared to vehicle-treated control (black diamond), in the treatment of LNCaP tumor xenografts. [Figure 6] Compared to the vehicle-treated control (black diamond), EC1169 (black triangle), EC1550 (black circle), and EC1551 (black square) at 2 μmol / kg, TIW, and 2 weeks, respectively, show no overall animal toxicity. [Figure 7] This shows the in vivo efficacy of EC1584 (inverted black triangle) and EC1588 (black triangle) at 2 μmol / kg, TIW, and 2 weeks, compared to vehicle-treated control (black circle), in the treatment of LNCaP tumor xenografts. [Figure 8] Compared to the vehicle-treated control (black circle), EC1584 (black inverted triangle) and EC1588 (black triangle) at 2 μmol / kg, TIW, and 2 weeks, respectively, do not show overall toxicity in animals. [Figure 9] This shows the in vivo efficacy of EC1169 (black circle) at 2 μmol / kg intravitreal wattage (TIW) for 2 weeks compared to docetaxel (black inverted triangle) at 10 mg / kg, BIW, 2 weeks, MTD (maximum tolerated dose) in the treatment of LNCaP tumor xenografts, and the in vivo efficacy of each compared to vehicle-treated controls (black square). [Figure 10] EC1169 (black circle) administered at 2 μmol / kg, TIW, over 2 weeks, showed substantially lower overall toxicity in animals compared to docetaxel (black inverted triangle) administered at 10 mg / kg, BIW, over 2 weeks, MTD. [Figure 11] This shows the in vivo efficacy of (black square) EC1718; (black triangle) EC1720; (black inverted triangle) EC1721; (black diamond) EC1719; and (white circle) EC167, administered at 2 μmol / kg, TIW, and over 2 weeks, compared to vehicle-treated control (black circle), in the treatment of LNCaP tumor xenografts. [Figure 12]EC1718 (black square); EC1720 (black triangle); EC1721 (black inverted triangle); EC1719 (black diamond); and EC1677 (white circle); show no general toxicity to animals when compared to vehicle treatment control (black circle).

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Detailed Description Some exemplary embodiments of the invention are described by the following clauses: 1. Formula: B-L-(D)n [Wherein, B includes, but is not limited to, urea or thiourea made from lysine and an amino acid such as lysine and aspartic acid, or glutamic acid, or homoglutamic acid, or one or more carboxylic acid derivatives thereof (wherein the urea or thiourea can bind to PSMA), L is a polyvalent linker, D is a drug radical, and n is an integer selected from 1, 2, 3, and 4] A conjugate represented by or a pharmaceutically acceptable salt thereof.

[0014] 2. Formula: B-L-(D)n [Wherein, B is a radical represented by the formula: TIFF2023098946000001.tif3289, L is a polyvalent linker, D is a drug radical, and n is an integer selected from 1, 2, 3, and 4] A conjugate represented by or a pharmaceutically acceptable salt thereof.

[0015] 3. The conjugate according to item 1 or 2, wherein L is a polyvalent linker containing an aminomethylphenylacetic acid diradical or an aminophenylacetic acid diradical, or both. 4. Formula: B-L-(D)n [In the formula, B is a PSMA-binding ligand, L is a polyvalent linker containing an aminomethylphenylacetic acid diradical or an aminomethylphenylacetic acid diradical, or both, D is a drug radical, and n is an integer selected from 1, 2, 3, and 4.] The compound represented by or a pharmaceutically acceptable salt thereof.

[0016] 5. The conjugates described in item 3, wherein B comprises, but is not limited to, urea or thiourea made from lysine and an amino acid, such as urea or thiourea made from lysine and aspartic acid, or glutamic acid, or homoglutamic acid, or one or more carboxylic acid derivatives thereof. 6. A conjugate according to any one of items 1 to 5, wherein B comprises urea or thiourea made from lysine and glutamate, or one or more carboxylic acid derivatives thereof. 7. A conjugate according to any one of items 1 to 5, wherein B comprises urea or thiourea made from lysine and glutamate. 8. A conjugate according to any one of items 1 to 5, wherein B comprises urea or thiourea made from L-lysine and L-glutamate, or one or more carboxylic acid derivatives thereof.

[0017] 9. The conjugate according to any one of items 1 to 5, wherein B comprises urea or thiourea made from L-lysine and L-glutamate. 10. A conjugate according to any one of items 1 to 5, wherein B comprises urea or thiourea made from lysine and glutamic acid. 11. A conjugate according to any one of items 1 to 5, wherein B comprises urea or thiourea made from D-lysine and D-glutamic acid. 12.B is D-lysine and formula: A conjugate according to any one of items 1 to 5, comprising urea or thiourea made from one of TIFF2023098946000002.tif2291. 13.B is D-lysine and formula: A conjugate according to any one of items 1 to 5, comprising urea or thiourea, made from TIFF2023098946000003.tif2022. 14. A compound according to any one of items 1 to 5, wherein B is urea. 15.B is as follows: A combination selected from TIFF2023098946000004.tif59122, as described in any one of items 1 to 5. 16.B is as follows: A combination described in any one of items 1-5, selected from TIFF2023098946000005.tif2257. 17.B is the formula: A conjugate as described in any one of items 1 to 5, as shown in TIFF2023098946000006.tif1826.

[0018] The combination according to any one of the preceding items, wherein n is 1, 2, or 3. The combination according to any one of the preceding items, wherein n is 1 or 2. The combination according to any one of the preceding items, wherein n is 1. A conjugate according to any one of the preceding paragraphs, wherein at least one drug is a contrast agent. A conjugate according to any one of the preceding paragraphs, wherein at least one drug is a diagnostic agent.

[0019] A conjugate according to any one of the preceding paragraphs, wherein at least one drug is a therapeutic agent. A conjugate according to any one of the preceding paragraphs, wherein at least one drug is a cytotoxic drug. A conjugate according to any one of the preceding paragraphs, wherein at least one drug is tubulicin. A conjugate according to any one of the preceding paragraphs, wherein at least one drug is a naturally occurring tubulicin. The conjugate according to any one of the preceding paragraphs, wherein at least one drug is tubulicin B.

[0020] At least one drug, formula: TIFF2023098946000007.tif3081[In the formula, n is 1-3; V is hydrogen, OR2, or halo, and W is hydrogen, OR2, or alkyl (where R2 is independently selected in each case from hydrogen, alkyl, and C(O)R3 (where R3 is alkyl, cycloalkyl, alkenyl, aryl, or arylalkyl, each optionally substituted)); however, if both V and W are OR2, R2 is not hydrogen; or V and W, together with the carbon they are bonded to, form a carbonyl group; X is a hydrogen atom, an alkyl group such as a C1-6 alkyl or C2-6 alkyl group, a C1-4 alkyl or C2-4 alkyl group, or an alkenyl group such as a C2-6 alkenyl or C2-4 alkenyl group, and each can be optionally substituted; Z is an alkyl or C(O)R4 (where R4 is an alkyl, CF3, or aryl); Ar is either an aryl or heteroaryl, and each can be optionally substituted; and R is either OH or R, and the carbonyl group to which it is attached is a carboxylic acid derivative. A conjugate according to any one of the preceding paragraphs, wherein a tubulicin represented by is described, and a pharmaceutically acceptable salt thereof is described.

[0021] The conjugate according to any one of the preceding items, wherein Ar is optionally substituted phenyl.

[0022] The conjugate according to any one of the preceding items, wherein Ar is a phenyl substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, cycloheteralkyl, cycloheteralkenyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl, each of which is optionally substituted.

[0023] The conjugate according to any one of the preceding items, wherein Ar is phenyl. The conjugate according to any one of the preceding items, wherein Ar is 4-hydroxyphenyl.

[0024] A compound according to any one of the preceding items, wherein X is CH2QR9 (where Q is -N-, -O-, or -S-; R9 is hydrogen or an alkyl, alkenyl, cycloalkyl, aryl, or arylalkyl, each optionally substituted) or C(O)R10. A combination according to any one of the preceding items, wherein Q is O. The conjugate according to any one of the preceding items, wherein R9 is an optionally substituted alkyl group.

[0025] The conjugate according to any one of the preceding items, wherein R9 is an alkyl group. The conjugate according to any one of the preceding items, wherein R10 is an optionally substituted alkyl group. The compound according to any one of the preceding items, wherein R10 is an alkyl group. At least one drug is given by the following formula: A combination described in any one of the above items, selected from TIFF2023098946000008.tif107160.

[0026] At least one drug, The composite described in any one of the preceding items, which is TIFF2023098946000009.tif3198.

[0027] At least one D is given by the formula: A conjugate according to any one of the preceding items, which is a radical represented by TIFF2023098946000010.tif2271.

[0028] At least one D is given by the formula: A compound according to any one of the preceding items, which is a radical represented by TIFF2023098946000011.tif2269.

[0029] At least one D is given by the formula: A conjugate according to any one of the preceding items, which is a radical represented by TIFF2023098946000012.tif2271.

[0030] At least one D is given by the formula: A conjugate according to any one of the preceding items, which is a radical represented by TIFF2023098946000013.tif2269.

[0031] At least one D is given by the formula: TIFF2023098946000014.tif2867 TIFF2023098946000015.tif2868 TIFF2023098946000016.tif2764 TIFF2023098946000017.tif2764[wherein n=1, 2, 3, 4, 5, or 6]. A compound according to any one of the preceding items, which is a radical represented by .

[0032] A compound according to any one of the preceding items, wherein L comprises an aminomethylphenylacetic acid diradical. A conjugate according to any one of the preceding items, wherein L comprises an aminophenylacetic acid diradical. The conjugate according to any one of the preceding items, wherein L forms urea or thiourea with lysine. The conjugate according to any one of the preceding items, wherein L forms urea with lysine. The conjugate according to any one of the preceding items, wherein L forms an amide or thioamide with lysine. The conjugate according to any one of the preceding items, wherein L forms an amide with lysine. The conjugate according to any one of the preceding items, wherein L comprises one or more aspartic acid diradicals. The conjugate according to any one of the preceding items, wherein L comprises two or more aspartic acid diradicals. The bond described in the preceding paragraph, wherein the aspartic acid diradical is an L-aspartic acid diradical.

[0033] A conjugate according to any one of the preceding items, wherein L contains a cysteine ​​diradical. A conjugate according to any one of the preceding items, wherein L comprises an L-cysteine ​​diradical. A compound according to any one of the preceding items, wherein L contains L-Asp-L-Asp-L-Cys. The conjugate according to any one of the preceding paragraphs, wherein L is a releaseable linker, such as a releaseable linker, which is cleaved under conditions that it encounters, is near, or is inside a pathogenic cell that expresses, preferentially expresses, or overexpresses PSMA.

[0034] A compound according to any one of the preceding items, wherein L contains a disulfide. A conjugate according to any one of the preceding items, wherein L comprises a cysteine ​​disulfide diradical. A conjugate according to any one of the preceding items, wherein L comprises an L-cysteine ​​disulfide diradical. A compound according to any one of the preceding items, wherein L contains L-Asp-L-Asp-L-Cys(SS). A compound according to any one of the preceding items, wherein L comprises a diradical of formula: OC(O)-N. A compound according to any one of the preceding items, wherein L comprises a diradical of formula: OC(O)-NH.

[0035] The conjugate according to any one of the preceding items, wherein L and at least one D together comprise a diradical represented by the formula:OC(O)-N. The conjugate according to any one of the preceding items, comprising L and at least one D together, comprising a diradical represented by the formula: OC(O)-NH. The compound according to any one of the preceding items, wherein L contains a diradical represented by the formula: S-(CH2)mO (where m is 2, 3, or 4). The compound according to any one of the preceding items, wherein L contains a diradical represented by the formula: S-(CH2)mOC(O)-N (where m is 2, 3, or 4). The compound according to any one of the preceding items, wherein L contains a diradical represented by the formula: S-(CH2)mOC(O)-NH (where m is 2, 3, or 4). The conjugate according to any one of the preceding items, wherein L and at least one D together comprise a diradical represented by the formula: S-(CH2)mOC(O)-N (where m is 2, 3, or 4). The conjugate according to any one of the preceding items, wherein L and at least one D together comprise a diradical represented by the formula: S-(CH2)mOC(O)-NH (where m is 2, 3, or 4). The compound according to any one of the preceding items, wherein the terminal sulfur atom forms a disulfide. The combination according to any one of the preceding items, wherein m is 2.

[0036] The compound according to any one of the preceding items, wherein L comprises a chain of at least about 7 atoms, at least about 8 atoms, at least about 9 atoms, at least about 10 atoms, at least about 11 atoms, at least about 12 atoms, at least about 13 atoms, at least about 14 atoms, or at least about 15 atoms.

[0037] The compound according to any one of the preceding items, wherein L comprises a chain of at least about 16 atoms, at least about 17 atoms, at least about 18 atoms, at least about 19 atoms, at least about 20 atoms, at least about 21 atoms, at least about 22 atoms, at least about 23 atoms, at least about 24 atoms, at least about 25 atoms, or at least about 26 atoms. The compound according to any one of the preceding items, wherein L comprises a chain of approximately 7 to approximately 35 atoms, approximately 7 to approximately 30 atoms, or approximately 7 to approximately 26 atoms.

[0038] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical shown in TIFF2023098946000018.tif1971.

[0039] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical as shown in TIFF2023098946000019.tif1980.

[0040] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000020.tif1878.

[0041] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000021.tif1870.

[0042] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000022.tif58155.

[0043] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000023.tif2877.

[0044] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000024.tif85159.

[0045] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000025.tif2877.

[0046] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000026.tif62158.

[0047] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000027.tif2877.

[0048] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000028.tif110129.

[0049] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000029.tif2776.

[0050] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000030.tif1326.

[0051] L is the formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000031.tif2331.

[0052] BL is, formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000032.tif3031.

[0053] BL is, formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000033.tif3031.

[0054] BL is, formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000034.tif3045.

[0055] BL is, formula: A conjugate according to any one of the preceding items, comprising a diradical represented by TIFF2023098946000035.tif3049.

[0056] formula: TIFF2023098946000036.tif26127 [In the formula, D is the radical of the drug] The compound represented by or its pharmaceutically acceptable salts, and / or hydrates, and / or solvates, and / or cocrystals thereof.

[0057] formula: TIFF2023098946000037.tif28160 [In the formula, D is the radical of the drug] The compound represented by or its pharmaceutically acceptable salts, and / or hydrates, and / or solvates, and / or cocrystals thereof.

[0058] formula: TIFF2023098946000038.tif28135 [In the formula, D is the radical of the drug] The compound represented by or its pharmaceutically acceptable salts, and / or hydrates, and / or solvates, and / or cocrystals thereof.

[0059] formula: TIFF2023098946000039.tif37170 [In the formula, D is the radical of the drug] The compound represented by or its pharmaceutically acceptable salts, and / or hydrates, and / or solvates, and / or cocrystals thereof.

[0060] A pharmaceutical composition comprising one or more compounds or conjugates described in any one of the preceding items. A pharmaceutical composition comprising one or more compounds or conjugates described in any one of the preceding items, for the treatment of a disease in a host animal caused by a population of pathogenic cells expressing PSMA. A single-dose or split-dose unit dose or unit administration form comprising one or more of the compounds or conjugates described in any one of the preceding items in a therapeutically effective amount for the treatment of a disease in a host animal caused by a population of pathogenic cells expressing PSMA.

[0061] Furthermore, the composition or unit dose or unit dosage form according to any one of the preceding items comprises one or more carriers, diluents or excipients, or a combination thereof.

[0062] A method for treating a disease in a host animal caused by a population of pathogenic cells expressing PSMA, comprising the step of administering to a patient a therapeutically effective amount of one or more compounds or conjugates described in any one of claims 1 to 73, or a composition comprising a unit dose or unit dosage form.

[0063] Use of one or more compounds, compositions, unit doses or unit dosage forms described in any one of the preceding paragraphs in the manufacture of a pharmaceutical product for the treatment of a disease in a host animal caused by a population of pathogenic cells expressing PSMA.

[0064] The composition, unit dose or unit dosage form, method or use according to any one of the preceding paragraphs, wherein the cells are prostate cancer cells.

[0065] The composition, unit dose or unit dosage form, method or use described in any one of the preceding paragraphs, wherein the disease is prostate cancer.

[0066] The composition, unit dose or unit dosage form, method or use described in any one of the preceding paragraphs, wherein the host animal is human.

[0067] In the enumeration of the embodiments and sections described above and below, it should be understood that all possible combinations of features, and all possible sub-concepts and secondary combinations, are described. For example, if B is limited to a binding ligand containing urea made from L-lysine and L-glutamate, it should be understood that L may be limited to a linker containing one or more aspartic acid diradicals, or a linker containing a cysteine ​​radical, or a radical containing L-Asp-L-Asp-L-Cys(SS), etc. Similarly, if D is limited to naturally occurring tubulosine, L may be limited to a linker containing the formula:S-(CH2)mOC(O)-N, or a linker containing a cysteine ​​disulfide diradical, or a linker containing an aminophenylacetic acid diradical, etc. Similarly, if B is limited to a binding ligand containing urea or thiourea made from lysine and glutamate, or one or more carboxylic acid derivatives thereof, then L may be limited to a linker containing one or more D-aspartate diradicals, D may be limited to tubulosine, or L may be limited to a linker containing a diradical represented by formula:OC(O)-N, D may be limited to a contrast agent, or L may be limited to a linker containing a diradical represented by formula:S-(CH2)mOC(O)-NH, D may be limited to a therapeutic agent, and so on. Other combinations, sub-concepts, and secondary combinations are also described by the collection of entries.

[0068] In another embodiment, at least one drug is a contrast agent. Exemplary contrast agents for the conjugates described herein include, but are not limited to, radioisotopes, such as radioisotopes of metals coordinated to a chelate group. Exemplary radiometallic isotopes include technetium, rhenium, gallium, gadolinium, indium, and copper, such as isotopes: 111In, 99mTc, 64Cu, 67Cu, 67Ga, 68Ga. Further exemplary examples of radionuclide contrast agents are described in U.S. Patent No. 7,128,893, the disclosure of which is incorporated herein by reference. Further exemplary chelate groups are given by formula: TIFF2023098946000040.tif2131[In the formula, R is independently selected in each case from H, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, etc., which are each optionally substituted.] These are tripeptides or tetrapeptides, such as the tripeptides shown, but are not limited to these. It should be understood that one R contains a heteroatom such as nitrogen, oxygen, or sulfur, and is the linker L bonding site. Exemplary examples include the following chelate groups: TIFF2023098946000041.tif56117[In the formula, X is oxygen, nitrogen, or sulfur, where X is bonded to a linker, and n is an integer between 1 and approximately 5] Write it down.

[0069] Examples of contrast agents include, but are not limited to, Oregon Green fluorescent agents such as Oregon Green 488 and Oregon Green 514; Alexa Fluoro fluorescent agents such as Alexa Fluoro 488 and Alexa Fluoro 647; Fluorescein and related analogues such as BODIPY F1 and BODIPY 505; Rhodamine fluorescent agents such as tetramethylrhodamine; and Dilite fluorescent agents such as Dilite 680 and Dilite 800, CW 800, IRdye 800CW, Texas Red, phycoerythrin, and others; however, these are not limited to such fluorescent agents. Further examples of fluorescent agents include the following formula: TIFF2023098946000042.tif3028[wherein X is oxygen, nitrogen, or sulfur, where X is bonded to the linker; Y is ORa, NRa2, or NRa3+; and Y' is O, NRa, or NRa2+; where each R is independently selected in each case from H, fluoro, sulfonic acid, sulfonate and its salts, etc.; and Ra is hydrogen or alkyl] Examples of compounds shown are given by the following formula: TIFF2023098946000043.tif2429[wherein X is oxygen, nitrogen, or sulfur, where X is bonded to a linker; and each R is independently selected in each case from H, alkyl, heteroalkyl, etc.; and n is an integer from 0 to about 4] Examples of compounds shown are:

[0070] Examples of contrast agents include, but are not limited to, PET contrast agents and FRET contrast agents. Examples of PET contrast agents include 18F, 11C, 64Cu, and 65Cu. Examples of PET contrast agents include 64Cu and 65Cu. In the case of 18F and 11C, it should be understood that the contrast isotope may be directly bound to the linker or present on a structure bound to the linker. For example, in the case of 18F, fluoroaryl groups such as fluorophenyl, difluorophenyl, and fluoronitrophenyl are described. For example, in the case of 11C, alkyl and alkylaryl groups are described.

[0071] In another embodiment, the drug can be any molecule capable of modulating or otherwise altering cellular function, such as a pharmaceutically active compound. Exemplary drugs include peptides, oligopeptides, retro-inverso-oligopeptides, proteins, protein analogs in which at least one non-peptide bond replaces a peptide bond, apolipoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, receptors and other membrane proteins; antigens and antibodies against them; haptens and antibodies against them; hormones, lipids, phospholipids, liposomes; toxins; antibiotics; analgesics; bronchodilators; beta-blockers; antibacterial agents; antihypertensive agents; antiarrhythmics, cardiac glycosides, anti-anginal agents. Examples of prohibited substances include, but are not limited to, cardiovascular drugs such as vasodilators; central nervous system drugs such as stimulants, psychotropic drugs, antimanic and depressant drugs; antiviral drugs; antihistamines; anticancer drugs such as chemotherapeutic agents; tranquilizers; antidepressants; H-2 antagonists; anticonvulsants; antiemetics; prostaglandins and prostaglandin analogs; muscle relaxants; anti-inflammatory substances; immunosuppressants; stimulants; decongestants; antiemetics; diuretics; antispasmodics; antiasthmatics; antiparkinsonian drugs; expectorants; antitussives; mucolytics; and minerals and nutritional additives.

[0072] Examples of chemotherapeutic agents include, but are not limited to, compounds that are cytotoxic, enhance tumor permeability, inhibit tumor cell proliferation, promote apoptosis in target cells, reduce anti-apoptotic activity, are used to treat diseases caused by infectious pathogens, enhance the endogenous immune response against pathogenic cells, or are useful in treating disease conditions caused by pathogenic cells. Such chemotherapeutic agents may act by any of a variety of mechanisms. For example, cytotoxic compounds can disrupt any broad range of cellular mechanisms that are important for cell survival and / or cell proliferation, and / or that cause cell death or apoptosis.

[0073] Further exemplary chemotherapeutic agents include adrenocorticoids and corticosteroids, alkylating agents, antiandrogens, antiestrogens, androgens, acramycin and acramycin derivatives, estrogens, antimetabolites, such as cytosine arabinoside, purine analogs, pyrimidine analogs, and methotrexate, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, tamoxifen, taxol, paclitaxel, paclitaxel derivatives, Taxotere®, cyclophosphamide, daunomycin, rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycin, discodermorid, microtubule inhibitors, epotilon, tubulosine, cyclopropylbenzo[e]indron, seco-cyclopropylbenzo[e]indron, O-Ac-seco-cyclopropylbenzo[ e) Indron, bleomycin and any other antibiotics; pinca alkaloids such as nitrogen mustard, nitrosurea, vincristine, vinblastine, vindesine, vinorelbine and their analogues and derivatives such as deacetylvinblastine monohydrazide (DAVLBH); colchicine, colchicine derivatives, allocorchicine, thiocolchicine, tritylcysteine, halichondrin B; dorastatins, e.g., dorastatin 10; amanitins, e.g., α-amanitin; camptothecin, irinotecan and other camptothecin derivatives; geldanamycin and geldanamycin derivatives; estramustine, nocodazole, MAP4, colsemid; inflammatory and anti-inflammatory agents; peptides and peptide-like signaling inhibitors; rapamycin, e.g., sirolimus and everolimus; and any other drugs or toxins.

[0074] In another embodiment, at least one drug is selected from cryptophycin, bortezomib, thiobortezomib, tubulisin, aminopterin, rapamycin such as sirolimus and everolimus, paclitaxel, docetaxel, doxorubicin, daunorubicin, α-amanitin, velcarin, didemnin B, geldanamycin, pluvalanol A, ispinesib, budesonide, dasatinib, epotilon, maytansine and tyrosine kinase inhibitors, as well as their analogs and derivatives.

[0075] Other drugs that may be included in the conjugates described herein include amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, and ribavirin.

[0076] In another embodiment, at least one drug is tubulicin. As used herein, the term “tubulicin” generally means the compounds described herein and their analogues and derivatives. Any corresponding pharmaceutically acceptable salts should also be understood to be included in the exemplary embodiments described herein. Exemplary derivatives of tubulicin include, but are not limited to, compounds that can be synthetically produced from the compounds described herein. Such derivatives may include compounds described herein that incorporate one or more protecting or protecting groups, such as prodrugs of the compounds described herein or compounds used in the production of other compounds described herein.

[0077] As described herein, the tubulicine compound may be an inhibitor of tubulicine polymerization or a DNA alkylating agent.

[0078] As an example of tubulosin, formula: TIFF2023098946000044.tif2363 and TIFF2023098946000045.tif2463[in the formula, n is 1-3; V is hydrogen, OR2, or halo, and W is hydrogen, OR2, or alkyl (where R2 is independently selected in each case from hydrogen, alkyl, and C(O)R3 (where R3 is alkyl, cycloalkyl, alkenyl, aryl, or arylalkyl, each optionally substituted)); however, if both V and W are OR2, R2 is not hydrogen; or V and W, together with the carbon they are bonded to, form a carbonyl group; X is a hydrogen atom, an alkyl group such as a C1-4 alkyl group, or an alkenyl group such as a C2-4 alkenyl group, and each can be optionally substituted; Z is alkyl or C(O)R4 (where R4 is alkyl, CF3, or aryl); or if Y is present, Z is alkyl; Y is O; Ar is an aryl or heteroaryl such as phenyl, and each can be optionally substituted; and R is either OH or R, and the carbonyl group to which it is attached is a carboxylic acid derivative such as an acyl hydrazide. Examples of compounds shown are listed, along with their salts, but are not limited to these.

[0079] In another embodiment, X is CH2QR9 (where Q is -N-, -O-, or -S-; R9 is hydrogen or optionally substituted alkyl, alkenyl, cycloalkyl, aryl, or arylalkyl) or C(O)R10 (where R10 is hydrogen or alkyl, alkenyl, cycloalkyl, aryl, or arylalkyl). In yet another embodiment, R9 and Q together form S(O)2R10, P(O)(OR10a)2 (where R10 and OR10a are independently selected in each case from hydrogen and optionally substituted alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, and arylalkyl, respectively, and R10a is a metal cation).

[0080] In another embodiment, X is H. Exemplary examples of such compounds and their preparations are described in J. Med. Chem. 10.1021 / jm701321p (2008), the disclosure of which is incorporated herein by reference.

[0081] In another embodiment, X is given by the formula: TIFF2023098946000046.tif1414[In the formula, R12 represents one or more substituents selected from alkyl, alkenyl, cycloalkyl, aryl, and arylalkyl groups, each of which may be optionally substituted.] This is a radical represented by . It should be understood that other olefins may be formed by isomerization depending on the reaction conditions and the properties of R12. For example, if R12 is alkyl, it is understood that under reaction conditions, the double bond may move to other carbon atoms along the alkenyl chain, such as forming terminal or ω-olefins.

[0082] In another embodiment, X is given by the formula: TIFF2023098946000047.tif1815[In the formula, R13 is C(O)R10, C(O)OR10, or CN (where R10 is independently chosen in each case)] It is a radical represented by [the symbol].

[0083] In another embodiment, X is CH2-OH.

[0084] In another embodiment, X is CH2-XA (where XA is a halogen, OS(O)2R10, OP(O)(OR10a)R10, or OP(O)(OR10a)2; where R10 and OR10a are independently in each case selected from hydrogen, optionally substituted alkyl, alkenyl, cycloalkyl, aryl, and arylalkyl, or R10a is a metal cation).

[0085] In another embodiment described in any of the embodiments described above, Ar is an optionally substituted aryl. In another embodiment described in any of the embodiments described above, Ar is of the formula: TIFF2023098946000048.tif1716[wherein R1 is hydrogen, or R1 independently represents 1 to 3 substituents selected from halo, nitro, carboxylate or derivative thereof, cyano, hydroxyl, alkyl, haloalkyl, alkoxy, haloalkoxy and OR6 (wherein R6 is hydrogen or optionally substituted alkyl, heteroalkyl, aryl, phenol protecting group, prodrug moiety, C(O)R7, P(O)(OR8)2 or SO3R8 (wherein R7 and R8 are independently selected in each case from hydrogen or optionally substituted alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and arylalkyl, respectively, or R8 is a metal cation))] It is a radical represented by [the symbol].

[0086] In another embodiment described in any of the embodiments described above, Z is methyl. In another embodiment described in any of the embodiments described above, R1 is H. In another embodiment described in any of the embodiments described above, R1 is OR6 at C(4), where R6 is hydrogen, alkyl, or COR7. In another embodiment described in any of the embodiments described above, V is hydrogen and W is OC(O)R3. In another embodiment described in any of the embodiments described above, V is hydrogen and W is acetyloxy.

[0087] In another embodiment described in any of the embodiments described above, the compounds of various formulas have the following absolute configuration at each of the indicated chiral carbon atoms: It has TIFF2023098946000049.tif2878.

[0088] Further exemplary tubulicins usable in the conjugates described herein include the following compounds and their pharmaceutically acceptable salts: TIFF2023098946000050.tif2777 TIFF2023098946000051.tif62131

[0089] In another embodiment, tubulicin is naturally occurring tubulicin. Natural tubulicin is generally a linear tetrapeptide consisting of N-methylpipecolic acid (Mep), isoleucine (Ile), a non-natural amino acid called tubuvalin (Tuv), and either a non-natural amino acid called tubutyrosine (Tut, a tyrosine analog) or a non-natural amino acid called tubuphenylalanine (Tup, a phenylalanine analog). In another embodiment, the formula is as follows: TIFF2023098946000052.tif2978 [wherein Ar, R, and R10 are as described in the various embodiments herein] The natural tubulicin shown and its analogues and derivatives, as well as its pharmaceutically acceptable salts, are described.

[0090] In another embodiment, the following general formula: TIFF2023098946000053.tif2779 TIFF2023098946000054.tif52128 The natural tubulicin and its medicinal salts are described.

[0091] It should be understood that conjugates of tubulicin or its analogs or derivatives may be formed at any position. For example, linker(L) may be at any of the following positions: TIFF2023098946000055.tif2880[In the formula, the symbol (*) indicates an arbitrary binding position] A tubulicin conjugate that binds to is described.

[0092] In another embodiment, compounds in which a conjugate is formed at each terminal carboxylic acid or terminal acylhydrazine derivative group of the tubulicine described herein are described herein.

[0093] Further tubulisins useful for producing the conjugates described herein are described in U.S. Patent Application Publication Nos. 2006 / 0128754 and 2005 / 0239713, the disclosures of which are incorporated herein by reference. Further tubulisins useful for producing the conjugates described herein are described in the concurrently continuing U.S. Patent Application Publication No. 2010 / 0240701, the disclosures of which are incorporated herein by reference. Tubulysin may also be produced as described in Peltier et al., "The Total Synthesis of Tubulysin D," J. Am. Chem. Soc. 128:16018-19 (2006), the disclosures of which are incorporated herein by reference.

[0094] In another embodiment, at least one drug is rapamycin. The term "rapamycin" as used herein includes sirolimus (rapamycin), temsirolimus, everolimus, and ridafololimus, as well as related compounds and formula: TIFF2023098946000056.tif6166[in the formula, YA is ORC or OCH2CH2ORC; One of RA, RB, or RC is a bond connected to L; and The other two of RA, RB, and RC are independently selected in each case from hydrogen, an optionally substituted heteroalkyl group, a prodrug-forming group, and C(O)RD (wherein RD is independently selected in each case from hydrogen and alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which is optionally substituted). It is understood to include the compounds indicated by and their pharmaceutically acceptable salts.

[0095] In another embodiment, at least one drug is a vinca alkaloid such as vincristine, vinblastine, vindesine, or vinorelbine, and its analogs and derivatives such as deacetylvinblastine monohydrazide (DAVLBH).

[0096] In another embodiment, at least one drug is mitomycin or an analog or derivative thereof.

[0097] In another embodiment, the conjugate described herein comprises at least two drugs, such as those described herein. In one variant, the drugs are the same. In another variant, at least two drugs are different. In another variant, two or more drugs are selected from vinca alkaloids, cryptophycin, bortezomib, thiobortezomib, tubulisin, aminopterin, rapamycin such as sirolimus or everolimus, paclitaxel, docetaxel, doxorubicin, daunorubicin, α-amanitin, vercarin, didemnin B, geldanamycin, pluvalanol A, ispinesib, budesonide, dasatinib, epotilon, maytansine, and tyrosine kinase inhibitors, as well as their analogs and derivatives.

[0098] As used herein, the term "linker" encompasses a chain of atoms that links the functional portions of two or more molecules to form a conjugate. For example, the atomic chain may be selected from C, N, O, S, Si, and P, or C, N, O, S, and P, C, N, O, and S. The atomic chain co-links the different functional capabilities of conjugates such as binding ligands, drugs, diagnostic agents, and imaging agents. Linkers can have varying lengths in a continuous backbone, ranging from approximately 2 to approximately 100 atoms. The atoms used to form the linker can be combined in all chemically related ways, including, for example, chains of carbon atoms that form alkylenes, alkenylenes, and alkylylene groups; chains of carbon and oxygen atoms that form ethers, polyoxyalkylene groups, or, when combined with carbonyl groups, esters and carbonates; chains of carbon and nitrogen atoms that form amines, imines, polyamines, hydrazines, hydrazones, or, when combined with carbonyl groups, amides, ureas, semicarbazides, carbazides; and chains of carbon, nitrogen, and oxygen atoms that form alkoxyamines, alkoxylamines, or, when combined with carbonyl groups, urethanes, amino acids, acyloxyamines, hydroxamic acids, etc. Furthermore, it should be understood that the atoms forming the chains in each of the exemplary embodiments described above can be saturated or unsaturated, and therefore can form single, double, or triple bonds, so as alkanes, alkenes, alkynes, imines, etc., can be radicals contained in the linker. Furthermore, it should be understood that the atoms forming the linker are also cyclized with each other or are part of a cyclic structure, and in the linker, cycloalkanes, cyclic ethers, cyclic amines, and other heterocyclic structures can form linkers such as arylenes and heteroarylenes. In this latter configuration, it should be understood that the length of the linker can be defined by any path through one or more cyclic structures. Exemplarily, the length of the linker is defined by the shortest path through each of the cyclic structures.It should be understood that the linker can be optionally substituted with any one or more open valencies along the chain of atoms, such as any substituent on any of the carbon, nitrogen, silicon, or phosphorus atoms. It should also be understood that the linker can link two or more functional parts of a molecule to form a conjugate with any open valency, and it is not necessary that any of the two or more functional parts forming the conjugate be bonded to any obvious end of the linker.

[0099] In another embodiment, the linker (L) is given by formula: TIFF2023098946000057.tif1986[wherein m1, m2, m3, n, p, q, and r are integers independently chosen from 0 to 8; however, at least one of m1, m2, m3, n, p, q, and r is not 0; AA is an amino acid; and the drug is arbitrarily bonded with one or more (*) atoms] It contains a radical represented by . It should be understood that the drug may be directly bound or bound via a further linker (L) portion. In another embodiment, AA is a natural amino acid having either a natural or unnatural stereoconfiguration. In another embodiment, one or more AAs are hydrophilic amino acids. In another embodiment, one or more AAs are Asp and / or Arg. In another embodiment, integer n is 1 or greater. In another embodiment, integer n is 2 or greater. In another embodiment, integer n is 3 or greater. In another embodiment, integer n is 4 or greater. In another embodiment, integer n is 5 or greater. In another embodiment, integer q is 1 or greater. In another embodiment, integer m1 is 1 or greater. In another embodiment, integer m1 is 1. In another embodiment, integer m2 is 1 or greater. In another embodiment, integer m2 is 1. In another embodiment, integer m3 is 1 or greater. In another aspect, the integer m3 is 1. In another aspect, the integer p is 1 or greater. In another aspect, the integer p is 1. In another aspect, the integer p is 2 or greater. In another aspect, the integer p is 2. In another aspect, the integer q is 1 or greater. In another aspect, the integer q is 1. In another aspect, the integer q is 2 or greater. In another aspect, the integer q is 2. In another aspect, the integer r is 1 or greater. In another aspect, the integer r is 1. In another aspect, the integer r is 2.

[0100] It should be understood that all combinations of the embodiments described above are described herein as further exemplary embodiments of the present invention. For example, in another embodiment, n is 1 or more and m1 is 1 or more; or n is 1 or more, m1 is 1 and q is 1; and so on. For example, in another embodiment, n is 1 or more and m2 is 1 or more; or n is 2 or more, m2 is 1 and q is 1; or n is 2 or more, m3 is 1, q is 1 and p is 1; and so on. For example, in another embodiment, n is 1 or more and m1 is 1 or more; or n is 2 or more, m3 is 1 and q is 1; or n is 2 or more, m2 is 1 and q is 1 and p is 1; or n is 2 or more, m1 is 1 and q is 1 and r is 1; or n is 2 or more, m3 is 1 and q is 1 and p is 1 and r is 1; and so on.

[0101] In another embodiment, the polyvalent linker is one or more divalent hydrophilic radicals described herein, also called linkers or spacer linkers. It should be understood that the various arrangements and / or orientations of hydrophilic linkers may be linear, branched, or both. For example, a hydrophilic linker may form the main chain of a linker that forms a conjugate of a ligand and one or more drugs. Alternatively, the hydrophilic portion of the linker may suspend or bind to the main chain of atoms linking the binding ligand B to one or more drugs D. In this latter arrangement, the hydrophilic portion may be proximal or distal to the main chain of atoms.

[0102] In another embodiment, the linker is generally linear, and the hydrophilic groups are generally arranged in a series to form a chain-like linker in the conjugate. In this other method, in this linear embodiment, the hydrophilic groups form some or all of the main chains of the linker.

[0103] In another embodiment, the linker is branched by a hydrophilic group. In this branched configuration, the hydrophilic group may be proximal or distal to the main chain. In each of these configurations, the linker is generally more spherical or cylindrical in shape. In one embodiment, the linker has a bottlebrush-like shape. In one embodiment, the main chain of the linker is formed of a linear system of amides, and the hydrophilic portion of the linker is formed by a parallel arrangement of branched side chains, such as those formed by linking monosaccharides, sulfonates, and their derivatives and analogs.

[0104] It is understood that the linker may be neutral or ionized under certain conditions, such as the physiological conditions encountered in vivo. For ionic compound linkers under selected conditions, the linker may be deprotonated to form an anion or protonated to form a cation. It is understood that one or more deprotonation or protonation events may occur. Furthermore, it is understood that the same linker may be deprotonated or protonated to form an internal salt or an amphoteric compound.

[0105] In another embodiment, the hydrophilic spacer linker is neutral, particularly neutral under physiological conditions, and the linker is neither significantly protonated nor deprotonated. In yet another embodiment, the hydrophilic spacer linker can be protonated to have one or more positive charges. It is understood that the protonation ability is condition-dependent. In one embodiment, the conditions are physiological conditions, and the linker is protonated in vivo. In yet another embodiment, the spacer encompasses both a region that is neutral and a region that can be protonated to have one or more positive charges. In yet another embodiment, the spacer encompasses both a region that can be deprotonated to have one or more negative charges and a region that can be protonated to have one or more positive charges. In this latter embodiment, it is understood that an amphoteric ion or internal salt may be formed.

[0106] In another embodiment, the linker, which can be deprotonated and negatively charged, includes aspartic acid, glutamic acid, and long-chain carboxylic acid groups, as well as sulfate esters such as alkyl esters of sulfate. In yet another embodiment, the linker region, which can be protonated and positively charged, includes amino groups such as ethylenediamine, propylenediamine, and butylenediamine lyaminoalkylene, and / or heterocyclic groups such as pyrrolidine, piperidine, and piperazine, and other amino groups, each optionally substituted. In yet another embodiment, the linker region, which is neutral, includes polyhydroxyl groups such as sugars, carbohydrates, saccharides, and inositol, and / or polyether groups such as polyoxyethylene and polyoxypropylene.

[0107] In another embodiment, the hydrophilic spacer linker described herein is formed primarily from carbon, hydrogen, and oxygen, and has a carbon / oxygen ratio of about 3:1 or less, or about 2:1 or less. In one embodiment, the hydrophilic linker described herein includes a plurality of ether functional groups. In another embodiment, the hydrophilic linker described herein includes a plurality of hydroxyl functional groups. Exemplary fragments and radicals that can be used to form such linkers include polyhydroxyl compounds such as carbohydrates, polyether compounds such as polyethylene glycol units, and acidic groups such as carboxyls and alkyl sulfates. In one variant, oligoamide spacers may also be included in the linker.

[0108] Examples of divalent hydrophilic linkers include carbohydrates such as saccharopeptides described herein, which possess both peptide and sugar characteristics; glucuronides, which can be incorporated by [2+3]Huisgen cyclization, also known as click chemistry; β-alkyl glycosides such as 2-deoxyhexapyranoses (2-deoxyglucose, 2-deoxyglucuronide, etc.); and β-alkylmannopyranosides. Examples of PEG groups include those of specific lengths ranging from about 4 to about 20 PEG groups. Examples of alkyl sulfate esters can also be introduced directly into the main chain by click chemistry. Examples of oligoamide spacers include EDTA and DTPA spacers, β-amino acids, etc.

[0109] In another embodiment, the polyvalent linker L is given by the following formula: TIFF2023098946000058.tif37144 TIFF2023098946000059.tif17154[In the formula, m is an integer chosen independently in each case from 1 to about 8; p is an integer chosen from 1 to about 10; and n is an integer chosen independently in each case from 1 to about 3] This includes one or more polyethers, such as linkers indicated by . In one embodiment, m is independently 1 to about 3 in each case. In another embodiment, n is 1 in each case. In yet another embodiment, p is independently 4 to about 6 in each case. Exemplarily, corresponding to the foregoing, the corresponding polypropylene polyether is intended herein and may be included in the conjugate as a hydrophilic spacer linker. Furthermore, it is understood that a mixture of polyethylene polyether and polypropylene polyether may be included in the conjugate as a hydrophilic spacer linker. Furthermore, cyclic variations of the aforementioned polyether compounds, such as tetrahydrofuranyl, 1,3-dioxane, 1,4-dioxane, etc., are intended herein.

[0110] In another embodiment, the polyvalent linker L contains multiple hydroxyl functional groups, such as linkers incorporating monosaccharides, oligosaccharides, polysaccharides, etc. It should be understood that the polyhydroxyl-containing spacer linker contains multiple -(CROH)- groups (where R is hydrogen or alkyl).

[0111] In another embodiment, the polyvalent linker L is one or more of the following fragments: TIFF2023098946000060.tif2526 TIFF2023098946000061.tif3137 TIFF2023098946000062.tif3036 TIFF2023098946000063.tif2739 TIFF2023098946000064.tif2845 TIFF2023098946000065.tif3149 TIFF2023098946000066.tif2646 TIFF2023098946000067.tif2747 TIFF2023098946000068.tif2746[wherein R is H, alkyl, cycloalkyl or arylalkyl, m is an integer from 1 to about 3, n is an integer from 1 to about 5 or from 2 to about 5, p is an integer from 1 to about 5, and r is an integer selected from 1 to about 3] This includes: In one embodiment, the integer n is 3 or 4. In another embodiment, the integer p is 3 or 4. In yet another embodiment, the integer r is 1.

[0112] In another embodiment, the polyvalent linker L is the following fragment: TIFF2023098946000069.tif2526 TIFF2023098946000070.tif2334 TIFF2023098946000071.tif2443[wherein R is H, alkyl, cycloalkyl or arylalkyl, m is an integer from 1 to about 3, n is an integer from 1 to about 5 or from 2 to about 5, p is an integer from 1 to about 5, and r is an integer selected from 1 to about 3] This includes: In one embodiment, the integer n is 3 or 4. In another embodiment, the integer p is 3 or 4. In yet another embodiment, the integer r is 1.

[0113] In another embodiment, the polyvalent linker L is one or more of the following cyclic polyhydroxy groups: TIFF2023098946000072.tif32150 TIFF2023098946000073.tif32150 TIFF2023098946000074.tif32150 TIFF2023098946000075.tif32150 TIFF2023098946000076.tif32150 TIFF2023098946000077.tif32150[In the formula, n is an integer between 2 and approximately 5, p is an integer between 1 and approximately 5, and r is an integer between 1 and approximately 4] This includes: In one embodiment, the integer n is 3 or 4. In another embodiment, the integer p is 3 or 4. In yet another embodiment, the integer r is 2 or 3. It is understood that all stereochemical forms of such a part of the linker are intended herein. For example, in the above formula, this part can be derived from ribose, xylose, glucose, mannose, galactose or other sugars, while retaining the stereochemical configuration of the side-chain hydroxyl and alkyl groups present in these molecules. Furthermore, it should be understood that a variety of deoxy compounds are also intended in the above formula. Exemplarily, compounds of the following formulas are intended: TIFF2023098946000078.tif39150[In each expression, n is equal to or less than r; for example, if r is 2 or 3, then n is 1 or 2, or 1, 2, 3]

[0114] In another embodiment, the polyvalent linker L is given by the following formula: TIFF2023098946000079.tif2440[In the formula, n and r are integers chosen from approximately 1 to 3, respectively] It contains one or more polyhydroxyl radicals represented by . In one embodiment, the linker is one or more of the following formulas: This includes polyhydroxyl compounds represented by TIFF2023098946000080.tif21153.

[0115] It is understood that all stereochemical forms of such parts of the linker are contemplated herein. For example, in the above formula, this part can be derived from ribose, xylose, glucose, mannose, galactose, or other sugars, while retaining the stereochemical configuration of the side-chain hydroxyl and alkyl groups present in these molecules.

[0116] In another embodiment, the polyvalent linker L comprises one or more polyhydroxyl groups detached from the linker's main chain. In one embodiment, such carbohydrate or polyhydroxyl groups are linked to the main chain by a triazole group, forming a triazole-bonded hydrophilic spacer linker. Exemplarily, the linker is given by the following formula: TIFF2023098946000081.tif52103[In the formula, n, m, and r are integers, each independently chosen from 1 to approximately 5] It includes the fragment represented by . In one exemplary embodiment, m is independently 2 or 3 in each case. In another embodiment, r is 1 in each case. In yet another embodiment, n is 1 in each case. In one variant embodiment, the group linking the polyhydroxyl group to the main chain of the linker is a different heteroaryl group, such as, but not limited to, pyrrole, pyrazole, 1,2,4-triazole, furan, oxazole, isoxazole, thienyl, thiazole, isothiazole, oxadiazole, etc. Similarly, a divalent six-membered ring heteroaryl group is intended. Other variant embodiments of the above exemplary hydrophilic spacer linker are given by the following formula: TIFF2023098946000082.tif6195[In the formula, n and r are integers, each independently chosen from 1 to about 5 in each case, and p is an integer chosen from 1 to about 4] This includes oxyalkylene groups such as those mentioned above.

[0117] In another embodiment, the polyvalent linker L comprises one or more carbohydrate groups or polyhydroxyl groups linked to the main chain by an amide group, forming an amide-bonded hydrophilic spacer linker. Exemplarily, such a linker is given by the following formula: TIFF2023098946000083.tif39150[In the formula, n is an integer selected from 1 to approximately 3, and m is an integer selected from 1 to approximately 22] It encompasses the fragment represented by . In one exemplary embodiment, n is 1 or 2. In another exemplary embodiment, m is selected from about 6 to about 10, and is exemplary 8. In one variant embodiment, the group linking the polyhydroxyl group to the main chain of the linker is a different functional group, such as, but not limited to, esters, ureas, carbamates, and acylhydrazones. Similarly, cyclic variant embodiments are also considered. Other variant embodiments of the above exemplary hydrophilic spacer linker are given by the following formula: It contains oxyalkylene groups as shown in TIFF2023098946000084.tif51150. [In the formula, n and r are integers, each independently chosen from 1 to about 5 in each case, and p is an integer chosen from 1 to about 4.]

[0118] In another embodiment, the polyvalent linker L is one or more of the following fragments: TIFF2023098946000085.tif45150 TIFF2023098946000086.tif102150 TIFF2023098946000087.tif89150[wherein R is H, alkyl, cycloalkyl or arylalkyl, m is an integer independently chosen from 1 to about 3, n is an integer from 1 to about 6, p is an integer from 1 to about 5, and r is an integer chosen from 1 to about 3] This includes: In one modified embodiment, the integer n is 3 or 4. In another modified embodiment, the integer p is 3 or 4. In yet another modified embodiment, the integer r is 1.

[0119] In another embodiment, the polyvalent linker L is one or more of the following fragments: TIFF2023098946000088.tif39150[wherein R is H, alkyl, cycloalkyl or arylalkyl, m is an integer independently chosen from 1 to about 3, n is an integer from 2 to about 6, p is an integer from 1 to about 5, and r is an integer chosen from 1 to about 3] This includes: In one modified embodiment, the integer n is 3 or 4. In another modified embodiment, the integer p is 3 or 4. In yet another modified embodiment, the integer r is 1.

[0120] In another embodiment, the polyvalent linker L is one or more of the following fragments: TIFF2023098946000089.tif51150 TIFF2023098946000090.tif83150 TIFF2023098946000091.tif89150[In the formula, m is an integer independently chosen from 1 to approximately 3, n is an integer from 1 to approximately 6, p is an integer from 1 to approximately 5, and r is an integer chosen from 1 to approximately 3] This includes: In one modified embodiment, the integer n is 3 or 4. In another modified embodiment, the integer p is 3 or 4. In yet another modified embodiment, the integer r is 1.

[0121] In another embodiment, the polyvalent linker L is one or more of the following fragments: TIFF2023098946000092.tif45150[In the formula, m is an integer independently chosen from 1 to approximately 3, n is an integer from 2 to approximately 6, p is an integer from 1 to approximately 5, and r is an integer chosen from 1 to approximately 3] This includes: In one modified embodiment, the integer n is 3 or 4. In another modified embodiment, the integer p is 3 or 4. In yet another modified embodiment, the integer r is 1.

[0122] In another embodiment, the polyvalent linker L is one or more of the following fragments: TIFF2023098946000093.tif51150 TIFF2023098946000094.tif51150 TIFF2023098946000095.tif45150[In the formula, m is an integer independently chosen from 1 to approximately 3, n is an integer from 1 to approximately 6, p is an integer from 1 to approximately 5, and r is an integer chosen from 1 to approximately 3] This includes: In one modified embodiment, the integer n is 3 or 4. In another modified embodiment, the integer p is 3 or 4. In yet another modified embodiment, the integer r is 1.

[0123] In another embodiment, the polyvalent linker L is connected to the main chain and the following formula: This is a combination with a branched side-chain motif, as exemplified by TIFF2023098946000096.tif25149 [wherein n is an integer independently chosen from 0 to about 3 in each case]. The above formula is intended to represent cyclic sugars with 4, 5, 6, and more members. Furthermore, the above formula can be modified to represent deoxy sugars, where it should be understood that one or more hydroxyl groups present in the formula are substituted with hydrogen, alkyl, or amino. Furthermore, it should be understood that the above formula is intended to represent a corresponding carbonyl compound in which one or more hydroxyl groups are oxidized to the corresponding carbonyl. Furthermore, in this exemplary embodiment, pyranose contains both carboxyl and amino functional groups and can (a) be inserted into the main chain and (b) provide a synthetic handle to the branched side chain in a variant of this embodiment. Any of the side-chain hydroxyl groups can be used to attach other chemical fragments, including additional sugars, to prepare the corresponding oligosaccharides. Other variations of this embodiment are also contemplated, including the insertion of pyranose or other sugars into the main chain at a single carbon, i.e., spiro configurations and similar configurations at paired carbons where two identical atoms are bonded to one atom. For example, one or two ends of a linker or drug D or binding ligand B can be linked to the sugar and inserted into the main chain in 1,1;1,2;1,3;1,4;2,3 or other configurations.

[0124] In another embodiment, the hydrophilic spacer linker described herein is formed primarily from carbon, hydrogen, and nitrogen and has a carbon / nitrogen ratio of about 3:1 or less or about 2:1 or less. In one embodiment, the hydrophilic linker described herein comprises a plurality of amino functional groups.

[0125] In another embodiment, the polyvalent linker L is one or more of the following formulas: TIFF2023098946000097.tif1964 TIFF2023098946000098.tif2170 TIFF2023098946000099.tif1876 TIFF2023098946000100.tif20106 TIFF2023098946000101.tif22157 TIFF2023098946000102.tif31150[In the formula, n is an integer independently selected from 1 to approximately 3 in each case] It contains an amino group represented by . In one embodiment, the integer n is 1 or 2 in each case. In another embodiment, the integer n is 1 in each case.

[0126] In another embodiment, the polyvalent linker L is a sulfate ester, such as an alkyl ester of sulfate. Exemplarily, the linker is given by the following formula: TIFF2023098946000103.tif53107[In the formula, n is an integer independently selected from 1 to approximately 3 in each case] It includes. For example, n is independently 1 or 2 in each case.

[0127] In linkers such as polyhydroxyl, polyamino, carboxylic acid, and sulfuric acid, which contain free hydrogen atoms bonded to heteroatoms, one or more free hydrogen atoms may be protected with appropriate hydroxyl, amino, or acid protecting groups, respectively, or blocked as corresponding prodrugs, the latter of which are selected for specific uses, such as prodrugs that release the parent drug under general or specific physiological conditions.

[0128] In another embodiment, the polyvalent linker is one or more of the following divalent radicals: TIFF2023098946000104.tif39150 TIFF2023098946000105.tif39150[In the formula, as stated above, n is an integer between 2 and approximately 5, p is an integer between 1 and approximately 5, and r is an integer between 1 and approximately 4] Includes.

[0129] In the embodiments described above, it should be further understood that the open sites, such as the (*) atom, are the binding sites for the binding ligand (B) or the delivered drug (D). Furthermore, it should be understood that the bond between B and any D, or both, may be direct or mediated by an intervening linker containing one or more radicals described herein. In addition, the (*) atom may form a releaseable linker having any drug D, or form other parts of linker L.

[0130] In another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxyl group-containing linkers. In yet another embodiment, the hydrophilic spacer linker comprises at least three carbohydrate-containing or polyhydroxyl group-containing linkers. In yet another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxyl group-containing linkers and one or more aspartic acid. In yet another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxyl compound group-containing linkers and one or more glutamic acid. In yet another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxyl group-containing linkers, one or more glutamic acid, one or more aspartic acid, and one or more beta-aminoalanine. In a series of modified embodiments, in each of the embodiments described above, the hydrophilic spacer linker also comprises one or more cysteine. In yet another series of modified embodiments, in each of the embodiments described above, the hydrophilic spacer linker also comprises at least one arginine.

[0131] In another embodiment, the polyvalent linker L includes one or more divalent 1,4-piperazine-containing hydrophilic spacer linkers that are part of a chain of atoms linking at least one binding ligand (L) to at least one drug (D). In one modified embodiment, the hydrophilic spacer linker includes one or more carbohydrate-containing or polyhydroxyl group-containing linkers. In another modified embodiment, the hydrophilic spacer linker includes one or more carbohydrate-containing or polyhydroxyl group-containing linkers and one or more aspartic acid. In yet another modified embodiment, the hydrophilic spacer linker includes one or more carbohydrate-containing or polyhydroxyl group-containing linkers and one or more glutamic acid. In a series of modified embodiments, in each of the embodiments described above, the hydrophilic spacer linker also includes one or more cysteine. In yet another series of modified embodiments, in each of the embodiments described above, the hydrophilic spacer linker also includes at least one arginine.

[0132] In another embodiment, the hydrophilic spacer linker comprises one or more oligoamide hydrophilic spacers, such as, but not limited to, aminoethylpiperazinylacetamide.

[0133] In another embodiment, the polyvalent linker L comprises a hydrophilic spacer linker containing one or more triazole-bonded carbohydrates or polyhydroxyl group-containing linkers. In another embodiment, the hydrophilic spacer linker comprises one or more amide-bonded carbohydrates or polyhydroxyl group-containing linkers. In another embodiment, the hydrophilic spacer linker comprises one or more PEG groups and one or more cysteine. In another embodiment, the hydrophilic spacer linker comprises one or more EDTE derivatives.

[0134] In another embodiment, the polyvalent linker L is given by formula: TIFF2023098946000106.tif6686[In the formula, * indicates a binding site to folic acid, ** indicates a binding site to the drug; F and G are independently 1, 2, 3, or 4] It contains the divalent radical shown by .

[0135] In another embodiment, the polyvalent linker L is given by formula: TIFF2023098946000107.tif6799 [In the formula, *, **, and *** represent the folate receptor binding site B and the binding site to one or more drugs D, respectively.] It contains the trivalent radical represented by . It should be understood that, if the drug is less, *, **, *** are substituted with hydrogen or heteroatoms. F and G are independently 1, 2, 3, or 4; W1 is NH or O. In another embodiment, m1 is 0 or 1.

[0136] In any embodiment described herein, heteroatom linkers such as -NR1R2-, oxygen, sulfur, and heteroatom linkers of formula:-(NHR1NHR2)-, -SO-, -(SO2)-, and -N(R3)O- [where R1, R2, and R3 are each independently selected from hydrogen, alkyl, aryl, arylalkyl, substituted aryl, substituted arylalkyl, heteroaryl, substituted heteroaryl, and alkoxyalkyl] may also be incorporated into the polyvalent linker L. It should be understood that the heteroatom linker may be used to covalently bind to any radical described herein, such as a drug radical D to the polyvalent linker, a ligand radical B to the polyvalent linker, or various di and polypolyvalent radicals from the polyvalent linker.

[0137] Exemplary further divalent radicals that can be used to form the linker portion are as follows: TIFF2023098946000108.tif117138

[0138] TIFF2023098946000109.tif155145

[0139] TIFF2023098946000110.tif195140

[0140] In another embodiment, the polyvalent linker L is a releaseable linker.

[0141] As used herein, the term “releaseable linker” means a linker that contains at least one bond that can be cleaved under physiological conditions when the compounds described herein are delivered to or into target cells. The linker itself may contain one or more cleavable, easily cleavable, or destructible bonds, or may form one or more cleavable, easily cleavable, or destructible bonds with the PSMA-binding ligand (B) and / or with one or more drugs (D). However, it is understood that the releaseable linkers described herein are not conveniently cleavable, easily cleavable, or destructible until the conjugate containing the releaseable linker reaches the intended target site or its vicinity. Therefore, the releaseable linkers described herein generally do not contain linkers that have bonds that are substantially cleavable, easily cleavable, or destructible under non-target conditions or in non-target tissues. Similarly, the releaseable linkers described herein do not contain linkers that have bonds that are substantially only cleavable, easily cleavable, or destructible under non-physiological conditions.

[0142] The term “releaseable linker” does not generally mean simply in vivo unstable binding to serum, plasma, gastrointestinal tract, or liver, unless these systems are targeted by cell surface receptor-binding ligands. However, after delivery and / or selective targeting, releaseable linkers may be cleaved under physiological conditions, such as by having one or more pH-unstable, acid-unstable, base-unstable, oxidatively unstable, metabolically unstable, biochemically unstable, and / or enzymatically unstable bindings, by any process involving at least one cleavable binding in the linker or in the covalent binding of the linker to B or any D. It is understood that such physiological conditions resulting in binding cleavage do not necessarily involve biological or metabolic processes, but rather may include standard chemical reactions, such as hydrolysis reactions occurring at physiological pH or as a result of compartmentalization into cellular organelles such as endosomes, which have a pH lower than cytoplasmic pH.

[0143] A cleavable bond is understood to be one that can link two adjacent atoms within a releaseable linker and / or link another linker or B and / or D as described herein at either end of the releaseable linker. If a cleavable bond links two adjacent atoms within a releaseable linker, after the bond is broken, the releaseable linker is divided into two or more fragments. Alternatively, if a cleavable bond is between a releaseable linker and another part such as an additional heteroatom, a spacer linker, another releaseable part of the linker, any D, or B, after the bond is broken, the releaseable linker is separated from the other part. A linker is a releaseable linker if it can deliver one or more drugs (D) in a manner that leaves no trace if it forms a cleavable, easily cleavable, or destructible bond with one or more drugs (D), where it should be understood that the one or more drugs (D) do not include any residual parts of the conjugate.

[0144] Exemplary radicals that contain a bond that can be cleaved by themselves, or that form a bond that can be cleaved with B and / or any D, include hemiacetals and their sulfur variations, acetals and their sulfur variations, hemiaminals, aminals, etc., or can be formed from methylene fragments substituted with at least one heteroatom, such as 1-alkoxyalkylenes, 1-alkoxycycloalkylenes, 1-alkoxyalkylene carbonyls, and 1-alkoxycycloalkylene carbonyls. Exemplary releaseable linkers described herein include polyvalent linkers such as carbonylarylcarbonyls, carbonyl(carboxyaryl)carbonyls, and carbonyl(biscarboxyaryl)carbonyls. Exemplary releaseable linkers described herein include polyvalent linkers such as alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl. Exemplary releaseable linkers described herein include oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, and oxysulfonylalkyl. Exemplary releaseable linkers described herein include polyvalent linkers such as iminoalkylidenyl, carbonylalkylideneiminyl, iminocycloalkylidenyl, and carbonylcycloalkylideneiminyl. Exemplary releaseable linkers described herein include polyvalent linkers such as alkylenthio, alkylenearylthio, and carbonylalkylthio. Each of the aforementioned fragments is optionally substituted with substituent X2 as defined herein.

[0145] The substituent X2 can be alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxyalkyl, alkylcarboxylate, alkylalkanoate, guanidinoalkyl, R4-carbonyl, R5-carbonylalkyl, R6-acylamino, and R7-acylaminoalkyl, where R4 and R5 are independently selected from amino acids, amino acid derivatives, and peptides, and R6 and R7 are independently selected from amino acids, amino acid derivatives, and peptides. In this embodiment, the heteroatom linker may contain nitrogen, and the substituent X2 and the heteroatom linker, together with the releaseable linker to which they are bound, form a heterocycle.

[0146] The heterocycles can be pyrrolidine, piperidine, oxazolidine, isoxazolidine, thiazolidinone, isothiazolidine, pyrrolidinenon, piperidinone, oxazolidinone, isoxazolidinone, thiazolidinone, isothiazolidinone, and succinimide.

[0147] Exemplary releaseable linkers include ketals, acetals, hemiaminals and aminals formed from methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylene carbonyl and 1-alkoxycycloalkylene carbonyl radicals, each optionally substituted; esters and amides formed from carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl and haloalkylene carbonyl radicals; alkylene(dialkylsilyl), a Oxysilanes and aminosilanes formed from alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl and (diarylsilyl)aryl radicals; including oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl, iminoalkylidenyl, carbonylalkylideneiminyl, iminocycloalkylidenyl, carbonylcycloalkylideneiminyl, alkylenthio, alkylenearylthio and carbonylalkylthio radicals.

[0148] Further exemplary linkers include hydrazones, acylhydrazone orthoformate, and carbamoyl derivatives.

[0149] Further exemplary linkers include disulfides and activated thioethers.

[0150] In any embodiment described herein, the releaseable linker may contain oxygen bonded to methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylene carbonyl, and 1-alkoxycycloalkylene carbonyl to form an acetal or ketal, each fragment optionally substituted with substituent X2 as defined herein. Alternatively, the methylene or alkylene may be substituted with an optionally substituted aryl.

[0151] In any embodiment described herein, the releaseable linker may contain nitrogen atoms bonded to methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylene carbonyl, and 1-alkoxycycloalkylene carbonyl to form a hemiaminal ether or aminal, each fragment optionally substituted with substituent X2 as defined herein. Alternatively, the methylene or alkylene may be substituted with optionally substituted aryl atoms.

[0152] In any embodiment described herein, the releaseable linker may contain oxygen that bonds to a sulfonyl alkyl to form an alkyl sulfonate.

[0153] In any embodiment described herein, the releaseable linker may contain nitrogen atoms bonded to iminoalkylidenyl, carbonylalkylideneiminyl, iminocycloalkylidenyl, and carbonylcycloalkylideneiminyl to form hydrazones, each optionally substituted with substituent X2 as defined herein. Alternative configurations include acylation of the hydrazone with carboxylic acid derivatives, orthoformic acid derivatives, or carbamoyl derivatives to form releaseable linkers containing a variety of acylhydrazones.

[0154] In any embodiment described herein, the releaseable linker may contain oxygen bonded to alkylene(dialkylsilyl), alkylene(alkylarylsilyl), (dialkylsilyl)aryl, alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl to form a silanol, each optionally substituted with substituent X2 as defined herein.

[0155] In any embodiment described herein, the releaseable linker may contain nitrogen that binds to carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, or carbonyl(biscarboxyaryl)carbonyl to form an amide or an amide having a drug nitrogen.

[0156] In any embodiment described herein, the releaseable linker may contain nitrogen that binds to carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, or carbonyl(biscarboxyaryl)carbonyl to form an ester or an ester with a drug oxygen.

[0157] It should be understood that divalent spacer linkers may be combined in any chemically relevant manner, directly or via interrupted heteroatoms, to construct the releaseable linkers described herein. Furthermore, it should be understood that the nature of the arrangement of the spacer and heteroatom linkers determines where the releaseable linker is cleaved in vivo. For example, two spacer linkers terminating at a sulfur atom, when combined, form a disulfide, which is the cleavable bond in the thus formed releaseable linker.

[0158] For example, in another embodiment, the polyvalent linker comprises a 3-thiosuccinimido-1-ylalkyloxymethyloxy moiety, where methyl is optionally substituted with an alkyl or substituted aryl.

[0159] In another embodiment, the polyvalent linker comprises a 3-thiosuccinimide-1-ylalkylcarbonyl, where the carbonyl forms an acylarizidine with the drug.

[0160] In another embodiment, the polyvalent linker includes a 1-alkoxycycloalkylene oxy moiety.

[0161] In another embodiment, the polyvalent linker comprises alkylene aminocarbonyl (dicarboxylarylene) carboxylate.

[0162] In another embodiment, the polyvalent linker comprises a dithioalkylcarbonyl hydrazide, where the hydrazide forms a hydrazone with the drug.

[0163] In another embodiment, the polyvalent linker comprises a 3-thiosuccinimido-1-ylalkylcarbonylhydrazide, where the hydrazide forms a hydrazone with the drug.

[0164] In another embodiment, the polyvalent linker comprises a 3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where the disubstituted silyl is substituted with an alkyl or optionally substituted with an aryl.

[0165] In another embodiment, the polyvalent linker includes a plurality of additional spacer linkers selected from natural amino acids and their stereoisomers.

[0166] In another embodiment, the polyvalent linker comprises a 2-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with the drug.

[0167] In another embodiment, the polyvalent linker comprises a 2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate with the drug and the aryl is optionally substituted.

[0168] In another embodiment, the polyvalent linker comprises a 4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate with the drug and the aryl is optionally substituted.

[0169] In another embodiment, the polyvalent linker comprises a 3-thiosuccinimod-1-ylalkyloxyalkyloxyalkylidene, where the alkylidene forms a hydrazone with the drug, each alkyl is independently selected, and the oxyalkyloxy is optionally substituted with an alkyl or optionally substituted with an aryl.

[0170] In another embodiment, the polyvalent linker comprises a 2-dithioalkyloxycarbonylhydrazide.

[0171] In another embodiment, the polyvalent linker comprises a 2- or 3-dithioalkylamino, where the amino forms a vinyl amide with the drug.

[0172] In another embodiment, the polyvalent linker comprises a 2-dithioalkylamino, where the amino forms a vinyl amide with the drug, and the alkyl is ethyl.

[0173] In another embodiment, the polyvalent linker comprises a 2- or 3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate with the drug.

[0174] In another embodiment, the polyvalent linker comprises 2-dithioalkylaminocarbonyl, where carbonyl forms a carbamate with the drug. In yet another embodiment, alkyl is ethyl.

[0175] In another embodiment, the polyvalent linker comprises a 2-dithioalkyloxycarbonyl, where the carbonyl forms a carbamate with the drug. In yet another embodiment, the alkyl is ethyl.

[0176] In another embodiment, the polyvalent linker comprises a 2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or carbamoylaziridine with the drug.

[0177] In another embodiment, the polyvalent linker comprises a 4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or carbamoylaziridine with the drug.

[0178] In another embodiment, the polyvalent linker described herein is of the formula: TIFF2023098946000111.tif22150 TIFF2023098946000112.tif22150[wherein n is an integer selected from 1 to about 4; Ra and Rb are each independently selected from hydrogen and alkyl groups such as lower alkyl groups such as optionally branched C1-C4 alkyl groups; or Ra and Rb together with the carbon atoms they bond to form a carbocyclic ring; R is an optionally substituted alkyl group, an optionally substituted acyl group or a suitably selected nitrogen protecting group; (*) indicates a bonding site for a drug, vitamin, contrast agent, diagnostic agent, other divalent linker or other part of the conjugate] It contains a divalent radical represented by .

[0179] In another embodiment, the polyvalent linker described herein is of the formula: TIFF2023098946000113.tif22150 TIFF2023098946000114.tif22150[wherein m is an integer selected from 1 to about 4; R is an optionally substituted alkyl group, an optionally substituted acyl group, or a suitably selected nitrogen protecting group; (*) indicates a bond site for a drug, vitamin, contrast agent, diagnostic agent, other divalent linker, or other part of the conjugate] It contains a divalent radical represented by .

[0180] In another embodiment, the polyvalent linker described herein is of the formula: TIFF2023098946000115.tif20150 TIFF2023098946000116.tif22150[wherein m is an integer selected from 1 to about 4; R is an optionally substituted alkyl group, an optionally substituted acyl group, or a suitably selected nitrogen protecting group; (*) indicates a bond site for a drug, vitamin, contrast agent, diagnostic agent, other divalent linker, or other part of the conjugate] It contains a divalent radical represented by .

[0181] In another embodiment, the compound described herein is of formula: TIFF2023098946000117.tif13150 TIFF2023098946000118.tif20150 TIFF2023098946000119.tif20150[In the formula, X is NH, O, or S] It includes one or more radical linkers selected from the following.

[0182] In another embodiment, the polyvalent linker described herein is of the formula: Contains a radical with the data type TIFF2023098946000120.tif26150.

[0183] In another embodiment, the polyvalent linker described herein is of the formula: TIFF2023098946000121.tif1550[In the formula, X is a heteroatom such as nitrogen, oxygen, or sulfur, n is an integer selected from 0, 1, 2, and 3, R is a substituent such as hydrogen or an alkoxy which has the ability to inductively stabilize a positive charge or by resonance on an aryl ring, and the symbol (*) indicates a bond site] It contains radicals having the following properties. It is understood that other substituents, such as (but not limited to) hydroxy, alkyl, alkoxy, alkylthio, and halo, may be present on the aryl ring, benzyl carbon, alkanoic acid, or methylene bridge.

[0184] In another embodiment, the polyvalent linker described herein comprises a radical selected from carbonyl, thionocarbonyl, alkylene, cycloalkylene, alkylene cycloalkyl, alkylene carbonyl, cycloalkylene carbonyl, carbonylalkylcarbonyl, 1-alkylene succinimido-3-yl, 1-(carbonylalkyl)succinimido-3-yl, alkylene sulfoxyl, sulfonylalkyl, alkylene sulfoxylalkyl, alkylene sulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl)succinimido-3-yl, and 1-(carbonyltetrahydrofuranyl)succinimido-3-yl, where each spacer linker is optionally substituted with one or more substituents X1; Here, each substituent X1 is independently selected from alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxyalkyl, alkylcarboxylate, alkylalkanoate, guanidinoalkyl, R4-carbonyl, R5-carbonylalkyl, R6-acylamino, and R7-acylaminoalkyl, where R4 and R5 are independently selected from amino acids, amino acid derivatives, and peptides, and R6 and R7 are independently selected from amino acids, amino acid derivatives, and peptides.

[0185] It should be understood that the compounds described herein may contain one or more chiral centers or may exist as multiple stereoisomers. In one embodiment, it should be understood that the invention described herein is not limited to specific stereochemical requirements, and that the compounds, and compositions, methods, uses, and pharmaceuticals containing them may be optically pure or any of various stereoisomer mixtures, such as other mixtures of racemic and enantiomers, other mixtures of diastereomers, etc. It should also be understood that such a mixture of stereoisomers may contain a single stereochemical configuration at one or more chiral centers, or a mixture of stereochemical configurations at one or more other chiral centers.

[0186] Similarly, the compounds described herein may contain geometric centers such as cis, trans, E, and Z double bonds. In another embodiment, it should be understood that the inventions described herein are not limited to specific phytochemical isomer requirements, and that the compounds, and compositions, methods, uses, and pharmaceuticals containing them, may be either pure or a mixture of various geometric isomers. It should also be understood that such a mixture of geometric isomers may contain a single stereochemistry in one or more double bonds, or a mixture of geometric isomers in one or more other double bonds.

[0187] In each of the embodiments described above and below, it should be understood that the formula not only encompasses and represents pharmaceutically acceptable salts of the compound, but also encompasses all hydrates and / or solvates of the compound. It is understood that certain functional groups, such as hydroxyl and amino, form complexes and / or coordination compounds with water and / or various solvents in various physical forms of the compound. Therefore, the formula described above should be understood as describing such hydrates and / or solvates, such as pharmaceutically acceptable solvates.

[0188] In each of the embodiments described above and below, it should be understood that the formula encompasses and represents each possible isomer, such as stereoisomers and geometric isomers, individually and in all possible mixtures. In each of the embodiments described above and below, it should be understood that the formula encompasses and represents all crystalline, partially crystalline, non-crystalline, and / or amorphous forms of the compound, as well as cocrystals.

[0189] In another embodiment, the compounds described herein are internalized into target pathogenic cells by binding to PSMA. More specifically, PSMA selectively and / or specifically binds to the conjugate, and internalization may occur, for example, via PSMA-mediated endocytosis. Once internalized, the conjugate, containing a releaseable linker, can complete the delivery of the drug into the target cell. While we do not wish to be bound to any particular theory, if the drug is toxic to normal cells or tissues, such a delivery system is thought to reduce toxicity to non-target cells and tissues, since the releaseable linker remains substantially or completely intact until the compounds described herein are delivered to the target cells. Thus, the compounds described herein act intracellularly by delivering the drug to intracellular biochemical processes, reducing the amount of exposure of the unbound drug to healthy cells and tissues of non-host animals.

[0190] The drug delivery conjugates described herein can be used for both human clinical medicine and veterinary applications. Therefore, the host animal harboring a population of pathogenic cells and to be treated with the compounds described herein may be human, or, in the case of veterinary applications, laboratory animals, agricultural animals, domestic animals, or wild animals. The present invention can be applied to host animals including, but are not limited to, humans, laboratory animals such as rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees, etc., domestic animals such as dogs, cats, and rabbits, etc., agricultural animals such as cattle, horses, pigs, sheep, goats, etc., and confined wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.

[0191] The drug delivery conjugate compounds described herein may be administered in combination therapy with any other known drug, whether or not the drug is targeted. Exemplary additional drugs include peptides, oligopeptides, retro-inverso oligopeptides, proteins, protein analogs in which at least one non-peptide bond substitutes a peptide bond, apolipoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, receptors, and other membrane proteins; antigens and antibodies against them; haptens and antibodies against them; hormones, lipids, phospholipids, liposomes; toxins; antibiotics; analgesics; bronchodilators; beta-blockers; antibacterial agents; antihypertensive agents; circulatory agents, for example. Examples include, but are not limited to, antiarrhythmics, glycoside cardiovascular drugs, antianginal drugs, and vasodilators; central nervous system drugs, such as stimulants, psychotropic drugs, antimanic drugs, and depressants; antiviral drugs; antihistamines; cancer drugs, such as chemotherapeutic agents; tranquilizers; antidepressants; H-2 antagonists; anticonvulsants; antiemetics; prostaglandins and prostaglandin analogs; muscle relaxants; anti-inflammatory substances; stimulants; decongestants; antiemetics; diuretics; antispasmodics; antiasthmatics; antiparkinsonian drugs; expectorants; antitussives; mucolytics; and minerals and nutritional additives.

[0192] As used herein, the term “alkyl” encompasses optionally branched chains of carbon atoms. As used herein, the terms “alkenyl” and “alkynyl” encompass optionally branched chains of carbon atoms, each containing at least one double or triple bond. It should be understood that an alkynyl may contain one or more double bonds. It should also be understood that in certain embodiments, alkyls of limited lengths such as C1-C24, C1-C12, C1-C8, C1-C6 and C1-C4, and C2-C24, C2-C12, C2-C8, C2-C6 and C2-C4 are advantageous. Exemplarily, alkyls of particularly limited lengths such as C1-C8, C1-C6 and C1-C4, and C2-C8, C2-C6 and C2-C4 are referred to as lower alkyls. In certain embodiments, it should be understood that alkenyl and / or alkynyl groups of limited lengths, such as C2-C24, C2-C12, C2-C8, C2-C6, and C2-C4, and C3-C24, C3-C12, C3-C8, C3-C6, and C3-C4, are also advantageous. Exemplarily, such alkenyl and / or alkynyl groups of particularly limited lengths, such as C2-C8, C2-C6, and C2-C4, and C3-C8, C3-C6, and C3-C4, are referred to as lower alkenyl and / or alkynyl groups. In this specification, it should be understood that shorter alkyl, alkenyl, and / or alkynyl groups may add lower lipophilicity to the compound and therefore will have different pharmacokinetic behavior. In the embodiments of the invention described herein, it should be understood that in each case, the description of alkyl means alkyl as defined herein, and optionally lower alkyl. In the embodiments of the present invention described herein, it should be understood that in each case, the term "alkenyl" means the alkenyl as defined herein and optionally a lower alkenyl.Examples of alkyl, alkenyl, and alkynyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, heptyl, octyl, and corresponding groups containing one or more double and / or triple bonds or combinations thereof.

[0193] As used herein, the term "alkylene" encompasses a divalent chain of optionally branched carbon atoms. As used herein, the terms "alkenylene" and "alkylene" encompass a divalent chain of optionally branched carbon atoms, each containing at least one double and / or triple bond. It should be understood that alkylene may contain one or more double bonds. It should also be understood that in certain embodiments, alkylenes having limited lengths such as C1-C24, C1-C12, C1-C8, C1-C6 and C1-C4, and C2-C24, C2-C12, C2-C8, C2-C6 and C2-C4 are referred to as lower alkylenes. It should also be understood that in certain embodiments, alkenylene and / or alkynylene groups of limited lengths such as C2-C24, C2-C12, C2-C8, C2-C6, and C2-C4, and C3-C24, C3-C12, C3-C8, C3-C6, and C3-C4, respectively, are advantageous. Exemplarily, such alkenylene and / or alkynylene groups of particularly limited lengths such as C2-C8, C2-C6, and C2-C4, and C3-C8, C3-C6, and C3-C4, are referred to as lower alkenylene and / or alkynylenes. In this specification, it will be understood that shorter alkylene, alkenylene, and / or alkynylene groups may add lower lipophilicity to the compound and therefore will have different pharmacokinetic behavior. In the embodiments of the present invention described herein, it should be understood that in each case, the descriptions of alkylene, alkenylene, and alkynylene mean alkylene, alkenylene, and alkynylene as defined herein, and optionally lower alkylene, alkenylene, and alkynylene. Exemplary alkylenes include, but are not limited to, methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, pentylene, 1,2-pentylene, 1,3-pentylene, hexylene, heptylene, and octylene.

[0194] As used herein, the term "cycloalkyl" encompasses a chain of carbon atoms that is optionally branched and has at least a portion of its structure as a ring. It should be understood that a cycloalkylalkyl is a part of a cycloalkyl. It should be understood that a cycloalkyl may be polycyclic. Exemplary cycloalkyls include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentylethy-2-yl, and adamantyl. As used herein, the term "cycloalkenyl" encompasses a chain of carbon atoms that is optionally branched and contains at least one double bond, and has at least a portion of its structure as a ring. It should be understood that one or more double bonds may be in the cyclic and / or acyclic portions of a cycloalkenyl. It should be understood that cycloalkenylalkyls and cycloalkylalkenyls are, respectively, parts of a cycloalkenyl. It should be understood that a cycloalkyl may be polycyclic. Examples of cycloalkenyls include, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl, and cycloheptenylpropenyl. It should be further understood that it is advantageous for the chain-forming cycloalkyl and / or cycloalkenyl to have limited lengths such as C3-C24, C3-C12, C3-C8, C3-C6, and C5-C6. In this specification, it is understood that shorter alkyl and / or alkenyl chain-forming cycloalkyl and / or cycloalkenyl may each add lower lipophilicity to the compound, and therefore will have different pharmacokinetic behaviors.

[0195] As used herein, the term "heteroalkyl" encompasses both carbon and at least one heteroatom, and is optionally branched. Exemplary heteroatoms include nitrogen, oxygen, and sulfur. In certain modified embodiments, exemplary heteroatoms include phosphorus and selenium. As used herein, the term "cycloheteroalkyl," including heterocyclyl and heterocycle, encompasses both carbon and at least one heteroatom, and is optionally branched, encompassing a chain of atoms in which at least a portion of the chain is cyclic. Exemplary heteroatoms include nitrogen, oxygen, and sulfur. In certain modified embodiments, exemplary heteroatoms include phosphorus and selenium. Exemplary cycloheteroalkyls include, but are not limited to, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, and quinuclidinyl.

[0196] As used herein, the term "aryl" encompasses polycyclic aromatic carbocyclic groups that may be optionally substituted. Examples of aromatic carbocyclic groups described herein include, but are not limited to, phenyl and naphthyl. As used herein, the term "heteroaryl" encompasses aromatic heterocyclic groups that may be optionally substituted. Examples of aromatic heterocyclic groups include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetradinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzoisoxazolyl, and benzisothiazolyl.

[0197] As used herein, the term "amino" encompasses NH2 groups, alkylaminos, and dialkylaminos (wherein the two alkyl groups in a dialkylamino may be the same or different, i.e., alkylalkylaminos). Exemplary examples include methylamino, ethylamino, dimethylamino, methylethylamino, and the like. Furthermore, the above-mentioned variations of the term amino are encompassed when amino is modified, such as aminoalkyl or acylamino, or modified by another term. Exemplary examples of aminoalkyls include H2N-alkyl, methylaminoalkyl, ethylaminoalkyl, dimethylaminoalkyl, and methylethylaminoalkyl. Exemplary examples of acylaminos include acylmethylamino and acylethylamino.

[0198] As used herein, the terms "amino and its derivatives" include, respectively, the aminos described herein, as well as alkylaminos, alkenylaminos, alkynylaminos, heteroalkylaminos, heteroalkenylaminos, heteroalkynylaminos, cycloalkylaminos, cycloalkenylaminos, cycloheteroalkylaminos, cycloheteroalkylaminos, cycloheteroalkenylaminos, arylaminos, arylalkylaminos, arylalkenylaminos, arylalkynylaminos, heteroarylaminos, heteroarylalkylaminos, heteroarylalkenylaminos, heteroarylalkynylaminos, acylaminos, and the like, each optionally substituted. The term "amino derivative" also includes urea, carbamates, and the like.

[0199] As used herein, the term "amino acid" generally refers to beta, gamma, and formula: -N(R)-(CR'R”)qC(O)- [In the formula, R is a hydrogen, alkyl, acyl, or a suitable nitrogen protecting group; R' and R'' are hydrogen or substituents independently chosen from each other in their respective appearances; and q is an integer such as 1, 2, 3, 4, or 5.] It means a longer amino acid such as the amino acid shown by . Exemplarily, each of R’ and / or R” is independently hydrogen or a side chain present in a natural amino acid, such as methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxymethyl, guanidinopropyl, etc., and derivatives and protected derivatives thereof, but is not limited thereto. The above formula includes all stereoisomers. For example, the amino acid may be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, etc.

[0200] As used herein, the term "amino acid derivative" generally means an amino acid as defined herein, where either or both of the amino group and / or the side chain are substituted. Exemplary amino acid derivatives include prodrugs and protecting groups of the amino group and / or the side chain, such as amines, hydroxy, carboxylic acids, and thiopro drugs and protecting groups. Further exemplary amino acid derivatives include substitution variants of the amino acids described herein, such as hydroxy groups, amides, ethers and esters of carbamates, and ureas of amino groups, esters, amides, and cyano derivatives of carboxylic acid groups, but are not limited thereto.

[0201] As used herein, the term "hydroxy and its derivatives" respectively includes optionally substituted OH, and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy, arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy, heteroarylalkenyloxy, heteroarylalkynyloxy, acyloxy, etc. The term "hydroxy derivative" also includes carbamates, etc.

[0202] As used herein, the terms "thio and its derivatives" include, respectively, optionally substituted SH, and alkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio, heteroalkynylthio, cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio, cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenylthio, arylalkynylthio, heteroarylthio, heteroarylalkylthio, heteroarylalkenylthio, heteroarylalkynylthio, acylthio, etc. The term "thio derivative" also includes thiocarbamates, etc.

[0203] As used herein, the term "acyl" includes, however, formyl and alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl, cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl, arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl, heteroarylalkynylcarbonyl, acylcarbonyl, and the like, which may be optionally substituted.

[0204] As used herein, the terms "carbonyl and its derivatives" include the groups C(O), C(S), C(NH) and their substituted amino derivatives.

[0205] As used herein, the term "carboxylic acid and its derivatives" includes the group CO2H and its salts, as well as their esters and amides, and CN.

[0206] As used herein, the term "sulfinic acid or its derivatives" includes SO2H and its salts, as well as its esters and amides.

[0207] As used herein, the term "sulfonic acid or its derivatives" includes SO3H and its salts, as well as its esters and amides.

[0208] The term "sulfonyl" as used herein includes, however, any substituted alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, heteroalkylsulfonyl, heteroalkenylsulfonyl, heteroalkynylsulfonyl, cycloalkylsulfonyl, cycloalkenylsulfonyl, cycloheteroalkylsulfonyl, cycloheteroalkenylsulfonyl, arylsulfonyl, arylalkylsulfonyl, arylalkenylsulfonyl, arylalkynylsulfonyl, heteroarylsulfonyl, heteroarylalkylsulfonyl, heteroarylalkenylsulfonyl, heteroarylalkynylsulfonyl, acylsulfonyl, etc.

[0209] As used herein, the term "phosphinic acid or its derivatives" includes P(R)O2H and its salts, as well as its esters and amides, where R is optionally substituted with alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, cycloheteroalkyl, cycloheteroalkenyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl.

[0210] As used herein, the term "phosphonic acid or its derivatives" includes PO3H2 and its salts, as well as its esters and amides.

[0211] As used herein, the term "hydroxylamino and its derivatives" includes NHOH, as well as optionally substituted alkyloxyl NH, alkenyloxyl NH, alkynyloxyl NH, heteroalkyloxyl NH, heteroalkenyloxyl NH, heteroalkynyloxyl NH, cycloalkyloxyl NH, cycloalkenyloxyl NH, cycloheteroalkyloxyl NH, cycloheteroalkenyloxyl NH, aryloxyl NH, arylalkyloxyl NH, arylalkenyloxyl NH, arylalkynyloxyl NH, heteroaryloxyl NH, heteroarylalkyloxyl NH, heteroarylalkenyloxyl NH, heteroarylalkynyloxyl NH, acyloxy, and the like.

[0212] As used herein, the terms "hydrazino and its derivatives" include alkyl NHNH, alkenyl NHNH, alkynyl NHNH, heteroalkyl NHNH, heteroalkenyl NHNH, heteroalkynyl NHNH, cycloalkyl NHNH, cycloalkenyl NHNH, cycloheteroalkyl NHNH, cycloheteroalkenyl NHNH, aryl NHNH, arylalkyl NHNH, arylalkenyl NHNH, arylalkynyl NHNH, heteroaryl NHNH, heteroarylalkyl NHNH, heteroarylalkenyl NHNH, heteroarylalkynyl NHNH, acyl NHNH, and the like, each of which can be optionally substituted.

[0213] As used herein, the term "optionally substituted" includes the substitution of a hydrogen atom on an optionally substituted radical with another functional group. Examples of such other functional groups include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylhetalkyl, heteroaryl, heteroarylalkyl, heteroarylhetalkyl, nitro, sulfonic acid and its derivatives, carboxylic acid and its derivatives. Exemplarily, any amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylhetalkyl, heteroaryl, heteroarylalkyl, heteroarylhetalkyl and / or sulfonic acid may be optionally substituted.

[0214] As used herein, the terms “optionally substituted aryl” and “optionally substituted heteroaryl” encompass the substitution of a hydrogen atom on an optional aryl or heteroaryl with another functional group. Examples of such other functional groups include, but are not limited to, amino, hydroxy, halo, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylhetalkyl, heteroaryl, heteroarylalkyl, heteroarylhetalkyl, nitro, sulfonic acid and its derivatives, carboxylic acid and its derivatives. Exemplarily, any amino, hydroxy, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylhetalkyl, heteroaryl, heteroarylalkyl, heteroarylhetalkyl and / or sulfonic acid may be optionally substituted.

[0215] Exemplary substituents include radical-(CH2)xZX(where x is an integer from 0 to 6, and ZX is halogen, hydroxy, alkanoyloxy, e.g., C1-C6 alkanoyloxy, optionally substituted alloyloxy, alkyl, e.g., C1-C6 alkyl, alkoxy, e.g., C1-C6 alkoxy, cycloalkyl, e.g., C3-C8 cycloalkyl, cycloalkoxy, e.g., C3-C8 cycloalkoxy, alkenyl, e.g., C2-C6 alkenyl, alkynyl, e.g., C2-C6 alkynyl, haloalkyl, e.g., C1-C6 haloalkyl, haloalkoxy, e.g., C1-C6 haloalkoxy, halocycloalkyl, e.g., C3-C8 halocycloalkyl, halocycloalkoxy, e.g., C3-C8 halocycloalkoxy, amino, C1-C6 alkylamino, (C1-C6 alkyl)(C1-C6 alkyl ) selected from amino, alkylcarbonylamino, N-(C1-C6 alkyl)alkylcarbonylamino, aminoalkyl, C1-C6 alkylaminoalkyl, (C1-C6 alkyl)(C1-C6 alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N-(C1-C6 alkyl)alkylcarbonylaminoalkyl, cyano, and nitro; or ZX may be -CO2R4 and -CONR5R6 (where R4, R5, and R6 are each independently selected from hydrogen, C1-C6 alkyl, aryl-C1-C6 alkyl, and heteroaryl-C1-C6 alkyl, respectively)), but is not limited to these.

[0216] As used herein, the term "leaving group" refers to a reactive functional group that generates an electrophilic site on an atom, to which a nucleophile can be attached in such a way that an electrophile can be added. Examples of leaving groups include, but are not limited to, halogens, optionally substituted phenols, acyloxy groups, and sulfonoxy groups. Such leaving groups may also be alkyls, acyls, etc. Such leaving groups may also be referred to as activating groups in this specification, such as when the leaving group is on an acyl. Furthermore, though not limited to these examples, conventional peptide, amide, and ester coupling agents such as PyBop, BOP-Cl, BOP, pentafluorophenol, and isobutylchloroformate form a variety of intermediates containing a leaving group as defined herein on a carbonyl group.

[0217] For example, the term “radical” as used herein with respect to PSMA-binding or targeted ligands and / or independently selected drugs means PSMA-binding or targeted ligands and / or independently selected drugs described herein, wherein one or more hydrogen atoms or atoms or groups such as alkyl groups are removed from a heteroatom to provide a radical for binding to a polyvalent linker L.

[0218] As used herein, the term “prodrug” generally refers to any compound that, when administered to a biological system, produces a biologically active compound as a result of one or more spontaneous chemical reactions, enzyme-catalyzed chemical reactions and / or metabolic chemical reactions, or a combination thereof. In vivo, prodrugs are typically acted upon by enzymes (such as esterases, amidases, and phosphatases), simple biochemistry, or other in vivo processes to release or regenerate a pharmaceutically active drug. This activation may occur via the action of endogenous host enzymes or non-endogenous enzymes administered to the host before, after, or during administration of the prodrug. Further details of prodrugs are described in U.S. Patent No. 5,627,165; and Pathalk et al., Enzymic protecting group techniques in organic synthesis, Stereosel. Biocatal. 775-797 (2000). It is understood that once the goals of targeted delivery, safety, and stability are achieved, the prodrug is conveniently converted back to the original drug, and any remaining open prodrug groups are rapidly removed.

[0219] Prodrugs can be produced from the compounds described herein by attaching a group that is ultimately cleaved in vivo to one or more functional groups present on the compound, such as -OH-, -SH, -CO2H, and -NR2. Exemplary prodrugs include, but are not limited to, carboxylic acid esters when the group is alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, acyloxyalkyl, or alkoxycarbonyloxyalkyl, and hydroxyl, thiol, and amine esters when the attached group is an acyl group, alkoxycarbonyl, aminocarbonyl, phosphate, or sulfate. Exemplary esters, also called active esters, include, but are not limited to, 1-indanyl, N-oxysuccinimide; acyloxyalkyl groups, such as acetoxymethyl, pivaloyloxymethyl, β-acetoxyethyl, β-pivaloyloxyethyl, 1-(cyclohexylcarbonyloxy)prop-1-yl, (1-aminoethyl)carbonyloxymethyl; alkoxycarbonyloxyalkyl groups, such as ethoxycarbonyloxymethyl, α-ethoxycarbonyloxyethyl, β-ethoxycarbonyloxyethyl; dialkylaminoalkyl groups such as dilow alkylaminoalkyl groups, such as dimethylaminomethyl, dimethylaminoethyl, diethylaminomethyl, diethylaminoethyl; 2-(alkoxycarbonyl)-2-alkenyl groups, such as 2-(isobutoxycarbonyl)pento-2-enyl, 2-(ethoxycarbonyl)buto-2-enyl; and lactone groups, such as phthalidyl, dimethoxyphthalidyl, etc.

[0220] Further exemplary prodrugs include chemical moieties such as amide or phosphorus groups that function to increase the solubility and / or stability of the compounds described herein. Further exemplary prodrugs for amino groups include (C3-C20)alkanoyl; halo-(C3-C20)alkanoyl; (C3-C20)alkenoyl; (C4-C7)cycloalkanoyl; (C3-C6)-cycloalkyl(C2-C16)alkanoyl; optionally substituted aroyl, e.g., unsubstituted aroyl or aroyl substituted with 1-3 substituents selected from halogen, cyano, trifluoromethanesulfonyloxy, (C1-C3)alkyl and (C1-C3)alkoxy (each optionally substituted with 1-3 halogen atoms); optionally substituted aryl(C2-C16)alkanoyl and optionally substituted heteroaryl(C2-C16)alkanoyl, e.g., unsubstituted or halogen Examples include, but are not limited to, aryl or heteroaryl radicals substituted with 1 to 3 substituents selected from (C1-C3)alkyl and (C1-C3)alkoxy (each optionally further substituted with 1 to 3 halogen atoms); and optionally substituted heteroarylalkanoyls having 1 to 3 heteroatoms selected from O, S, and N in the heteroaryl portion and 2 to 10 carbon atoms in the alkanoyl portion, such as unsubstituted or heteroaryl radicals substituted with 1 to 3 substituents selected from halogen, cyano, trifluoromethanesulfonyloxy, (C1-C3)alkyl and (C1-C3)alkoxy (each optionally further substituted with 1 to 3 halogen atoms). The exemplified groups are illustrative and not exhaustive and can be produced by conventional processes.

[0221] The prodrug itself may not have significant biological activity, but it is understood that after in vivo administration, it can undergo one or more spontaneous chemical reactions, enzyme-catalyzed chemical reactions, and / or metabolic chemical reactions, or combinations thereof, to produce a compound described herein that is biologically active or a precursor of a biologically active compound. However, in some cases, the prodrug is understood to be biologically active. It is also understood that prodrugs can often be useful in improving drug efficacy or safety, such as through improved oral bioavailability, pharmacodynamic half-life, etc. A prodrug also has the meaning of a derivative of a compound described herein that simply masks undesirable drug properties or includes a group that improves drug delivery. For example, one or more compounds described herein may exhibit undesirable properties that can be pharmacologic, pharmaceutical, or pharmacokinetic barriers in clinical drug applications, such as being free-blocked or minimized, having poor oral drug absorption, lack of site specificity, chemical instability, toxicity, and poor patient compliance (unpleasant taste, bad odor, pain at the injection site, etc.) and others. It is understood herein that prodrugs, or other strategies using reversible derivatives, are useful in optimizing the clinical application of drugs.

[0222] In all cases disclosed herein, the description of a range of integers for a variable should be understood to describe all individual members of the described range and all possible sub-ranges for the variable. For example, the description that n is an integer from 0 to 8 describes the individual values of the range, 0, 1, 2, 3, 4, 5, 6, 7, and 8, and the selectable values such as n is 0, or n is 1, or n is 2. Further, the description that n is an integer from 0 to 8 also describes each and every sub-range that may be based on further embodiments, such as n being an integer from 1 to 8, 1 to 7, 1 to 6, 2 to 8, 2 to 7, 1 to 3, 2 to 4.

[0223] As used herein, the term “composition” generally means any product obtained directly or indirectly from any product containing a specific amount of a specific component, and from a specific combination of a specific component. It should be understood that the compositions described herein may be prepared from the isolated compounds described herein, or their salts, solutions, hydrates, solvates, and other forms thereof. It should also be understood that compositions may be prepared from various amorphous, non-amorphous, partial-crystalline, crystalline, and / or other morphological forms of the compounds described herein. It should also be understood that compositions may be prepared from various hydrates and / or solvates of the compounds described herein. Therefore, such pharmaceutical compositions describing the compounds described herein should be understood to encompass individual or any combination of various morphological forms and / or solvate or hydrate forms of the compounds described herein. Furthermore, it should be understood that compositions may be prepared from various cocrystals of the compounds described herein.

[0224] Exemplary, a composition may include one or more carriers, diluents, and / or excipients. The compounds described herein or compositions containing them may be formulated in therapeutically effective doses in any conventional dosage form suitable for the method described herein. The compounds described herein or compositions containing them, such formulations, can be administered using known procedures (generally, see Remington: The Science and Practice of Pharmacy, (21st edition, 2005)) and in a wide range of conventional routes and dosage forms for the method described herein.

[0225] As used herein, the term “therapeutic dose” means the amount of an active compound or drug that elicits a biological or pharmacokinetic response in a tissue system, animal or human, as determined by researchers, veterinarians, physicians, or other and / or clinicians, such as the relief of symptoms of the disease or disorder being treated. In one aspect, a therapeutic dose is the amount that can treat or alleviate a disease or symptoms of a disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it should be understood that the total positional daily dose of the compounds and compositions described herein may be determined by the attending physician within the bounds of sound medical judgment. A specific therapeutic dose level for any particular patient will vary depending on a variety of factors, including the disorder being treated and its severity; the activity of the particular compound used; the particular composition used; the patient’s age, weight, overall health, sex, and diet; the timing, route of administration, and rate of elimination of the particular compound used; the duration of treatment; any drugs used in combination with or concurrently with the particular compound used; and similar factors well known to researchers, veterinarians, physicians, or other clinicians in the art.

[0226] It is understood that the effective therapeutic dose, whether monotherapy or combination therapy, may be conveniently selected in relation to the toxicity of either compound or other undesirable side effects that may occur during administration of one or more of the compounds described herein. Furthermore, it is understood that the co-therapies described herein may allow for the administration of compounds exhibiting such toxicity or other undesirable side effects at lower doses, where lower doses are defined as amounts below the toxicity threshold or below the therapeutic dose window that would be administered without co-therapy.

[0227] In addition to the exemplary dosages and administration protocols described herein, it should be understood that the effective dose of any one or a mixture of the compounds described herein can be readily determined by the participating diagnostician or physician by the use of known techniques and / or by observing results obtained under similar circumstances. In determining the effective dose or dosage, many factors will be considered by the participating diagnostician or physician, including, but are not limited to, the species of mammal such as human, its size, age and overall health, the specific disease or disorder being present, the degree or involvement or severity of the disease or disorder, the individual patient's response, the specific compound being administered, the mode of administration, the bioavailability characteristics of the formulation being administered, the chosen administration plan, the use of concomitant medications and other relevant circumstances.

[0228] The dosage of each compound in a claimed combination depends on several factors, including the method of administration, the condition being treated or prevented, the severity of the condition, and the patient's age, weight, and overall health. Furthermore, pharmacogenetic information (the effect of genotype on the pharmacokinetic, pharmacodynamic, or potency profile of the treatment) for a particular patient may influence the medication used.

[0229] It should be understood that in the methods described herein, individual components of concurrent or combined administration may be administered simultaneously, concurrently, sequentially, separately, or as a single pharmaceutical formulation by any appropriate means. When concurrently administered compounds or compositions are administered in separate dosage forms, the number of doses administered per day for each compound may be the same or different. Compounds or compositions may be administered via the same or different routes of administration. Compounds or compositions may be administered simultaneously in divided or single dosage forms, in the same or different number of doses, according to a concurrent or alternating dosing plan during the course of treatment.

[0230] As used herein, the term “administer” encompasses all means of introducing the compounds and compositions described herein to a patient, including but not limited to oral (PO), intravenous (IV), intramuscular (IM), subcutaneous (SC), transdermal, inhalation, oral cavity, intraocular, sublingual, vaginal, and rectal administration. The compounds and compositions described herein may be administered in unit dosage forms and / or formulations, including conventional non-toxic, pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0231] Examples of oral administration forms include tablets, capsules, elixirs, and syrups.

[0232] Exemplary routes for parenteral administration include intravenous, intra-arterial, intraperitoneal, epidural, intraurethral, ​​intrasternal, intramuscular, and subcutaneous routes, as well as any other parenteral administration routes recognized in the Art.

[0233] Administration includes topical use, such as when administered locally to a site of disease, injury, or malfunction, or to a specific organ or tissue system. Exemplary topical administration may be performed during invasive surgery or other procedures where the disease, injury, or malfunction is accessible. Alternatively, topical administration may be performed using parenteral delivery, in which the compound or composition described herein accumulates locally without being distributed generally to a number of other non-target sites in the patient being treated. Furthermore, it is understood that topical administration may be performed directly at the site of injury or locally to the surrounding tissue. Similar variations relating to topical delivery to specific tissue types, such as organs, are also described herein. Exemplary, the compound may be administered directly to the nervous system, including, but not limited to, intracerebral, intraventricular, intracerebrospinal, intracisional, intracisional, intrathecal, intracisional and / or perispinal delivery routes, by delivery via intracranial or intravertebral needles and / or catheters, with or without the use of a pumping device.

[0234] Depending on the disease, route of administration, and / or whether the compound and / or composition described herein is administered topically or systemically, a wide range of acceptable dosages, such as doses ranging from approximately 1 μg / kg to approximately 1 g / kg, are intended herein. Dosage may be single or divided, and may be administered according to a wide range of protocols, such as qd (once daily), bid (twice daily), tid (three times daily), or every other day, once weekly, once monthly, or once quarterly. In each of these cases, it is understood that the therapeutically effective dose described herein corresponds to the example of administration or to the total daily, weekly, monthly, or quarterly dose determined by the administration protocol.

[0235] In the preparation of pharmaceutical compositions of the compounds described herein, one or more compounds in a therapeutically effective amount in any of the various systems described herein may be mixed with one or more excipients, diluted with one or more excipients, or encapsulated in a carrier such as a capsule, sachet, paper, or other container. The excipients may act as diluents and may be solid, semi-solid, or liquid materials, acting as a vehicle, carrier, or medium for the active ingredient. Thus, the pharmaceutical composition may be a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol (as a solid or in a liquid medium), ointment, soft and hard gelatin capsule, suppository, sterile injection, or sterile packaging powder. Depending on the selected dose and dosage form, the composition may contain the active ingredient in a range of about 0.1% to about 99.9%.

[0236] The effective use of the compounds, compositions, and methods described herein for treating or mitigating diseases caused by pathogenic cells expressing PSMA may be based on animal models, such as disease models of mice, dogs, pigs, and non-human primates. For example, human prostate cancer may be characterized by loss of function and / or progression over the subcutaneous tissue, which are understood to be induced in animals such as mice and other surrogate test animals. In particular, a mouse model described herein, in which cancer cells such as LNCaP cells are implanted subcutaneously, can be used to evaluate the compounds, therapeutic methods, and pharmaceutical compositions described herein to determine the therapeutic effective dose described herein.

[0237] The compounds, linkers, intermediates, and conjugates described herein may be produced using conventional processes as described in International Patent Applications WO 2009 / 002993, WO 2004 / 069159, WO 2007 / 022494, and WO 2006 / 012527, and U.S. Patent Application No. 13 / 837539 (filed March 15, 2013). The disclosures of each of the above-mentioned documents are incorporated herein by reference in their entirety.

[0238] Each reference cited herein is incorporated herein by reference.

[0239] The following examples further illustrate specific embodiments of the present invention, but these exemplary examples should not be construed as limiting the invention in any way. [Examples]

[0240] TIFF2023098946000122.tif68160

[0241] Example: Compound 104 In a 250 mL round-bottom flask, H-Glu(OtBu)-OtBu·HCl(1) (4.83 g, 16.3 mmol) and 4-nitrophenyl chloroformate (102) (3.47 g, 17.2 mmol) were dissolved in dichloromethane (50 mL) and stirred under argon on an ice bath. Diisopropylethylamine (6.28 mL, 36.1 mmol) was slowly added dropwise, and the reaction mixture was stirred on an ice bath for 5 minutes, then warmed to room temperature and stirred for 30 minutes. H-Lys(Z)-OtBu·HCl(103) (7.01 g, 18.8 mmol) was added gradually, followed by the addition of diisopropylethylamine (6.54 mL, 37.5 mmol), and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and then purified by silica gel chromatography using ethyl acetate / petroleum ether to obtain 104 (8.76 g, 86%, ESI m / z = 622.54 [M+H]+).

[0242] Example: Compound 105 104 (8.76 g, 14.1 mmol) was dissolved in anhydrous methanol (100 mL) and slowly poured down the wall of a 250 mL round-bottom flask containing 10 wt% (100 mg) palladium / carbon. A hydrogen gas balloon was attached to the flask using a three-way stopcock adapter, the air in the flask was evacuated under reduced pressure, then replaced with hydrogen gas (3x), and the mixture was stirred at room temperature under hydrogen gas for 1 hour. Anhydrous, untreated Celite (~20 g) was added to the reaction mixture and stirred for 5 minutes. The reaction mixture was filtered and concentrated under reduced pressure to obtain 105 (6.86 g, quantitative, ESI m / z = 488.46 [M+H]+).

[0243] TIFF2023098946000123.tif2994

[0244] Example: Compound 107 Boc-4-aminomethylphenylacetic acid (106) (2.00 g, 7.5 mmol) was dissolved in a solution of trifluoroacetic acid (9.75 mL) and triisopropylsilane (0.25 mL), stirred at room temperature for 30 minutes, then concentrated under reduced pressure and co-evaporated with dichloromethane (3x), and then placed under vacuum to obtain 4-aminomethylphenylacetic acid (107) (quantitative).

[0245] TIFF2023098946000124.tif40161

[0246] Example: Compound 108 To a stirred solution of 4-nitrophenyl chloroformate (102) (1.01 g, 5.0 mmol) in anhydrous dimethylformamide (10 mL), a solution of 105 (2.45 g, 5.0 mmol) and diisopropylethylamine (0.88 mL, 5.0 mmol) in anhydrous dimethylformamide (10 mL) was slowly added dropwise. The reaction mixture was stirred under argon at room temperature for 30 minutes. The reaction mixture was cooled in an ice bath, and a suspension of 7 (~1.25 g, ~7.5 mmol) and diisopropylethylamine (1.76 mL, 10.1 mmol) in anhydrous dimethylformamide (10 mL) was slowly added dropwise to the reaction vessel. The reaction mixture was then warmed to room temperature and stirred under argon for 30 minutes. The reaction mixture was purified by preparative HPLC in 10-100% acetonitrile / 0.1% formic acid to obtain compound 8 (0.56 g, 16%, 1H NMR: consistent with the structure of compound 108; ESI m / z = 679.50 [M+H]+).

[0247] TIFF2023098946000125.tif153163

[0248] Example: Preparation of ligand 7 containing a coupling group TIFF2023098946000126.tif5486

[0249] Example: Peptide 109 Table 1: Reagents for Peptide 109 Synthesis TIFF2023098946000127.tif57134

[0250] H-Cys(4-methoxytrityl)-2-chlorotrityl resin (0.87 mmol) was loaded into a peptide synthesis vessel and washed with isopropyl alcohol (3 x 10 mL), followed by dimethylformamide (3 x 10 mL). Then, Fmoc-Asp(OtBu)-OH (2.0 equivalents), diisopropylethylamine (4.0 equivalents), and PyBOP (2.0 equivalents) in dimethylformamide were added to the vessel. Argon was passed through for 1 hour, the coupling solution was drained, and the resin was washed with dimethylformamide (3 x 10 mL) and isopropyl alcohol (3 x 10 mL). A Kaiser test was performed to assess the completion of the reaction. Before each amino acid coupling, Fmoc deprotection was performed using 20% ​​piperidine in dimethylformamide (3 x 10 mL). The above sequence was repeated to complete the two coupling steps. The resin was dried under argon for 30 minutes.

[0251] TIFF2023098946000128.tif3079

[0252] Example: Peptide 110 Table 2; Reagents for Peptide 110 Synthesis TIFF2023098946000129.tif62149

[0253] 109 (0.18 mmol) was loaded into a synthesis vessel and washed with isopropyl alcohol (3 x 10 mL), followed by dimethylformamide (3 x 10 mL). Fmoc deprotection was performed using 20% ​​piperidine in dimethylformamide (3 x 10 mL). A Kaiser test was performed to assess the completion of the reaction. Next, 108 (1.2 equivalents), diisopropylethylamine (4.0 equivalents), and PyBOP (2.0 equivalents) in dimethylformamide were added to the vessel. Argon was passed through for 1 hour, the coupling solution was drained, and the resin was washed with dimethylformamide (3 x 10 mL) and isopropyl alcohol (3 x 10 mL). A Kaiser test was performed to assess the completion of the reaction. Peptides were cleaved from the resin using a cleavage mixture consisting of dithiothreitol (114 mg, 0.74 mmol) dissolved in a solution of trifluoroacetic acid (19 mL), H2O (0.5 mL), and triisopropylsilane (0.5 mL). One-third of the cleavage mixture was introduced, and argon was aerated for 30 minutes. The cleavage mixture was drained into a clean flask. The resin was aerated two more times with the additional cleavage mixture for 30 minutes each time, and drained into a clean flask. The drained cleavage mixture was then concentrated and purified by preparative HPLC in 0-30% acetonitrile / 0.1% formic acid to obtain 110 (66.9 mg, 43%, 1H NMR: consistent with the structure of 110; ESI m / z = 844.57 [M+H]+).

[0254] Examples: Similarly, the following compounds are prepared as described herein. TIFF2023098946000130.tif3164EC1080 TIFF2023098946000131.tif4098EC1067 TIFF2023098946000132.tif3992EC1100 TIFF2023098946000133.tif32119EC1167 TIFF2023098946000134.tif38125EC1168 (Accurate mass: 797.27; Molecular weight: 797.72) TIFF2023098946000135.tif3884EC1170 (Accurate mass: 510.20; Molecular weight: 510.49) TIFF2023098946000136.tif39121EC1302 TIFF2023098946000137.tif39135EC1303 TIFF2023098946000138.tif34129EC1307 D-Asp-D-Asp TIFF2023098946000139.tif59162 Example: EC1169 (Compound 112) In a 25 mL round-bottom flask, 16 (47 mg, 0.04 mmol) was dissolved in dimethyl sulfoxide (2 mL). A solution of 110 (36 mg, 0.04 mmol) in 20 mM pH 7 sodium phosphate buffer (2 mL) was added dropwise with stirring for 30 minutes at room temperature under argon ventilation. The reaction mixture was purified by preparative HPLC (10-100% acetonitrile / 50 mM NH4HCO3 pH 7) to obtain 112 (56.6 mg, 74%, 1H NMR: structure matching EC1169; ESI m / z = 895.58 [M+2H]2+).

[0255] Example: Synthesis of 3-nitro-2-disulfenethanol 2 TIFF2023098946000140.tif22109 A 500 mL three-neck flask was dried, purged with argon, and then fitted with an additive funnel. 3-nitro-2-sulfenylchloridopyridine 1 (5.44 g, 27.11 mmol, 1.4 equivalents) was added to the flask and dissolved in 200 mL of CH2Cl2. The solution was cooled to 0°C. Mercaptoethanol (1.33 mL, 18.98 mmol) was diluted in 50 mL of CH2Cl2 and placed in the additive funnel. Then, the 2-mercaptoethanol solution was slowly added dropwise over 15 minutes. The reaction was monitored by TLC (in 5% CH3OH / CH2Cl2, Rf 0.4). The solvent was removed under reduced pressure and the flask was dried. The crude product was purified with silica gel (5% CH3OH / CH2Cl2). The fractions were collected, the solvent was evaporated using a rotary evaporator, and the flask was dried. 3.4 g of 3-nitro-2-disulfeneethanol 2 was obtained (77% yield).

[0256] Example: Synthesis of 4-nitrophenyl-(3'-nitropyridine-2'-yl)disulfenylethyl carbonate 3 TIFF2023098946000141.tif25144 A 250 mL round-bottom flask was dried and purged with argon. 3-nitro-2-disulfenethanol 2 (3.413 g, 14.69 mmol) was added and dissolved in 45 mL of CH2Cl2. Triethylamine (2.9 mL, 20.57 mmol, 1.4 equivalents) and 4-nitrophenyl chloroformate (3.663 g, 17.63 mmol, 1.2 equivalents) were added, and the mixture was stirred overnight under argon. The mixture was concentrated under reduced pressure and dried. The residue was purified with silica (30% siRNA / petroleum ether), the fraction was collected, the solvent was removed under reduced pressure, and the fraction was dried. 2.7 g of 4-nitrophenyl-(3'-nitropyridine-2'-yl)disulfenethyl carbonate 3 was obtained (47% yield).

[0257] Example: Synthesis of 2-(Boc-tube tyrosine (Tut))hydrazine carboxylic acid (3'nitropyridyl-2'-yl) disulfanyl ethyl ester 6 TIFF2023098946000142.tif66117 10.67 g (33 mmol) of Boc-Tut acid 4 was dissolved in 100 mL of anhydrous THF, and 17.24 g (33 mmol) of PyBop and 17.50 mL (99 mmol, 3.0 equivalents) of DIPEA were added. The reaction mixture was stirred for several minutes, and 1.0 mL (31.68 mmol, 0.96 equivalents) of hydrazine was added and stirred for 15 minutes. LC-MS analysis (X-bridge shield RP18, 3.5 μm column; gradient 10%~100% acetonitrile, 6 minutes, pH 7.4 buffer) confirmed the formation of hydrazide 5. 14.47 g (36.3 mmol, 1.1 equivalents) of 4-nitrophenyl-(3'-nitropyridine-2'-yl)disulfenylethyl carbonate 2 was added. The resulting clear solution was stirred at room temperature for 24 hours. LC-MS analysis (X-bridge shield RP18, 3.5 μm column; gradient 30%~100% acetonitrile, 9 minutes, pH 7.4 buffer) showed >98% conversion. The reaction mixture was diluted with HCl (~1.0 L) and washed with saturated NH4Cl (400 mL), saturated NaHCO3 solution (3 x 300 mL), and brine (300 mL). The organic layer was dried over Na2SO4 (100 g) and concentrated under reduced pressure. The crude product was loaded onto a Teledyne Redisep gold silica column and eluted using a CombiFlash chromatography system with MeOH / CH2Cl2 (330 g column; 0~10% gradient). The fractions were collected, the solvent was removed under reduced pressure, and the mixture was dried. 16.10 g of 2-(Boc-Tut)hydrazinecarboxylic acid (3'nitropyridyl-2'-yl) disulfanyl ethyl ester 6 was obtained (82% yield).

[0258] Example: Synthesis of azidomethylbutyrate dipeptide 9 TIFF2023098946000143.tif64113

[0259] Dipeptide 7 (10.83 g, 27.25 mmol) was dissolved in 100 mL of dichloromethane, and imidazole (2.05 g, 1.1 equivalents) was added. The reaction mixture was stirred at room temperature to dissolve all solids, and cooled in an ice bath for 10 minutes. TESCl (4.8 mL, 1.05 equivalents) was added dropwise at 0°C, and the mixture was stirred under argon and warmed to room temperature over 1.5 hours. TLC (3:1 hexane / siRNA) showed complete conversion. The reaction product was filtered to remove the imidazole HCl salt. 125 mL of dichloromethane was added to the filtrate, and the resulting solution was extracted with 250 mL of brine. The brine layer was extracted with 125 mL of dichloromethane. The combined organic phases were washed with 250 mL of brine, separated, dried over 45.2 g of Na2SO4, and filtered. The resulting solution was concentrated under reduced pressure, co-evaporated with toluene (2 x 5 mL), and dried overnight under high vacuum to obtain 14.96 g of crude product 8.

[0260] Crude product 8 was used without further purification. The TES-protected dipeptide was dissolved in 100 mL of THF (anhydrous, inhibitor-free), cooled to -45°C, and stirred at -45°C for 15 minutes before adding KHMDS (0.5 M toluene solution, 61 mL, 1.05 equivalents) dropwise. After the addition of KHMDS, the reaction mixture was stirred at -45°C for 20 minutes, and chloromethyl butyrate (4.4 mL, 1.1 equivalents) was added. The reaction mixture was stirred at -45°C for a further 20 minutes. The reaction was stopped by adding 25 mL of MeOH, and the mixture was warmed to room temperature. 250 mL of siRNA and 250 mL of brine were added to the reaction mixture to separate the organic phase. The solvent was evaporated to reduce the volume of the solution. The solution was passed through 76.5 g of silica in a 350 mL sintered glass funnel. The silica plug was washed with 500 mL of siRNA / petroleum ether (1:4). The filtrate and washings were concentrated to obtain an oily residue, which was dried under high vacuum to yield 16.5 g of product 9 as a bright yellow waxy substance.

[0261] Example: Synthesis of tripeptide methyl ester 10 TIFF2023098946000144.tif33157 Based on 16.5 g of alkylated dipeptide 9 (26.97 mmol), 5.51 g of N-methyl pipecolate (MEP) (1.4 equivalents) and 7.63 g of pentafluorophenol (1.5 equivalents) were placed in a 300 mL hydrogenation flask. Then, 115 mL of NMP was added, followed by 7.78 g of EDC (1.5 equivalents). The mixture was stirred overnight at room temperature. 16.5 g of alkylated dipeptide 9 was dissolved in 16.5 mL of NMP, the solution was transferred to the hydrogenation flask, the remaining 9 was washed with 8 mL of NMP and transferred to the hydrogenation flask. Anhydrous 10% Pd / C (1.45, 0.05 equivalents) was added. The reaction mixture was vacuumed and hydrogenated three times, and the flask was shaken under hydrogen (~35 psi) for 3.5 hours. The reaction reaction was analyzed by HPLC. The reaction mixture was filtered through 40 g of Celite in a 350 mL sintered glass funnel and washed with 250 mL of siRNA. The filtrate and washings were transferred to a separatory funnel and washed with 1% NaHCO3 / 10% NaCl solution (200 mL x 3). The organic layer was isolated and dried over 45.2 g of Na2SO4. The volume was filtered and rotated under reduced pressure. A viscous brown residue was obtained and dried overnight under high vacuum to obtain 19.3 g of crude product. The crude product was dissolved in 10 mL of dichloromethane, divided into two parts, and purified by 330 g of Teledyne Redisep gold silica column. The two purified fractions were combined, evaporated, and dried under high vacuum to obtain 7.64 g of compound 10 as a pale yellow solid (total yield: 39%, through 3 steps from compound 7).

[0262] Example: Synthesis of tripeptide acid 11 TIFF2023098946000145.tif38148 Methyl ester 10 (6.9 g, 9.7 mmol) was dissolved in 1,2-dichloromethane (193 mL) and placed in a round-bottom flask equipped with a stirring bar and condenser. Trimethyltine hydroxide (24.6 g, 14 equivalents) was added to this solution. The mixture was heated at 70°C for 5 hours. LC-MS analysis showed that the desired product had formed and <15% of the starting methyl ester 10 remained. The reaction mixture was cooled in an ice bath for 30 minutes. The resulting precipitate was then filtered off. The filtrate was stored overnight at -20°C. The filtrate was then divided into two parts, each subjected to the following chromatographic procedure.

[0263] Each portion was concentrated under reduced pressure and then placed under high vacuum for 30 minutes. The concentrate was then rapidly dissolved in acetonitrile (95 mL). Ammonium bicarbonate solution (95 mL; 50 mM, pH=7) was then added to this solution. This solution was loaded into a Biotage SNAP C18 reversed-phase cartridge (400 g, KP-C18-HS) and eluted with 50 mM ammonium bicarbonate and acetonitrile (1:1~100% ACN) using a Biotage chromatography system. The fractions were analyzed by LC-MS. The pure fractions were combined and ACN was removed under reduced pressure. The resulting aqueous suspension was extracted with dimethyl(3 X). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Purification of the two portions resulted in a clean recovery of 11 (4.6 g, 65%).

[0264] Example: Synthesis of Acetyltripeptide 13 TIFF2023098946000146.tif69152 In a round-bottom flask, tripeptide acid 11 (3.9 g, 5.6 mmol) was dissolved in THF (23 mL). To this solution, 3 HF·TEA complex (1.8 mL, 2 equivalents) was added. The reaction mixture was stirred at room temperature for 1 hour. LC-MS analysis showed complete conversion to the desired des-TES product 12. The solvent was removed under reduced pressure, and the residue was placed under high vacuum for 40 minutes. The resulting residue was then dissolved in pyridine (26 mL), and acetic anhydride (7.9 mL, 15 equivalents) and DMAP (25 mg) were added. The reaction mixture was stirred at room temperature for 1 hour. LC-MS analysis showed complete conversion to the desired acetyltripeptide acid 13. Next, a 1:1 solution of 1,4-dioxane / water (150 mL) was added to the reaction mixture. The reaction mixture was stirred for 1 hour, at which point the solvent was removed under high vacuum rotational evaporation. Toluene was added to the residue, and the solvent was removed under reduced pressure (80 mL, 3X). The resulting crude 13 was dried overnight under high vacuum. The crude substance was then dissolved in ACN (72 mL). Sodium phosphate buffer (50 mM, pH=7.8, 288 mL) was then added, and the pH of the resulting suspension was adjusted to neutral using saturated sodium bicarbonate solution. This solution was loaded into a Biotage SNAP C18 reversed-phase cartridge (400 g, KP-C18-HS), and eluted with water and acetonitrile (20% ACN~65% ACN) using a Biotage chromatography system. The fractions were analyzed by LC-MS. The clean fractions were combined, ACN was removed, and the aqueous solution was placed in a freeze-dryer to obtain purified acetyl tripeptide 13 (2.5 g, 71%).

[0265] Example: Synthesis of 2-(tubulisin B)hydrazinecarboxylic acid (3'nitropyridyl-2'-yl) disulfanyl ethyl ester 16 TIFF2023098946000147.tif105155

[0266] Activated Boc-Tut-fragment 6 (2.63 g, 4.42 mmol, 1.1 equivalents) was treated with TFA / CH2Cl2 (42 mL; 1:1) and stirred for 30 minutes. LC-MS analysis (X-bridge shield RP18, 3.5 μm column; gradient 10%-100% acetonitrile, 6 minutes, pH 7.4 buffer) confirmed product formation. The TFA was removed under reduced pressure and co-evaporated with CH2Cl2 (3 x 30 mL), and the activated Tut-derivative 14 was dried under high vacuum for 18 hours. In another flask, tripeptide acid 13 (2.51 g, 4.02 mmol) was dissolved in 70 mL of CH2Cl2 (anhydrous), and 1.48 g (8.04 mmol, 2.0 equivalents) of pentafluorophenol in 5 mL of CH2Cl2 was added, followed by 8.74 g (20.1 mmol, 5.0 equivalents) of DCC resin. The resulting reaction mixture was stirred at room temperature for 20 hours. LC-MS analysis (X-bridge shield RP18, 3.5 μm column; gradient 10%-100% acetonitrile, 6 minutes, pH 7.4 buffer) showed >99% conversion. The DCC resin was filtered off, CH2Cl2 was removed under reduced pressure, and the pentafluorophenol activation product 15 was dried under high vacuum for 10 minutes. The residue was dissolved in 16.7 mL of DMF, and DIPEA (12.6 mL, 72.36 mmol, 18.0 equivalents) was added. Tut-fragment trifluoroacetate 14 in DMF (8.5 mL) was slowly added over 5 minutes. The resulting clear solution was stirred at room temperature for 1 hour. LC-MS analysis (X-bridge shield RP18, 3.5 μm column; gradient 10%-100% acetonitrile, 6 minutes, pH 7.4 buffer) confirmed product formation. The reaction mixture was diluted with Depositphotos (700 mL), washed with brine (300 mL, 2 x 100 mL), dried over Na₂SO₄ (75 g), concentrated, and dried for 15 hours. The crude product was dissolved in CH₂Cl₂ (25 mL), loaded onto a Teledyne Redisep gold silica column, and eluted using a CombiFlash chromatography system with MeOH / CH₂Cl₂ (330 g column; 0-5% gradient). The fractions were collected, the solvent was removed by evaporation using a rotary evaporator, and the mixture was dried.3.91 g of 2-(tubulosine B)hydrazinecarboxylic acid (3'nitropyridyl-2'-yl) disulfanyl ethyl ester 16 was obtained (89% yield).

[0267] Example: Preparation of 2-(tubulicin B)hydrazinecarboxylic acid (pyrido-2-yl) disulfanyl ethyl ester 3 TIFF2023098946000148.tif56153

[0268] Examples: Similarly, the following compounds are prepared as described herein. TIFF2023098946000149.tif33162EC1555 TIFF2023098946000150.tif31162EC1568

[0269] Examples: Further tubulicins described herein may be isolated from natural sources, such as bacteria and other ferments, but not limited to these. Alternatively, tubulicins described herein may be produced by conventional methods, such as those described in PCT International Publications WO 2009 / 055562, WO 2012 / 019123, and WO 2013 / 149185, and concurrently pending U.S. application 13 / 841078 (each of which disclosures is incorporated herein by whole-word reference).

[0270] Example: Another preparation of EC1169 (compound 112) TIFF2023098946000151.tif101122

[0271] Examples: The following representative example compounds are provided to better illustrate the inventions described herein and may be prepared according to the synthetic methods described for the above examples and / or by conventional methods. TIFF2023098946000152.tif37162EC1069 TIFF2023098946000153.tif33140EC1183

[0272] TIFF2023098946000154.tif30158EC1192(C78H112N14O28S3, Precision weight: 1788.69, Molecular weight: 1790.00) TIFF2023098946000155.tif30160EC1197(C77H110N14O28S3; Precision weight: 1774.68; Molecular weight: 1775.97) TIFF2023098946000156.tif24160EC1241(C79H114N14O28S3, Precision weight: 1802.71, Molecular weight: 1804.03) TIFF2023098946000157.tif24160EC1268 (C78H112N14O28S3, Precision weight: 1788.69, Molecular weight: 1790.00) TIFF2023098946000158.tif24163EC1269 (C78H112N14O28S3, Precision weight: 1788.69, Molecular weight: 1790.00)

[0273] TIFF2023098946000159.tif40163EC1308(C78H112N14O28S3, Mass:1788.6933, MW:1789.9959) TIFF2023098946000160.tif30160EC1309 TIFF2023098946000161.tif26159EC1310 TIFF2023098946000162.tif35163 EC1385 TIFF2023098946000163.tif29165 EC1386

[0274] TIFF2023098946000164.tif39159EC1387 TIFF2023098946000165.tif35160EC1388 TIFF2023098946000166.tif35163EC1437 TIFF2023098946000167.tif26158EC1452 TIFF2023098946000168.tif30157EC1550

[0275] TIFF2023098946000169.tif33158EC1551 TIFF2023098946000170.tif23163EC1584 (C78H112N14O28S3, Precision weight: 1788.69, Molecular weight: 1790.00) TIFF2023098946000171.tif43161EC1588 TIFF2023098946000172.tif25162EC1677 (C78H114N14O27S3, Precision weight: 1774.71, Molecular weight: 1776.01)

[0276] TIFF2023098946000173.tif23163EC1718(C77H112N14O27S3, Precision weight: 1760.70, Molecular weight: 1761.99) TIFF2023098946000174.tif27161EC1719 (C79H116N14O27S3, Precision weight: 1788.73, Molecular weight: 1790.04)

[0277] TIFF2023098946000175.tif29162EC1720 (C80H118N14O27S3, Precision weight: 1802.75, Molecular weight: 1804.07) TIFF2023098946000176.tif33162EC1721(C81H120N14O27S3, Precision weight: 1816.76, Molecular weight: 1818.09)

[0278] Method Example: PSMA affinity analysis LNCaP cells were seeded in 12-well Corning Cellbind plates and allowed to form an adherent monolayer overnight in RPMI / HIFCS. The used incubation medium was supplemented with 10% HIFCS, with and without increasing concentrations of the test compound, such as unlabeled PMPA or the compounds specified herein, such as EC1169 or EC1568, and replaced with RPMI containing a standard PSMA-binding ligand such as 100 nM 3H-PMPA, or a competing compound such as EC0652, Re-EC652, or 99mTc-EC0652, or a negative control intermediate without a PSMA-binding ligand used as a negative control. Cells were incubated at 37°C for 1 hour, then rinsed three times with 0.5 mL of PBS. 500 microliters of 1% sodium dodecyl sulfate in PBS were added to each well; after 5 minutes, the cell lysates were collected and transferred to individual tubes or vials containing 5 mL of scintillation cocktail, and then the radioactivity was counted. In FFRPMI (no competitor), cells exposed only to a standard PSMA-binding ligand such as 3H-PMPA or a competing compound such as 99mTc-EC0652 are designated as negative controls, while cells exposed to a standard PSMA-binding ligand such as 3H-PMPA + 1 mM unlabeled PMPA or a competing compound such as 99mTc-EC0652 + Re-EC0652 serve as positive controls. The degradation per minute (DPM) measured in the latter samples (representing nonspecific binding of the label) is subtracted from the DPM values ​​from all samples. Relative affinity is defined as the inverse molar ratio of the compound required to replace 50% of the standard PSMA-binding ligand such as 3H-PMPA or the competing compound such as 99mTc-EC0652 bound to PSMA on LNCaP cells. The relative affinity of a standard PSMA-binding ligand such as PMPA or a competing compound such as Re-EC0652 to PSMA is set to 1.

[0279] Method and Example: Dose-response assay for PSMA+LNCaP cells LNCaP cells were seeded in 24-well Corning Cellbind plates and allowed to form a nearly dense monolayer overnight in RPMI / HIFCS. Thirty minutes before adding the test compound, such as the compounds described herein, the used medium was aspirated from all wells and replaced with fresh RPMI. After rinsing once with 1 mL of fresh RPMI / HIFCS, 1 mL of medium containing the increasing concentration of the test compound was added to each well (4 wells per sample). The test compound-treated cells were pulsed at 37°C for 2 hours, rinsed four times with 0.5 mL of medium, and then chased in 1 mL of fresh medium for up to 70 hours. The used medium was aspirated from all wells and replaced with fresh medium containing 5 μCi / mL of 3H-thymidine. After incubation for a further 4 hours at 37°C, the cells were washed three times with 0.5 mL of PBS and then treated with 0.5 mL of ice-cold 5% trichloroacetic acid per well. After 15 minutes, trichloroacetic acid is aspirated, and cells are solubilized for 15 minutes by adding 0.5 mL of 0.25 N sodium hydroxide. 450 microliters of each solubilized sample are transferred to a scintillation vial containing 3 mL of Ecolume scintillation cocktail and then counted using a liquid scintillation counter. The final tabular results are expressed as the percentage of 3H-thymidine uptake compared to the untreated control.

[0280] Method and Example: In vivo activity against PSMA+-expressing tumors transplanted into mice Male nu / nu mice aged 4-7 weeks (Harlan Sprague Dawley, Inc., Indianapolis, IN) were kept on a standard 12-hour light-dark cycle throughout the experiment and given free access to rodent diet #2918 (Harlan Teklad, Madison, WI). LNCaP cells were grown in RPMI at 37°C in a 5% CO2 / 95% air-humidified atmosphere with 10% HIFCS, harvested, and resuspended on ice in Matrigel solution (50% RPMI + 50% high-concentration Matrigel, BD#354248) to a harvest concentration of 1 x 10⁶ cells / 50 μL. The cell solution and injection needle (28 gauge) were kept on ice until injection, and 50 μL of the cell solution was injected subcutaneously into the dorsomedial region. Mice are divided into groups of 5, 7, or 9, and a freshly prepared test compound solution is injected into the lateral tail vein in 200 μL of phosphate-buffered saline (PBS) under sterile conditions. Intravenous (iv) treatment is typically initiated when the LNCaP tumors reach a size of approximately 100–150 mm3. Control group mice receive no treatment. After each subcutaneously injected tumor has grown, the tumors are measured three times per week during treatment and twice per week after treatment until the tumor volume reaches 1500 mm3. Using a vernier caliper, the tumors are measured in two vertical directions, and their volumes are calculated as 0.5 x L x W² (where L = measurement on the longest axis (nm) and W = measurement on the axis perpendicular to L (nm)). As a general measure of overall toxicity, body weight change is determined on the same schedule as tumor volume measurement. The maximum percentage weight loss attributable to treatment on any given day is determined for each mouse. Animal survival is monitored daily. Dying animals are euthanized by CO2 asphyxiation.

[0281] Examples: Relative affinity of the compounds described herein compared to the PSMA inhibitors DUPA and PMPA. PMPA has been reported to be one of the best-affinity ligands for PSMA, or to be the best-affinity ligand. The data shown in Figures 1 and 2 demonstrate that the compounds described herein exhibit higher affinity for PSMA than PMPA. TIFF2023098946000177.tif2937PMPA TIFF2023098946000178.tif2647DUPA TIFF2023098946000179.tif38150EC0652

[0282] Unexpectedly, the ligands described herein were found to have a higher affinity for PSMA than PMPA, which has been reported to be the ligand with the highest affinity. Furthermore, unexpectedly, the ligand conjugates described herein were found to have an even higher affinity for PSMA.

[0283] Data for further exemplary compounds described herein are shown in the table below. TIFF2023098946000180.tif104143

[0284] Examples: Dose-response of compounds described herein to PSMA+LNCaP cells As a measure of cytotoxicity, a standard 3H-thymidine uptake assay was used. The data shown in Figure 3 reveal that EC1169 has an IC50 of 13 nM, indicating dose-response cytotoxicity to cells in vitro. Figure 4 shows the corresponding dose-response cytotoxicity and IC50 values ​​for (black inverted triangle) EC1718, IC50 17.9 nM; (black diamond) EC1677, IC50 20.9 nM; (black triangle) EC1719, IC50 37.5 nM; (black circle) EC1720, IC50 54.2 nM; and (black square) EC1721, IC50 65.6 nM.

[0285] Examples: Further compounds described herein are shown below in relation to LNCaP cells (2 hours to 72 hours) as measured by 3H-thymidine-uptake cells. TIFF2023098946000181.tif67132

[0286] Examples: Activity of the compounds described herein against in vivo PSMA+ tumors As shown in Figure 5, treatment of nude mice with PSMA-positive LNCaP human xenografts with EC1169 (black triangle), EC1550 (black circle), and EC1551 (black square), each at 2 μmol / kg, TIW, for 2 weeks, resulted in complete remission in all test animals. Each compound was compared to the vehicle treatment control (black diamond). Complete remission was observed when the tumor did not appear to be growing net during the 14-day treatment period (vertical dotted lines indicate the last treatment day). As described herein, it should be understood that the transplant contains cancer cells in the matrix (total volume 100-150 mm3). Since the matrix remains throughout the entire observation period, the reduction in tumor size may not necessarily be determined by external measurements. Surprisingly, it was also found that treatment with the compounds described herein resulted in cures. For example, EC1169 resulted in cures in 2 / 7 of the test animals. Healing is observed when the tumor does not appear to grow during the entire 85-day observation period. The data shown in Figure 5 are the average measurements for each cohort. Therefore, it should be understood that the increase in tumor volume that begins around 40–45 days represents regrowth in the remaining test animals.

[0287] Examples: Total toxicity of the compounds described herein As shown in Figure 6, the observed effects of EC1169 (black triangle), EC1550 (black circle), and EC1551 (black square) did not result in weight loss or tumor organ tissue degradation.

[0288] Examples: Activity of the compounds described herein against in vivo PSMA+ tumors Similarly, as shown in Figure 7, treatment of nude mice with PSMA-positive LNCaP human xenografts with EC1584 (black inverted triangle) and EC1588 (black triangle) at 2 μmol / kg TIW for 2 weeks resulted in complete remission in all test animals. Each compound was compared to the vehicle treatment control (black circle). Surprisingly, treatment with EC1588 was found to result in cure in 3 / 7 of the test animals.

[0289] Examples: Total toxicity of the compounds described herein As shown in Figure 8, the observed efficacy of EC1584 (black inverted triangle) and EC1588 (black triangle) did not result in weight loss or tumor organ tissue degradation.

[0290] Examples: Activity of the compounds described herein compared to conventional chemotherapeutic agents As shown in Figure 9, treatment of mice with LNCaP tumors with docetaxel (the most active chemotherapeutic agent approved for prostate cancer) (black inverted triangle) at 10 mg / kg, BIW, 2 weeks, MTD, was found to produce only minimal antitumor activity, even when administered at its MTD, with only 1 / 4 cured. Furthermore, as shown in Figure 10, the minimal observed efficacy of docetaxel was accompanied by high overall toxicity, as evidenced by severe weight loss (18%). EC1169 (black circle), administered at 2 μmol / kg, TIW, 2 weeks, was more active and less toxic compared to docetaxel against PSMA+ LNCaP tumors. Figure 9 shows that treatment with EC1169 resulted in complete remission in all test animals and a 2 / 5 cure rate. Figure 10 also shows that the higher efficacy demonstrated by EC1169 was not accompanied by substantially lower toxicity than docetaxel, which significantly provides a wider treatment window. The efficacy of each compound was compared against vehicle-treated controls (black squares).

[0291] Examples: Figure 11 shows the in vivo efficacy of (black square) EC1718; (black triangle) EC1720; (black inverted triangle) EC1721; (black diamond) EC1719; and (white circle) EC1677 compared to (black circle) untreated controls. All compounds were administered at 2 μmol / kg, TIW for 2 weeks, starting on day 21 post-tumor transplantation (PTI). The dotted line indicates the last treatment day. The data show that the compounds described herein are effective in reducing tumor growth in vivo compared to untreated animals. Furthermore, (black square) EC1718 resulted in a 1 / 7 cure rate; (black inverted triangle) EC1721 resulted in a 1 / 7 cure rate; (black diamond) EC1719 resulted in a 2 / 7 cure rate; and (white circle) EC1677 resulted in a 4 / 7 cure rate, where no tumor regrowth was observed in those animals during the observation period. Furthermore, as shown in Figure 12, the compounds described herein did not exhibit overall toxicity to test animals. While we do not wish to be bound by any particular theory, for EC1718, the weight change observed in Figure 12 on day 81 is thought to be due to the effect of tumor size.

[0292] Examples: Specificity of the compounds described herein PSMA-negative KB tumors did not respond well to EC1169 therapy, which supports the conclusion that the compounds described herein exhibit target specificity to PSMA-expressing cells.

[0293] Examples: Hematological toxicity The conjugates described herein demonstrate significantly improved hematological toxicity. EC1169, EC1584, and EC1588 were administered intravenously to rats at 0.33 and 0.51 μmol / kg twice weekly (BIW) for two weeks. Hematological toxicity in erythrocytes and leukocytes was significantly lower than in untreated controls.

Claims

1. Formula: BL-D [In the formula, B has the structure: 【Chemistry 1】 It contains a urea structure consisting of lysine and glutamic acid, L is a polyvalent linker comprising a chain of at least 8 atoms; D is the radical of the drug. or a pharmaceutically acceptable salt thereof.

2. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein D is a naturally occurring tubulysin.

3. D is 【Chemistry 2】 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the drug is selected from the group consisting of:

4. B has the structure: 【Transformation 3】 The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

5. B has the structure: 【Chemistry 4】 The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

6. B has the structure: 【Transformation 5】 The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

7. 7. The conjugate of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein L comprises a chain of at least 9 atoms.

8. 7. The conjugate of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein L comprises a chain of at least 10 atoms.

9. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, selected from the following compounds: 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】

10. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof for treating a disease in a host animal caused by a pathogenic cell population expressing PSMA.

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