Methods and compositions for treating cancer using prmt5 inhibitors

Abstract

Conjugates of the formula T-L-A wherein T is a ligand targeting prostate specific membrane antigen (PSMA), L is a linker, and A is a radiosensitizer; a pharmaceutical composition comprising the conjugate and a pharmaceutically acceptable carrier; and methods of treating prostate cancer or metastases thereof in a patient using radiotherapy. A conjugate of formula T-L-A can be administered prior to or after administration of a conjugate of formula T-L-X, wherein X is or comprises a radiotherapy or (radiation) imaging agent. Conjugates of formula T-L-(X) A, pharmaceutical compositions comprising the same, and methods of use are also disclosed.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Application No. 63 / 535,767, filed August 31, 2023; and the benefit of U.S. Application No. 63 / 560,987, filed March 4, 2024, each incorporated herein by reference as fully set forth herein. Technical Field

[0003] This disclosure generally relates to prostate-specific membrane antigen (PSMA) ligands, radiosensitizers, conjugates, compositions, and methods of use in imaging and treatment of prostate cancer.

[0004] Government support terms

[0005] This invention was made with government support from the U.S. Army Medical Research Acquisition Activity (licenses W81XWH-12-1-0346, W81XWH-13-1-0398, and W81XWH-16-1-0394) and the National Institutes of Health (license CA212403). The government holds certain rights to this invention. Background Technology

[0006] Treatment-induced neuroendocrine differentiation (NED) is an emerging mechanism of therapy resistance and a process leading to the development of neuroendocrine prostate cancer (NEPC). In NED, a subpopulation of androgen receptor (AR)-positive prostate cancer cells transdifferentiate into AR-negative neuroendocrine-like (NE-like) cells in response to all existing therapies, including radiation therapy (RT), androgen therapy (ADT), and chemotherapy. Recent increases in treatment-induced NEPC (t-NEPC) following the use of potent next-generation AR signaling inhibitors, abiraterone and enzalutamide, further establish the clinical significance of treatment-induced NED. NEPC is now recognized as an aggressive variant of castration-resistant prostate cancer (CRPC) and accounts for 17-25% of all CRPC. There is currently no effective treatment to manage this deadly end-stage disease. Therefore, developing new therapeutic approaches to prevent treatment-induced NED not only sensitizes prostate cancer cells to existing therapies but also prevents disease progression. In view of the foregoing, it is an object to provide such a treatment. From the description, this and other objects and advantages and features of the invention will be apparent. SUMMARY

[0007] Studies by the present inventors have identified DNA repair enzymes (e.g., protein arginine methyltransferase 5 (PRMT5) and ataxia-telangiectasia mutated (ATM)) as modulators of fractionated ionizing radiation (FIR)-induced NED. The present inventors hypothesize that at least PRMT5 epigenetically reprograms treatment-induced NED through cofactor switching and that targeting PRMT5 can inhibit treatment-induced NED and sensitize prostate cancer cells to therapy.

[0008] To this end, the present disclosure relates to a conjugate of the formula T-L-A, wherein:

[0009] T is a ligand targeting prostate-specific membrane antigen (PSMA),

[0010] L is a linker, and

[0011] A comprises a DNA repair enzyme inhibitor, wherein the DNA repair enzyme inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor, an ataxia-telangiectasia mutated (ATM) inhibitor, or a DNA protein kinase (DNA-PK) inhibitor.

[0012] The radiosensitizer can be, among others, a DNA repair enzyme inhibitor (e.g., a single-strand DNA repair enzyme inhibitor or a double-strand DNA repair enzyme inhibitor). Some examples of DNA repair enzymes include a protein arginine methyltransferase 5 (PRMT5) inhibitor and an ataxia-telangiectasia mutated (ATM) inhibitor. Some examples of PRMT5 inhibitors include JNJ-64619178 or GSK3326595. An example of an ATM inhibitor includes AZD0156.

[0013] The conjugate of Formula T-L-A can be used in a method of treating prostate cancer or metastasis thereof in a patient using radiation therapy, the method comprising administering to the patient the conjugate of Formula T-L-A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the conjugate of Formula T-L-A, thereby treating prostate cancer or metastasis thereof in the patient. In such a method, the conjugate of Formula T-L-A can be used in combination with a conjugate of Formula T-L-X, or a pharmaceutically acceptable salt thereof, wherein:

[0014] T is a ligand targeting prostate-specific membrane antigen (PSMA),

[0015] L is a linker, and

[0016] X is a radiotherapeutic or radioimaging agent. Also disclosed are compounds of Formula T-L-(X)A. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings generally illustrate the various embodiments, by way of example, and not by way of limitation, in which:

[0018] Figure 1 . PRMT5 regulates expression of androgen receptor (AR) and DNA damage response (DDR) genes in prostate cancer cells. Protein arginine methyltransferase 5 (PRMT5) epigenetically activates transcription of AR and genes involved in DDR, such as double-strand break (DSB) repair and G2 checkpoint genes, promoting prostate cancer growth and survival, radioresistance, and tumor recurrence.

[0019] Figure 2. Development of SMTD for cancer treatment. A. Shows the application of... 177 Representative images from mCRPC patients before and after two treatments with Lu-PSMA-617. B. Structure of the novel conjugate for targeted delivery to cancer cells expressing luteinizing hormone-releasing hormone receptor (LHRHR). Red, small molecule LHRHR antagonist; green, near-infrared dye; blue, linker. C through D. Representative images showing specific tumor targeting in OVCAR3 xenograft tumors in mice. 100-fold overdose of the competitor (unlabeled conjugate) to demonstrate specific uptake of the conjugate by LHRHR.

[0020] Figure 3. PRMT5 promotes CRPC cell growth by epigenetically activating AR expression. A, Growth curves of 22Rv1 cells incubated for 6 days with 10 µM PRMT5 inhibitor (BLL3.3) or an equal volume of carrier (dimethyl sulfoxide; DMSO) (MTT assay). B to C, Representative Western blot analysis (B) and quantification (C) of protein expression in cell lysates from A on day 6. D, qPCR analysis of gene expression in cells from A on day 6. E, Growth curves of 22Rv1 cells with doxycycline-inducible PRMT5 knockdown (22Rv1-shPRMT5#1) incubated for 6 days in the presence (Dox (+)) or absence (Dox (-)) (MTT assay). F to H, Representative Western blot analysis (F) and quantification (G) of protein expression in cell lysates from E on day 6. H, qPCR analysis of gene expression in cells from E on day 6. I, Growth curves (MTT assay) of 22Rv1 cells (22Rv1-shSC) with doxycycline-induced scrambled control expression incubated for 6 days in the presence (Dox (+)) or absence (Dox (-)). J to L, Representative Western blot analysis (J) and quantification (K) of protein expression in cell lysates from I on day 6. L, qPCR analysis of gene expression in cells from I on day 6. M, Flow cytometry analysis of cells on day 6 after PI staining (sub-G1 cells were gated out). N, ChIP-qPCR assay of PRMT5 binding to the proximal or distal region of the AR promoter (N). O, ChIP-qPCR assay of enrichment of histone markers at the proximal promoter region of AR. For MTT, Western blot, cell cycle, and qPCR analyses, statistical significance of group differences was determined for “DMSO vs BLL3.3” or “Dox (-) vs Dox (+)”. For ChIP-qPCR, values ​​were normalized relative to the corresponding IgG controls, and statistical significance of group differences was determined for “specific IP vs IgG IP”. For all experiments, results are presented as mean ± SD from three independent experiments. Student's t-test and Welch correction were performed to determine statistical significance of group differences. .

[0021] Figure 4PRMT5 functions as a major regulator of DDR. In response to radiation, PRMT5 expression is upregulated and forms a complex with pICln to catalyze H4R3me2s to promote transcription of the indicated DDR genes involved in DSB repair and G2 arrest.

[0022] Figure 5. Radiation enhances PRMT5 expression in cells, xenograft tumors, and prostate cancer tissues. A. Prostate cancer cell lines shown were subjected to the indicated cumulative dose of FIR (2 Gy / day, 5 days / week), and PRMT5 expression and H4R3me2s levels were determined by Western blotting. B. Radiation-resistant sublines (IRR1, 2, 3) isolated from LNCaP cells maintained higher levels of PRMT5 and H4R3me2s after 40 Gy FIR. C. High PRMT5 expression in the nucleus of recurrent prostate cancer tissue after RT failure (RT-recurrent) compared to primary prostate cancer tissue (RT-primary).

[0023] Figure 6. PRMT5 knockdown sensitized LNCaP xenograft tumors to FIR and reduced tumor recurrence. LNCaP-shPRMT5 and LNCaP-SC cell lines were injected into nude mice to establish xenograft tumors. Treatment was administered for 4 weeks with 40 Gy of FIR (5 Gy / application, 10 Gy / week) and PRMT5 knockdown (KD) induced by doxycycline (1 mg / mL) in drinking water. Tumor growth was monitored for 7 months after the 4-week treatment period until all tumors in the SC group showed a tumor burden (>1,500 mm). 3 A. Schematic diagram of treatment strategy and tumor regeneration monitoring. B. Tumor growth over time. P < 0.0001 (two-way ANOVA and Sidak multiple comparison test). C. Tumor recurrence (> 400 mm) 3 The percentage of ), P < 0.0001 (one-tailed Fisher exact test). D. Tumor-free survival. P < 0.0001 (Kaplan-Meier estimate, log-rank test).

[0024] Figure 7. JNJ-64619178 is a potent PRMT5 inhibitor in prostate cancer cells. A. CRPC cells 22Rv1 were treated with 10 µM JNJ-64619178 (JNJ) or dimethyl sulfoxide (DMSO) for a specified number of days, and cell growth was measured by MTT assay. B. At the end of day 6, total cell lysates were prepared from A for Western blotting of full-length AR (AR-FL), AR-V7, PRMT5, and β-actin. C. Quantitative expression levels of AR-FL, AR-V7, and PRMT5 from B. D. Similar experiments were performed as described in A, and mRNA expression of AR-FL, AR-V7, and PRMT5 was quantified by qRT-PCR. E. LNCaP cells were pretreated with a specified concentration of JNJ or DMSO for 4 days, followed by irradiation (IR) at 2 Gy (IR+) or without IR (IR-). DSB repair (γH2AX foci) at 6 hours post-IR was determined by immunofluorescence. The quantitative mean number of foci per cell is shown. At least 60 cells were counted for each treatment condition. F. Similar experiments were performed, and the expression of the PRMT5 target genes shown was quantified by qRT-PCR. Involucrin (IVL), a target gene repressed by PRMT5, was used as a control. All bars are mean ± SD of three independent experiments. Brown-Forsythe and Welch ANOVA were used in E, followed by Dunnett's T3 multiple comparison test for statistical analysis. Student's t-test was used to determine the statistical significance between DMSO and JNJ in A, B, C, D, and F. ).

[0025] Figure 8 Enhanced PSMA-based TRT strategies for mCRPC treatment. PSMA-based TRT molecules. 177 Lu-PSMA-617 will be conjugated to the novel radiosensitizer JNJ-64619178 (JNJ). Following specific binding of PSMA to PSMA via PSMA-617 (617), the conjugate will be internalized into PSMA-expressing mCRPC cells via endocytosis. JNJ will be released into the cytoplasm and specifically bind to PRMT5 to inhibit PRMT5 activity, leading to downregulation of AR and DDR gene expression and impaired DSB repair. Therefore, the inclusion of the radiosensitizer is expected to enhance DSB repair by impairing its function. 177 Lu's effectiveness.

[0026] Figure 9 Is using 177Absolute tumor volume measurement graph of combination therapy with Lu PSMA-617 20 and compound 22.

[0027] Figure 10 Is using 177 Weight measurement chart of combination therapy with Lu PSMA-617 20 and compound 22.

[0028] Figure 11 Is using 177 Survival curves of combination therapy with Lu PSMA-617 20 and compound 22.

[0029] Figure 12 This image illustrates the effects of compounds 20, 21, and 23 on radiation-induced DNA double-strand breaks (DSBs) repair in LNCaP cell lines after staining with γH2AX for 1 hour at 1 nM. DAPI is a nuclear staining agent.

[0030] Figure 13 This is a graph illustrating the effects of compounds 20, 21, and 23 on radiation-induced DNA double-strand break (DSB) repair in LNCaP cell lines after staining with γH2AX at 1 nM for 1 hour. LNCaP cell lines were treated with the compounds by IR 2 Gy for 1 hour. Foci were counted in each cell. This graph was generated using data from three independent experiments. γH2AX is a marker of DNA breakage.

[0031] Figure 14 This is a graph illustrating the effects of compounds 20, 21, and 23 on radiation-induced DNA double-strand break (DSB) repair in the DU145 cell line. The DU145 cell line was treated with the compounds at IR 2 Gy for 1 hour. Foci were counted in each cell. This graph was generated using data from three independent experiments. γH2AX is a marker of DNA breakage.

[0032] Figure 15 This is a graph illustrating the effects of compounds 20, 21, and 23 on radiation-induced DNA double-strand break (DSB) repair in the PC-3 cell line. The PC-3 cell line was treated with the compounds at IR 2 Gy for 1 hour. Foci were counted in each cell. This graph was generated using data from three independent experiments. γH2AX is a marker of DNA breakage.

[0033] Figure 16 This graph was generated using data from the tumor regression analysis study described in this article. The arrows indicate the dates of beam radiation.

[0034] Figure 17 It is a weight measurement chart. Detailed Implementation

[0035] Reference will now be made to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the exemplary subject matter is not intended to limit the claims to the disclosed subject matter.

[0036] Conjugates or compounds of the formulas TLA and TLX are provided (where “conjugate” and “compound” are used interchangeably), wherein each L may be the same or different. T is a ligand targeting prostate-specific membrane antigen (PSMA), L is a linker, X is a radiotherapy agent or (radio)imaging agent or contains a radiotherapy agent or (radio)imaging agent, and A contains a DNA repair enzyme inhibitor, wherein the DNA repair enzyme inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor, an ataxia-telangiectasia mutation (ATM) inhibitor, or a DNA protein kinase (DNA-PK) inhibitor.

[0037] The conjugates TLA and TLX are typically administered at different times and at different frequencies, both of which can be adjusted by the physician. For example, the two conjugates may often be administered at different times because they compete with the same target (e.g., PSMA), thus affecting the uptake of each conjugate at the target site. The two conjugates may also often be administered at different frequencies. For example, TLX may be administered at a lower frequency because it contains a radiation therapy agent (e.g., once every 6 weeks as a cycle), while TLA may be repeatedly administered during treatment to maintain inhibition of DNA repair enzymes that repair DNA damage caused by TLX irradiation.

[0038] Regardless of whether the TLA compound contains a PRMT5 inhibitor or an ATM inhibitor, the compound will prevent irradiation-induced DNA break repair by the TLX compound, which contains a chelated radioisotope / radiotherapy agent. Therefore, when A is a PRMT5 or ATM inhibitor, the TLA compound is expected to improve the efficacy of the TLX compound by preventing the repair of double-strand DNA breaks.

[0039] T may include the following parts: .

[0040] T may contain: Where R 1 and R 2 The carboxylic acid group is independently selected from hydrogen or optionally substituted groups, such as malonic acid, succinic acid, glutamic acid, 5-aminohexanoic acid, and adipic acid groups, wherein the carboxylic acid group is connected by an α-coated carbon atom relative to the carboxylic acid with an R-shaped group. 1 and R 2 Nitrogen linkage.

[0041] T can be a urea of ​​(a) an aminodicarboxylic acid or a derivative thereof and (b) an aminodicarboxylic acid or a derivative thereof, wherein (a) and (b) may be the same or different. An example of such a group of T includes: The asterisk indicates the carbon atom α of the carboxylic acid relative to the 5-aminohexanoic acid group.

[0042] L can be any suitable adapter. In one instance, L can be a "non-releasable adapter" or a "non-cleavable adapter." A "non-releasable adapter" or "non-cleavable adapter" refers to an adapter that cannot be cleaved under extracellular physiological conditions (e.g., pH-instable, acid-instable, oxidatively unstable, or enzyme-instable bonds). However, such an adapter may contain bonds that can be cleaved after entering the cell.

[0043] In another instance, L can be a "releasable linker". A "releasable linker" is a linker containing at least one bond that can be broken under physiological conditions (e.g., a bond that is pH unstable, acid unstable, oxidatively unstable, or enzyme unstable). The releasable group also includes a photochemically cleavable group. Some examples of photochemically cleavable groups include 2-(2-nitrophenyl)-ethyl-2-ol groups containing a linker of o-nitrobenzyl, diphenylacetone, trans-o-cinnamoyl, m-nitrophenyl, or benzylsulfonyl (see, for example, Dorman and Prestwich, Trends Biotech. 18:64-77 (2000); Greene and Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, New York (1991); and U.S. Patent Nos. 5,143,854; 5,986,076; 5,917,016; 5,489,678; and 5,405,783, the entire teachings of which are expressly incorporated herein by reference).

[0044] L may comprise an atomic chain of about 3 atoms to about 30 atoms (e.g., about 3 atoms to about 7 atoms, about 5 atoms to about 15 atoms, about 5 atoms to about 25 atoms, about 5 atoms to about 12 atoms, about 7 atoms to about 15 atoms, about 7 atoms to about 12 atoms, about 7 atoms to about 15 atoms, or about 10 atoms to about 30 atoms). L may comprise an atomic chain of about 5 Å to about 45 Å. L may comprise a peptide. L may comprise one or more phenylalanine residues, each of which may be optionally substituted independently. L may comprise at least one phenylalanyl-phenylalanyl group, wherein at least one phenyl group may be optionally substituted independently. L may comprise a polyethylene oligomer. n (POEG) n ), polyethylene glycol n (PEG)n ), or mixtures thereof, wherein n = 1 to 36.

[0045] In some embodiments, L may contain at least one linker group, each linker group being selected from polyethylene glycol (PEG), alkyl groups, sugars, and peptides. In some embodiments, the linker is a dual linker based on PEG (e.g., PEGylated), alkyl groups, sugars, and peptides.

[0046] The linker can be any suitable linker. For example, in some embodiments, the linker is a hydrophilic linker, such as one or more linkers comprising amino acids (same or different), alkyl chains, PEG monomers, PEG oligomers, PEG polymers, or any combination thereof. In some embodiments, the linker comprises peptidoglycan, polysaccharide, or anionic oligomers. For linkers comprising one or more PEG units, unless otherwise specified, all carbon and oxygen atoms of the PEG unit are part of the backbone. The “backbone” of linker L can be the shortest chain of continuous atoms forming covalent bonds between T and X and / or T and A. In some embodiments, multivalent linkers have a branched backbone, wherein each branch serves as part of the backbone linker up to the end.

[0047] The L group described herein may have any suitable length and chemical composition. For example, the chain length of L may be at least about 7 atoms. In one variation, the length of L is at least about 10 atoms. In another variation, the length of L is at least about 14 atoms. In yet another variation, the length of L is about 7 to about 31 atoms, about 7 to about 24 atoms, or about 7 to about 20 atoms. In yet another variation, the length of L is about 14 to about 31 atoms, about 14 to about 24 atoms, or about 14 to about 20 atoms. In yet another variation, the chain length of L may be at least 7 atoms, at least 14 atoms, at least 20 atoms, at least 25 atoms, at least 30 atoms, at least 40 atoms, 1 to 15 atoms, 1 to 5 atoms, 5 to 10 atoms, 5 to 20 atoms, 10 to 40 atoms, or 25 to 100 atoms. An example of an L group with a chain length of 1 to 5 atoms is a group of the following formula:

[0048]

[0049] Where R 1 It can be H, alkyl, arylalkyl, -alkyl-S-alkyl or arylalkyl, or a side chain of any naturally or non-naturally occurring amino acid, etc.; and the number represents the atoms counted as part of the chain, which in this example is 3 atoms. R 1Some examples include H (glycine), alkyl groups (e.g., alanine, valine, isoleucine, and leucine), -alkyl-S-alkyl groups (e.g., methionine), arylalkyl groups (e.g., phenylalanine, tyrosine, tryptophan, and naphthylalanine), etc. (and R) 1 The atoms that are connected can be chiral and can have any suitable relative configuration, such as D-configuration or L-configuration.

[0050] The atoms used to form L can be combined in all chemically relevant ways, such as chains of carbon atoms forming alkylene groups, chains of carbon and oxygen atoms forming polyoxyalkylene groups, chains of carbon and nitrogen atoms forming polyamines, etc. Furthermore, it should be understood that the bonds connecting the atoms in the chain can be saturated or unsaturated, such that alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, etc., can be divalent groups included in L. Additionally, it should be understood that the atoms forming the linker can also cyclize each other to form saturated or unsaturated divalent cyclic groups in the linker, such as groups of the following formula:

[0051] ,

[0052] Each X 2 Independently CH2, N (when it exists with X) 2 When the bond is connected (NH or O), and each X 3 Independently N, C (when there exists a relationship with X) 3 (When the bond is linked) or CH. In each of the foregoing and other L groups described herein, the chain forming the linker may be substituted or unsubstituted.

[0053] As an alternative or supplement to the chain length, L may have any suitable substituents that can affect the hydrophobicity or hydrophilicity of L. Thus, for example, L may have hydrophobic side chain groups, such as alkyl, cycloalkyl, aryl, arylalkyl, or similar groups, each optionally substituted. If L contains one or more amino acids, L may contain hydrophobic amino acid side chains, such as one or more amino acid side chains from phenylalanine (Phe) and tyrosine (Tyr), including their substituted variants, as well as analogues and derivatives of such side chains.

[0054] L may contain a portion that is neutral under physiological conditions. However, L may contain portions that can be protonated or deprotonated to carry one or more positive charges or one or more negative charges, respectively. Alternatively, L may contain a neutral portion and a portion that can be protonated to carry one or more positive charges. Some examples of neutral portions include polyhydroxy groups such as sugars, carbohydrates, glycosides, inositol, etc., and / or polyether groups such as polyoxyethylene groups, including polyethylene oxide, polypropylene oxide, etc. Some examples of portions that can be protonated to carry one or more positive charges include amino groups, such as polyaminoalkylene groups, including ethylenediamine, propylenediamine, butanediamine, etc., and / or heterocycles, including pyrrolidine, piperidine, piperazine, and other amino groups, each of which may optionally be substituted. Some examples of portions that can be deprotonated to carry one or more negative charges include carboxylic acids, such as aspartic acid, glutamic acid, and longer-chain carboxylic acid groups, and sulfate esters, such as alkyl esters of sulfuric acid.

[0055] Exemplary polyoxyethylene groups include those with a specific length range of about 4 to about 20 polyoxyethylene (e.g., polyethylene glycol) groups. Exemplary alkyl sulfates can also be directly introduced into the backbone using click chemistry. Exemplary L groups comprising polyamines include L groups comprising EDTA and DTPA groups:

[0056]

[0057] (Poly)peptides:

[0058]

[0059] β-amino acids, etc.

[0060]

[0061] and its combinations, where each R 2 Independently, H, alkyl, arylalkyl, heterocyclic alkyl, ureyl, aminoalkyl, alkylthio, or amide alkyl groups are used, for example, in the side chains of naturally occurring amino acids such as alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, methionine, lysine, arginine, and histidine. Non-naturally occurring amino acids are also considered in this document.

[0062] The L group may have any suitable molecular weight, for example, about 30 g / mol to about 1,000 g / mol, about 30 g / mol to about 300 g / mol, about 100 g / mol to about 500 g / mol, or about 150 g / mol to about 600 g / mol.

[0063] The terms “non-releasable adapter” or “non-cleavable adapter” are used interchangeably. As used herein, it refers to an adapter that is non-cleavable under extracellular physiological conditions, such as pH instability, acid instability, oxidative instability, or enzyme instability. However, such an adapter may contain bonds that are cleavable after entering the cell.

[0064] L may comprise carbonyl, thiocarbonyl, alkylene, cycloalkylene, aminoalkylene, alkylcycloalkylene, alkylcycloalkylene carbonyl, aminoalkylcycloalkylene carbonyl, alkylene carbonyl, cycloalkylene carbonyl, carbonylalkylene, 1-alkylsuccinimide-3-yl, 1-(carbonylalkyl)succinimide-3-yl, alkylsulfonyl, sulfonylalkyl, alkylsulfonylalkyl, alkylsulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl)succinimide-3-yl or 1-(carbonyltetrahydrofuranyl)succinimide-3-yl, each optionally substituted, and combinations thereof. In this example, L may also comprise additional nitrogen (e.g., -NR). 3 -, where R 3 L may be H or alkyl, such that L comprises an alkylene carbonyl group, a cycloene alkylene carbonyl group, a carbonyl alkyl carbonyl group, or a 1-(carbonylalkyl)succinimide-3-yl group, each of which may optionally be substituted and bonded to nitrogen to form an amide. Alternatively, L may also comprise a sulfur atom and an alkylene or cycloene alkyl group, each of which may optionally be substituted with a carboxyl group and may bond to sulfur to form a thiol. In another example, L comprises a sulfur atom and a 1-alkylene succinimide-3-yl and a 1-(carbonylalkyl)succinimide-3-yl group bonded to sulfur to form a succinimide-3-yl thiol.

[0065] L may contain alkyleneaminoalkylenecarbonyl, alkylene-thio-(carbonylalkylsuccinimide-3-yl), alkylenecycloalkylenecarbonyl, aminoalkylenecycloalkylenecarbonyl, alkylenecarbonyl, cycloalkylenecarbonyl, and combinations thereof, as further illustrated by the following formula:

[0066] , where the asterisk indicates the point of connection with a group present in L, T, A or X; and where x and y are each independently 1, 2, 3, 4 or 5.

[0067] L can have any suitable type of atom in the chain, including C (e.g., -CH2-, C(O)) and N (e.g., NH, NR). 4 , where R 4These include, for example, H, alkyl, alkylaryl, etc.), O (e.g., -O-), P (e.g., -OP(O)(OH)O-), and S (e.g., -S-). For example, the atoms used to form L can be combined in all chemically relevant ways, such as chains of carbon atoms forming alkyl groups, chains of carbon and oxygen atoms forming polyoxyalkyl groups, chains of carbon and nitrogen atoms forming polyamines, etc., including rings, such as those forming aryl and heterocyclic groups (e.g., triazoles, etc.). (e.g., azoles). Furthermore, the bonds connecting atoms in the chain within L can be saturated or unsaturated, allowing for the inclusion of divalent groups such as alkanes, alkenes, alkynes, cycloalkanes, aromatic hydrocarbons, and imides within L. Additionally, the chain forming L can be, for example, -N(R... 4 )2 groups are substituted or not substituted.

[0068] Other examples of L include L groups comprising the following groups: 1-alkylsuccinimide-3-yl, carbonyl, thiocarbonyl, alkyl, cycloalkyl, alkylcycloalkyl, alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-alkylsuccinimide-3-yl, 1-(carbonylalkyl)succinimide-3-yl, alkyl sulfoxide, sulfonylalkyl, alkyl sulfoxide alkyl, alkylsulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl)succinimide-3-yl, and 1-(carbonyltetrahydrofuranyl)succinimide-3-yl, wherein each group may be substituted or unsubstituted. Any of the above groups may be L or may be included as part of L. In some cases, any of the above groups may be used in combination (or more than once) (e.g., -alkyl-C(O)-alkyl) and may also contain additional nitrogen (e.g., alkyl-C(O)-NH-, -NH-alkyl-C(O)-, or -NH-alkyl-), oxygen (e.g., -alkyl-O-alkyl-), or sulfur (e.g., -alkyl-S-alkyl-). Some examples of such L groups are alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimide-3-yl, and succinimide-3-ylthiol, wherein each group may be substituted or unsubstituted.

[0069] L may contain one or more of the following groups in any suitable combination: , where R a It is an aryl group (such as phenyl and naphthyl) or a heteroaryl group.

[0070] If, for example, release of A in vivo is desired, then a conjugate of formula TLA may contain a releasable connector of L. Releasable connectors of L are well known in the art.

[0071] L can be a "releaseable linker" that can be cleaved by enzymes. The enzyme can be a cathepsin, metalloproteinase, esterase, phosphatase, or pyrophosphatase. L can be cleaved by reactive oxygen species (ROS). L can be a p-aminophenol ether. L can be cleaved through hypoxia activation. L can be a quinone, nitroaromatic, aliphatic N-oxide, or heteroaromatic N-oxide.

[0072] One or more cleavable bonds may be present within the cleavable linker and / or at one or both ends of the cleavable linker. It should be understood that such physiological conditions leading to bond cleavage include, for example, standard chemical hydrolysis reactions occurring at physiological pH or due to compartmentalization into organelles (e.g., endosomes with a pH lower than the cytosol pH). Illustratively, the divalent linker can be cleaved under other physiological or metabolic conditions, for example, through the action of a glutathione-mediated mechanism. It should be understood that the instability of the cleavable bond can be modulated by including functional groups or segments in the divalent linker L that can assist or promote such bond cleavage, also known as ortho-assisted action. The instability of the cleavable bond can also be modulated by, for example, substitution changes at or near the cleavable bond, such as including α-branching adjacent to the cleavable disulfide bond, increasing the hydrophobicity of the substituent on the silicon in the portion having a hydrolyzable silicon-oxygen bond, homologating the alkoxy group to form a portion of a hydrolyzable ketal or acetal, etc. Additionally, it should be understood that the divalent linker L may contain additional functional groups or fragments that can assist or promote further fragmentation of the PSMA-binding drug linker conjugate after the bond breakage of the releasable linker (when present).

[0073] In one instance, L may contain one or more releasable linkers, which are cleaved under the conditions described herein via a chemical mechanism involving β-elimination. Such releasable linkers include β-thio, β-hydroxy, and β-amino substituted carboxylic acids and their derivatives, such as esters, amides, carbonates, carbamates, and ureas. Such linkers also include 2- and 4-thioaryl esters, carbamates, and carbonates.

[0074] An example of a releasable connector includes a connector of the following type:

[0075]

[0076] Where n is an integer selected from 0, 1, 2, and 3, and R 5 It is H or alkyl, R 6 It is hydrogen or a substituent, including substituents that can inductively or resonantly stabilize the positive charge on the aryl ring, such as alkoxy groups. The releasable linker can be further substituted.

[0077] Assisted cleavage of the releasable portion of L may include involving benzyl Intermediate, benzylene intermediate, lactone cyclization, oxygen Mechanisms such as intermediates and β-elimination. Besides fragmentation following the cleavage of the releasable portion of L, the initial cleavage of the releasable linker can also be promoted through an adjacent auxiliary mechanism. Therefore, in the examples of the releasable portion of L given above, the cyclizable hydroxyalkanoic acid is cleaved via, for example, oxygen... Ions promote the cleavage of methylene bridges and facilitate bond cleavage or subsequent fragmentation following bond cleavage of releasable joints. Alternatively, acid-catalyzed oxygenation of methylene bridges... Ion-assisted cleavage can initiate the cascade fragmentation of this illustrative divalent linker or its fragments. Alternatively, acid-catalyzed hydrolysis of carbamates can promote the β-elimination of cyclizable hydroxyalkanoic acids, and via, for example, oxygen... Ions promote the cleavage of methylene bridges. It should be understood that other chemical mechanisms of bond breaking or cleavage under metabolic, physiological, or cellular conditions can trigger such cascade fragmentation.

[0078] Illustrative mechanisms for cutting divalent joints include the following 1,4 and 1,6 fragmentation mechanisms for carbonates and urethanes:

[0079]

[0080] Among them, Nuc - It is an exogenous or endogenous nucleophile, glutathione, or bioreducing agent, etc., and R 7 One of Z is connected to T (or X) via other parts of a divalent connector, and the other is connected to X (or T) via other parts of a divalent connector. 7 The positions of Z and Z can be interchanged, such that the resulting products are, for example, ZS-Nuc and HO-R. 7 or H2N-R 7 .

[0081] Although the fragmentation mechanism described above is a concerted mechanism, any number of discrete steps can occur to achieve the final cleavage of the divalent linker into the final product shown. For example, bond cleavage can also occur via acid-catalyzed elimination of the urethane moiety, which can be facilitated by stabilization provided by the disulfide or aryl group of β-sulfur as illustrated in the examples above. In those variations of this embodiment, the releasable linker is the urethane moiety. Alternatively, fragmentation can be initiated by nucleophilic attack on the disulfide group, resulting in cleavage to form a thiolate. The thiolate can intermolecularly displace the carbonic acid or urethane moiety and form the corresponding thiohexacyclopropane. In the case of a divalent linker containing a benzyl group, following the illustrative cleavage of the disulfide bond, the resulting phenyl thiolate can be further fragmented to release the carbonic acid or urethane moiety moiety by forming a resonance-stabilized intermediate. In any of these cases, the releasable nature of the illustrative divalent linker can be achieved by any mechanism that can be associated with the present chemical, metabolic, physiological, or biological conditions.

[0082] Therefore, as described above, the releasable connector may contain a disulfide group. Other examples of releasable connectors contained in L may include divalent groups comprising alkylaziridin-1-yl, alkylcarbonylaziridin-1-yl, carbonylalkylaziridin-1-yl, alkylsulfonylaziridin-1-yl, sulfonylalkylaziridin-1-yl, sulfonylalkylaziridin-1-yl, or alkylsulfonylaziridin-1-yl, wherein each releasable connector may optionally be substituted. Other examples of releasable connectors may include divalent groups comprising methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylene carbonyl, 1-alkoxycycloalkylene carbonyl, carbonylaryl carbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(dicarboxyaryl)carbonyl, haloalkylene carbonyl, alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, (diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl, iminoalkylene, carbonylalkylimino, iminocycloalkyl, carbonylcycloalkylimino, alkylthio, alkylarylthio, or carbonylalkylthio, wherein each releasable connector may optionally be substituted.

[0083] Other examples of the releasable connectors included in L may include an oxygen atom and a methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylene carbonyl, or 1-alkoxycycloalkylene carbonyl, wherein each releasable connector may optionally be substituted. Alternatively, the releasable connector may include an oxygen atom and a methylene, wherein the methylene may be substituted with an aryl group, and the releasable connector may bond with oxygen to form an acetal or ketal. Furthermore, the releasable connector may include an oxygen atom and a sulfonyl alkyl group, and the releasable connector may bond with oxygen to form an alkyl sulfonate.

[0084] Other examples of releasable connectors included in L may include nitrogen (e.g., -NR) 5 -, where R 5 (H or alkyl) and iminoalkylene, carbonylalkylimino, iminocycloalkylene, and carbonylcycloalkylimino, wherein each releasable linker may optionally be substituted and the releasable linker may be bonded to nitrogen to form a hydrazone. In an alternative configuration, the hydrazone may be acylated with a carboxylic acid derivative, an orthoformate derivative, or a carbamoyl derivative to form a variety of acylhydrazone releasable linkers.

[0085] Other examples of releasable connectors included in L may include an oxygen atom and an alkylene (dialkylsilyl), alkylene (alkylarylsilyl), alkylene (diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, or (diarylsilyl)aryl, wherein each releasable connector may optionally be substituted and the releasable connector may be bonded to oxygen to form a silanol.

[0086] Other instances of releasable connectors included in L may include two separate nitrogen atoms (e.g., -NR) 5 -) and carbonylaryl carbonyl, carbonyl (carboxyaryl) carbonyl, carbonyl (dicarboxyaryl) carbonyl, and can release the linker to bond with heteroatom nitrogen to form amides, and also through amide bonds with Z or R 7 Bonding.

[0087] Other examples of releasable connectors included in L may include oxygen atoms, nitrogen (e.g., -NR) 5 -) and carbonyl aryl carbonyl, carbonyl (carboxyaryl) carbonyl, carbonyl (dicarboxyaryl) carbonyl, and can release the linker to form amides, and also through the amide bond with Z or R 7 Bonding.

[0088] The linker L may also contain a group that improves tumor uptake of conjugates of formula TLA, although conjugates of formula TLX may also contain such a group linked to L. Some examples of groups that improve tumor uptake of conjugates of formula TLA and / or TLX include albumin-binding groups of the following formula:

[0089] For example, the following groups:

[0090] Such a group can be attached to L by any suitable substituent present on L, including -NR. 4 - group, where R 4 It is an H or alkyl group, or an -O- group; or it is formed by the presence of -NR on the L. 4 - The group of the group is connected to L, where R 4 It is an H or alkyl group, or an -O- group.

[0091] A may contain a radiosensitizer. The radiosensitizer may be selected from topoisomerase inhibitors, hypoxia-activated anthraquinones (AQ4N), alkylating agents, and DNA repair enzyme inhibitors. Topoisomerase inhibitors may be camptothecin or topotecan. Alkylating agents may be temozolomide. DNA repair enzyme inhibitors may be PRMT5, ATM, or DNA-PK inhibitors.

[0092] A may contain a radiosensitizer for inhibiting double-stranded DNA repair, such as a protein arginine methyltransferase 5 (PRMT5) inhibitor. An example of a PRMT5 inhibitor that can be used as a radiosensitizer base is JNJ-64619178, which has the following formula:

[0093] ;

[0094] It is GSK3326595, which has the following formula:

[0095] ;as well as

[0096] It is GSK336871, which has the following formula:

[0097]

[0098] Or the medicinal salt of each of the aforementioned.

[0099] Therefore, for example, conjugates of formula TLA include the formula:

[0100] Conjugates or their pharmaceutically acceptable salts, for example:

[0101] Conjugates or their medicinal salts;

[0102] as well as

[0103] Conjugates or their pharmaceutically acceptable salts, for example:

[0104] Conjugates or their medicinal salts.

[0105] These conjugates are examples of dual-targeting conjugates of prostate-specific membrane antigen (PSMA) as targeted radionuclide therapy (TRT) and PRMT5 as a PRMT5 inhibitor. The first conjugate contains a fragment of JNJ-64619178, and the second conjugate contains a fragment of GSK3326595.

[0106] Other PRMT5 inhibitors that may be included in A include those described in Zhu et al., Journal of Computer-Aided Molecular Design 33: 775-785 (2019) (which is incorporated by reference as fully elaborated herein), and include JNJ-64619178 and GSK3326595: .

[0107] A may contain radiosensitizers for inhibiting double-stranded DNA repair, such as ataxia-telangiectasia mutation (ATM) inhibitors. Some examples of ATM inhibitors that can be used as a radiosensitizer base include Cinobufagin, AZD0156, AZD1390, AZ32, KU-60019, KU-55933, Wortmannin, CP-466722, AZ31, chloroquine diphosphate, Mirin, Torin 2, CGK 733, and ETP-46464. Other ATM inhibitors include AZD-7648, VE-821, Camonsertib, Lartesertib, Ro 90-7501, ATM Inhibitor-1, ATM Inhibitor-2, ATM Inhibitor-3, ATM Inhibitor-4, AO11, and ATR-IN-15.

[0108] Therefore, for example, conjugates of formula TLA include the formula: Conjugates or their pharmaceutically acceptable salts, for example: Conjugates or their medicinal salts.

[0109] A may contain a radiosensitizer, such as a DNA protein kinase (DNA-PK) inhibitor. Some examples of DNA-PK inhibitors that can be used as the basis for radiosensitizers include Nedisertib, AZD7648, KU57748, Torin 2, PI-103, NU7026, Samotolisib, PIK 90, CC-115, PIK-75 hydrochloride, VX-984, KU-0060648, BAY-8400, DNA-PK-IN-2, DNA-PK-IN-1, DNA-PK-IN-4, DNA-PK-IN-5, DNA-PK-IN-6, XRD-0394, NU5455, STL127705, PI-103 hydrochloride, LTURM34, ZL-2201 free base, DNA-PK-IN-14, DNA-PK-IN-10, DNA-PK-IN-11, and DNA-PK-IN-13.

[0110] An example of a conjugate of formula TLX includes a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0111] ,For example

[0112]

[0113] It is sometimes referred to as "PSMA-617".

[0114] X may contain a radioactive isotope that binds to a chelating agent, such that X is a radiotherapy agent or (radio) imaging agent, or contains a radiotherapy agent or (radio) imaging agent, wherein the chelating agent is selected from:

[0115] DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or its derivatives;

[0116] TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) or its derivatives;

[0117] SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) or its derivatives;

[0118] NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) or its derivatives;

[0119] NETA (4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl)acetic acid or a derivative thereof;

[0120] TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphonic acid) or its derivatives;

[0121] HBED (N,NO-bis(2-hydroxybenzyl)-ethylenediamine-N,NO-diacetic acid) or its derivatives;

[0122] 2,3-HOPO (3-hydroxypyridin-2-one) or its derivatives;

[0123] PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadecane-1(15),11,13-triene-3,6,9-triacetic acid) or its derivatives;

[0124] DFO (deferriphosphate) or its derivatives;

[0125] DTPA (diethylenetriaminepentaacetic acid) or its derivatives;

[0126] OCTAPA (N,NO-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,NO-diacetic acid) or its derivatives; or H2-MACROPA (N,N'-bis[(6-carboxy-2-pyridylmethyl]-4,13-diaza-18-crown-6) or its derivatives;

[0127] H2dedpa (1,2-[[carboxyl)-pyridin-2-yl]-methylamino]ethane or a derivative thereof; and

[0128] EC20-head contains β-l-diaminopropionic acid, aspartic acid, and cysteine.

[0129] X may contain radioactive isotopes selected from the following: .

[0130] In view of the above, a conjugate of formula TL-(X)A is also provided, wherein:

[0131] T is a ligand that targets prostate-specific membrane antigen (PSMA).

[0132] L stands for connector.

[0133] A contains a DNA repair enzyme inhibitor, wherein the DNA repair enzyme inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor, an ataxia-telangiectasia mutation (ATM) inhibitor, or a DNA protein kinase (DNA-PK) inhibitor; and

[0134] X is a radiotherapy agent or a radioimaging agent.

[0135] A and X can be connected to L.

[0136] The conjugate may also contain an albumin-binding group. The albumin-binding group may have the following formula: .

[0137] The albumin-binding group may be linked to L via a substituent or group present on L, wherein the albumin-binding group is optionally linked to L as described above. T is as defined above. L is as defined above. A is as defined above.

[0138] An example of a compound of formula TL-(X)A is a compound of the following formula: For example, the following compounds: .

[0139] The above compounds can be synthesized according to methods known in the art.

[0140] Pharmaceutical compositions comprising the above-described conjugates (e.g., conjugates of formula TLA, formula TLX, and combinations thereof) and a pharmaceutically acceptable carrier are also provided. Pharmaceutical compositions comprising conjugates of formula TL-(X)A are also provided. The pharmaceutical compositions can be prepared according to methods known in the art. Combinations of conjugates or combinations of conjugates with other active agents may also be combined with a pharmaceutically acceptable carrier.

[0141] A method for radiographic imaging of a patient with prostate cancer or its metastases is also provided. This method includes (i) administering a conjugate of formula TLX or a pharmaceutical composition containing thereof to the patient, and (ii) obtaining radiographic images of the patient. Even a method for radiographic imaging of a patient with prostate cancer or its metastases is provided. This method includes (i) administering a conjugate of formula TL-(X)A or a pharmaceutical composition containing thereof to the patient, and (ii) obtaining radiographic images of the patient.

[0142] It even provides methods for treating patients with prostate cancer or its metastases using radiation therapy. These methods include administering conjugates (such as conjugates of formula TLA, formula TLX, and combinations thereof) or pharmaceutical compositions containing them to the patient. The method may also include administering immune checkpoint inhibitors to the patient. Immune checkpoint inhibitors can be selected from T lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein 1 (PD-1) inhibitors, programmed cell death ligand 1 (PD-L1) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, T cell immunoglobulin and mucin-domain containing-3 (TIM-3) inhibitors, V-domain Ig suppressor of T cell activation (VISTA), B- and T- lymphocyte attenuator (BTLA), and T cell immunoglobulin and ITIM domain (TIGIT) inhibitors. Immune checkpoint inhibitors can be selected from nivolumab (Opdivo), pembrolizumab (Keytruda), ipilimumab (Yervoy), atezolizumab, avelumab, and durvalumab.

[0143] It even provides a method for treating patients with prostate cancer or its metastases using radiation therapy. This method involves administering a conjugate of formula TL-(X)A or a pharmaceutical composition containing it to the patient.

[0144] PSMA is not entirely confined to prostate tissue. It is expressed in other cancers, more specifically in the neovascularization associated with these cancers (Silver et al., Clin Cancer Res 3: 81-85 (1997); and Liu et al., Cancer Res 57: 3629-3634 (1997)). Persistent and intense expression of PSMA has been reported in the neovascularization of conventional (clear cell) kidney cells, bladder transitional cells, testicular embryos, neuroendocrine cells, colon, and mammary glands (Chang et al., Clin Cancer Res 5: 2674-2681 (1999), ibid.). Anti-PSMA mAbs also persistently bind to duodenal epithelial (brush border) cells and proximal tubular cells in the kidneys (Liu et al. (1997), ibid.; and Chang et al. (1999), ibid.). Therefore, the aforementioned approach to treating patients (i.e., those with prostate cancer or its metastases) could be used to treat other cancers.

[0145] Conjugates of formula TLA, formula TLX, and combinations thereof can also be used in these methods, provided that "compounds for targeted radionuclide therapy (TRT)" are used. Therefore, conjugates of formula TLA, formula TLX, and combinations thereof can be used for: methods for treating cancer patients, comprising the step of administering a therapeutically effective amount of a compound for targeted radionuclide therapy (TRT), wherein the compound for targeted radionuclide therapy (TRT) also acts as a protein arginine methyltransferase 5 (PRMT5) inhibitor; methods for treating patients with metastatic castration-resistant prostate cancer (mCRPC), comprising the step of administering a therapeutically effective amount of a compound that dually targets PSMA as targeted radionuclide therapy (TRT) and PRMT5 as a PRMT5 inhibitor; methods for treating cancer patients, comprising the step of administering a therapeutically effective amount of targeted radionuclide therapy (TRT) and a therapeutically effective amount of a PRMT5 inhibitor; or methods for treating mCRPC patients, comprising the step of administering a therapeutically effective amount of a PSMA-based TRT and a therapeutically effective amount of a PRMT5 inhibitor, wherein the TRT is optionally a PSMA-based TRT. This method can improve the efficacy of targeted radiotherapy and reduce toxicity caused by higher radiation doses taken up by other PSMA-expressing tissues (mainly salivary glands).

[0146] Also provided are pharmaceutical compositions for treating patients with mCRPC comprising compounds that dually target PSMA as a TRT and PRMT5 as a PRMT5 inhibitor, and one or more diluents, excipients, or carriers, wherein said compounds have a formula or are pharmaceutically acceptable salts thereof; pharmaceutical compositions for treating cancer patients comprising the steps of administering a therapeutically effective amount of TRT and a therapeutically effective amount of a PRMT5 inhibitor; and pharmaceutical compositions for treating cancer patients comprising the steps of administering a therapeutically effective amount of TRT and a therapeutically effective amount of a PRMT5 inhibitor as a radiosensitizer. In some embodiments, JNJ-64619178 may be a PRMT5 inhibitor, but it may be replaced by any other type of PRMT5 inhibitor. JNJ-64619178 is also used as a radiosensitizer, and it may be replaced by any other type of radiosensitizer (e.g., AR inhibitors or those that inhibit key DNA damage response proteins). If the proposed method is superior to existing treatments (surgery, radiation therapy, or radiation therapy plus androgen derivation therapy), the proposed method may be used for hormone-naïve prostate cancer expressing PSMA. If the target molecule PSMA-617 is replaced by other target molecules, the proposed approach can be used to treat any other type of cancer. The empirically proven effects of JNJ-64619178 or any other PRMT5 inhibitor can also be used alone in combination with other radiotherapy and chemotherapy. PRMT5 also promotes DSB repair induced by chemotherapy (e.g., etoposide). Therefore, PRMT5 inhibitors such as JNJ-64619178 or other PRMT5 inhibitors can also be used as chemosensitizers in combination cancer therapies.

[0147] The terms "substituted" or "substituent" and "functional group" refer to groups that can be substituted or replaced on a molecule or on another group (e.g., an aryl or alkyl group). Some examples of substituents include, but are not limited to, halogens (e.g., F, Cl, Br, and I), OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azide, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, etc. Each R can be independently hydrogen, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclic, heteroaryl, or heteroarylalkyl, wherein any alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclic, heteroaryl, or heteroarylalkyl, or two R groups bonded to a nitrogen atom or to an adjacent nitrogen atom can form a heterocyclic group together with one or more nitrogen atoms, which can be monosubstituted or independently polysubstituted.

[0148] As used in this article, the term "alkyl" refers to an alkyl group having 1 to 40 carbon atoms (C1-C4). 40 ), 1 to 20 carbon atoms (C1-C 20 ), 1 to 12 carbon atoms (C1-C 12 Alkyl groups are substituted or unsubstituted straight-chain and branched monovalent or divalent alkyl and cycloalkyl groups having 1 to 8 carbon atoms (C1-C8) or, in some embodiments, 1 to 6 carbon atoms (C1-C6). Some examples of straight-chain alkyl groups include those having 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Some examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl. The term "alkyl" as used herein covers n-alkyl, isoalkyl, and trans-isoalkyl, as well as other branched forms of alkyl groups. Representative substituted alkyl groups may be substituted once or more with any of the groups listed herein, such as amino, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen groups.

[0149] As used in this article, the term "alkenyl" refers to an alkyl group having at least one double bond and having 1 to 40 carbon atoms (C1-C4). 40 ), 1 to 20 carbon atoms (C1-C 20 ), 1 to 12 carbon atoms (C1-C 12 Alkenes can be substituted or unsubstituted straight-chain and branched monovalent or divalent alkenyl and cycloalkenyl groups having 1 to 8 carbon atoms (C1-C8) or, in some embodiments, 1 to 6 carbon atoms (C1-C6). Examples of straight-chain alkenyl groups include those having 1 to 8 carbon atoms, such as -CH=CH-, -CH=CHCH3, and -CH2CH=CHCH2- groups, wherein the double bonds may have an E-configuration or a Z-configuration. And when multiple bonds are present, each double bond may independently have an E-configuration or a Z-configuration. Examples of branched alkenyl groups include, but are not limited to, -CH=C(CH3)- and CH2C=CH(CH3) groups. Representative substituted alkenyl groups may be substituted once or more with any of the groups listed herein, such as amino, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen groups.

[0150] As used herein, the term "cycloalkyl" refers to a substituted or unsubstituted cycloalkyl group, such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the cycloalkyl group may have 3 to about 8-12 ring members, while in other embodiments, the number of ring carbon atoms is 3 to 4, 5, 6, or 7. The cycloalkyl group may have any number of carbon atoms, such as 3 to 8 carbon atoms (C3-C8), 3 to 6 carbon atoms (C3-C6), and 4 to 8 carbon atoms (C4-C8). Cycloalkyl groups also include polycyclic cycloalkyl groups, such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and caretenyl; and fused rings, such as, but not limited to, decalinyl.

[0151] As used herein, the term "cycloalkylalkyl" means a substituted or unsubstituted alkyl group as defined herein, wherein the hydrogen or carbon bond of the alkyl group as defined herein is replaced by a cycloalkyl bond as defined herein. Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylalkyl groups.

[0152] As used herein, the term "alkylcycloalkyl" means a substituted or unsubstituted cycloalkyl group as defined herein, wherein the hydrogen atoms of the cycloalkyl group as defined herein are replaced by alkyl bonds as defined herein. Representative alkylcycloalkyl groups include, but are not limited to, alkylcyclopropyl groups.

[0153] As used herein, the term "acyl" refers to a group containing a carbonyl moiety, wherein the group is bonded by a carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which may be a subset of a substituted or unsubstituted alkyl, aryl, aralkylcycloalkyl, cycloalkylalkyl, heterocyclic, heterocyclic alkyl, heteroaryl, heteroaryl, or heteroarylalkyl group. In the specific case where the carbonyl carbon atom is bonded to hydrogen, the group is "formyl," which is an acyl as defined herein. An acyl may contain 0 to approximately 12-40, 6-10, 1-5, or 2-5 additional carbon atoms bonded to the carbonyl. Acryloyl is an example of an acyl. Acyls may also contain heteroatoms as defined herein. Nicotinyl (pyridyl-3-carbonyl) is an example of an acyl as defined herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl. When a group containing a carbon atom bonded to the carbonyl carbon atom contains a halogen, the group is called a "haloacyl" group. An example is the trifluoroacetyl group.

[0154] The term "heterocyclic carbonyl" is an example of an acyl group bonded to a substituted or unsubstituted heterocyclic group, as defined herein by the term "heterocyclic group". An example of a heterocyclic carbonyl group is a prolyl group, wherein the prolyl group may be D- or L-prolyl.

[0155] As used herein, the term "aryl" refers to a substituted or unsubstituted cyclic aromatic hydrocarbon that does not contain heteroatoms in its ring. Therefore, aryl includes, but is not limited to: phenyl, azulel, heptapenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylene, pyrene, napthacenyl, phenylene, biphenylene, anthracene, and naphthyl. In some embodiments, the aryl group contains about 6 to about 14 carbons (C6-C5) in the cyclic moiety of the group. 14 ) or 6 to 10 carbon atoms (C6-C 10 Aryl groups can be unsubstituted or substituted, as defined herein. The terms "aryl" and "aryl group" encompass fused rings, including those containing fused aromatic and non-aromatic groups. Therefore, "aryl" and "aryl group" include groups of the following formula: Each of them can be substituted or unsubstituted, such as hydroxylated.

[0156] Representative substituted aryl groups can be monosubstituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl groups or 2 to 8-substituted naphthyl groups, which can be substituted by carbon or non-carbon groups (such as those listed herein).

[0157] The terms “aralkyl” and “arylalkyl” refer to an alkyl group as defined herein, wherein the hydrogen or carbon bond of the alkyl group is replaced by an aryl bond as defined herein. Representative aralkyl groups include benzyl and phenethyl, as well as fused (cycloalkylaryl)alkyl groups, such as 4-ethyl-indenyl. Arylene is an alkenyl group as defined herein, wherein the hydrogen or carbon bond of the alkyl group is replaced by an aryl bond as defined herein.

[0158] The term "heterocyclic group" or "heterocycle" refers to a substituted or unsubstituted aromatic and non-aromatic cyclic compound containing three or more ring members, one or more of which (e.g., 1, 2, or 3) are heteroatoms, such as, but not limited to, N, O, and S. Therefore, a heterocyclic group can be a cycloheteroalkyl or heteroaryl group, or, if polycyclic, any combination thereof. In some embodiments, the heterocyclic group contains 3 to about 20 ring members, while other such groups have 3 to about 15 ring members. In some embodiments, the heterocyclic group includes heterocyclic groups containing 3 to 8 carbon atoms (C3-C8), 3 to 6 carbon atoms (C3-C6), 3 to 5 carbon atoms (C3-C5), or 6 to 8 carbon atoms (C6-C8). A heterocyclic group represented as a C2-heterocyclic group can be a 5-ring having two carbon atoms and three heteroatoms, a 6-ring having two carbon atoms and four heteroatoms, etc. Similarly, the C4-heterocyclic group can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, etc. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. The heterocyclic ring can also contain one or more double bonds, for example, in the groups 3,6-dihydro-2H-pyran and 3,4-dihydro-2H-pyran, which respectively have the following formulas: Each of them can be replaced.

[0159] A heterocyclic ring is one embodiment of a heterocyclic group. The phrase "heterocyclic group" includes fused rings, which include those comprising fused aromatic and non-aromatic groups. Representative heterocyclic groups include, but are not limited to, tetrahydro-2H-thiaran-1,1-dioxide having the following formula: It can be substituted; having the following formula: 4a,5,6,7-tetrahydro-4H-pyrrolo[1,2-d][1,3,4] Diazine group: It can be substituted with: pyrrolyl, pyrrolidone (e.g., pyrrolidone-2-one), azacyclic butyl, piperidinyl, piperazinyl, morpholinyl, chromanyl, indole-2-one, isoindole-2-one, furanyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiopheneyl, tetrahydrofuranyl, pyrroleyl. azole group, Diazolyl, imidazolyl, imidazo[1,2-a]pyridyl (having the following formula): It can be substituted; triazolyl, tetrazolyl, benzo[] Azolinyl, thiazolinyl, benzothiazolinyl, and benzimidazolinyl. An example of an indole-1,4-dimethyl ketone group includes a group having the following general formula: , where R is defined as in this paper.

[0160] Some examples of isoindoline ketone groups include groups having the following general formula:

[0161] , where R is defined as in this paper.

[0162] benzo[a] Some examples of the zolylinyl group include groups having the following general formula:

[0163] , where R is defined as in this paper.

[0164] Some examples of benzothiazolinyl groups include groups having the following general formula:

[0165] , where R is defined as in this paper.

[0166] In some implementations, benzo[a] The R group in the zolyl and benzothiazolyl groups is an N(R)2 group. In some embodiments, each R is hydrogen or an alkyl group, wherein the alkyl group is substituted or unsubstituted. In some embodiments, the alkyl group is substituted with a heterocyclic group (e.g., with pyrrolidinyl).

[0167] The term "heterocyclic alkyl" refers to an alkyl group as defined herein, wherein the hydrogen or carbon bond of the alkyl group as defined herein is replaced by a heterocyclic group as defined herein. Representative heterocyclic alkyl groups include, but are not limited to, furan-2-ylmethyl, furan-3-ylmethyl, pyridin-3-ylmethyl, tetrahydrofuran-2-ylmethyl, and indole-2-ylpropyl.

[0168] The term "heterocyclic alkoxy" refers to an alkyl group as defined herein, wherein the hydrogen or carbon bond of the alkyl group as defined herein is replaced by a heterocyclic group as defined herein, and the alkyl group is connected to an oxygen atom. Representative heterocyclic alkoxy groups include, but are not limited to, -O-(CH2). q Heterocyclic groups, where q is an integer from 1 to 5. In some embodiments, the heterocyclic alkyl alkoxy group includes -O-(CH2). q Morpholinyl, for example -O-CH2CH2-morpholinyl.

[0169] The term “heteroarylalkyl” means an alkyl group as defined herein, wherein the hydrogen or carbon bond of the alkyl group is replaced by a heteroaryl bond as defined herein.

[0170] The term "alkoxy" refers to an oxygen atom bonded to an alkyl group (including cycloalkyl groups) as defined herein. Examples of straight-chain alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, and hexoxy. Examples of branched-chain alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, and isohexoxy. Examples of cyclic alkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. An alkoxy group may contain 1 to approximately 12-20 or approximately 12-40 carbon atoms bonded to an oxygen atom, and may also contain double or triple bonds, and may also contain heteroatoms. For example, allyloxy is an alkoxy group as understood herein. Methoxyethoxy is also an alkoxy group as understood herein, as is methylenedioxy when two adjacent atoms in the structure are substituted with methylenedioxy groups.

[0171] The terms "amine," "amino group," "ammonia," and "amino group" refer to -NH2, -NHR, -NR2, or -NR3. + Substituents of the form (where each R is defined herein), and the protonated form of each (except -NR3). + In addition, it cannot be protonated. Therefore, any compound substituted with an amino group can be considered an amine. In this context, "amino" can refer to a primary, secondary, tertiary, or quaternary amino group.

[0172] "Alkylamino" includes monoalkylamino, dialkylamino, and trialkylamino. Examples of "alkylamino" are -NH-alkyl and -N(alkyl)2.

[0173] An example of a “cycloalkylamino” group is -NH-cycloalkyl and -N(cycloalkyl)2.

[0174] An example of a “cycloalkyl heterocyclic amino” group is -NH-(heterocyclic cycloalkyl), in which the heterocyclic group is linked to nitrogen and the cycloalkyl group is linked to the heterocyclic group.

[0175] An example of a "heterocyclic cycloamino" group is -NH- (cycloalkyl heterocycle), in which the cycloalkyl group is linked to nitrogen and the heterocyclic group is linked to the cycloalkyl group.

[0176] The term "amide group" refers to a group of the formula -C(O)NR2, where R is defined herein.

[0177] Unless otherwise stated, the terms “halogenated,” “halogen,” and “halide” refer, either on their own or as part of another substituent, to a fluorine, chlorine, bromine, or iodine atom.

[0178] The term "haloalkyl" includes monohaloalkyl, polyhaloalkyl (where all halogen atoms may be the same or different), and perhaloalkyl (where all hydrogen atoms are replaced by halogen atoms such as fluorine). Some examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, -CF(CH3)2, etc.

[0179] The term “treatment” and its variations (in relation to a disease or condition) are methods for obtaining a beneficial or desired outcome, which includes, and preferably includes, clinical outcomes, and includes, but is not limited to, one or more of the following: improving a disease-related symptom, curing a disease, reducing the severity of a disease, delaying disease progression, alleviating one or more disease-related symptoms, improving the quality of life of patients with a disease, prolonging survival, and / or prophylactic or preventative treatment.

[0180] "Effective amount" means any amount sufficient to achieve the desired biological effect. In conjunction with the teachings provided herein, effective prophylactic or therapeutic regimens can be planned that do not cause significant undesirable toxicity but are effective in treating a particular subject, by selecting from a variety of active conjugates or compounds and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and mode of administration. The effective amount for any particular application can vary depending on factors such as the disease or condition being treated, the specific compound being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can determine the effective amount of a particular compound and / or other therapeutic agent empirically without excessive experimentation. A maximum dose may be used, i.e., the highest safe dose based on some medical judgment. Multiple doses may be used daily to achieve appropriate systemic levels of the compound. Appropriate systemic levels can be determined, for example, by measuring the patient's peak or sustained plasma levels. "Dose" and "dosage" are used interchangeably herein.

[0181] Typically, for human subjects, the daily oral dose of the compound ranges from about 0.01 mg / kg / day to 1,000 mg / kg / day. Oral doses of 0.5 to 50 mg / kg administered once or more daily may produce therapeutic results. The dosage may be appropriately adjusted depending on the route of administration to achieve the desired local or systemic drug levels. For example, intravenous administration may involve lower dose variations ranging from one to several orders of magnitude daily. If the response is insufficient at such doses, even higher doses (or more effective doses achieved through different, more localized delivery routes) may be used up to the extent tolerated by the patient. Multiple daily doses may be considered to achieve appropriate systemic levels of the compound.

[0182] For therapeutic use, a “therapeutic effective amount” (or “effective amount”) of a compound refers to the amount of the compound in a formulation that, when administered as part of a desired dosing regimen (in mammals, such as humans), relieves symptoms, improves condition, or delays the onset of disease symptoms based on clinically acceptable criteria (e.g., at a reasonable benefit / risk ratio applicable to any medical treatment) for the treatment of the disorder or condition or for cosmetic purposes.

[0183] For any compound, the therapeutically effective dose can be initially determined from animal models. The therapeutically effective dose can also be determined from human data of compounds that have been tested in humans and compounds known to exhibit similar pharmacological activity, such as other relevant active agents. Higher doses may be required for parenteral administration. The administered dose can be adjusted based on the relative bioavailability and potency of the compound being administered. Adjusting the dose to achieve maximum potency based on the methods described above and other methods known in the art is entirely within the capabilities of a person skilled in the art.

[0184] The formulation can be administered in a pharmaceutically acceptable solution, which may conventionally contain a pharmaceutically acceptable concentration of salts, buffers, preservatives, compatible carriers, excipients, and optional other therapeutic ingredients. For therapeutic use, an effective amount of the compound can be administered to the subject by any means of delivering the compound to the desired surface. Administration of the pharmaceutical composition can be accomplished by any means known to those skilled in the art. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection (e.g., injection into a tumor or abscess), mucosal (e.g., ocular surface), inhalation, and topical administration.

[0185] For intravenous and other parenteral routes of administration, compounds can be formulated as lyophilized formulations, lyophilized formulations of active compounds embedded or encapsulated in liposomes, lipid complexes in aqueous suspensions, or salt complexes. Lyophilized formulations are typically reconstituted in a suitable aqueous solution (e.g., in sterile water or saline) shortly before administration.

[0186] For oral administration, compounds can be readily formulated by combining the active compound with a pharmaceutically acceptable carrier known in the art. Such a carrier allows the compound to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the intended recipient. Pharmaceutical formulations for oral use can be obtained as solid excipients, optionally by grinding the resulting mixture, and, if desired, by processing the mixture into granules after adding suitable adjuvants to obtain tablets or dragees with cores. Suitable excipients are particularly fillers, such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose formulations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinyl pyrrolidone (PVP). If desired, a disintegrant such as cross-linked PVP, agar, or alginate or its salts (e.g., sodium alginate) may be added. Optionally, oral formulations may also be formulated in saline or buffer solutions (e.g., EDTA for neutralizing internal acidic conditions) or may be administered without any carrier.

[0187] Oral formulations of the compounds were also considered. The compounds could be chemically modified to make oral delivery of the derivatives effective. Typically, the chemical modifications considered involve the attachment of at least one moiety to the compound itself, wherein said moiety allows for (a) inhibition of acid hydrolysis; and (b) uptake from the stomach or intestine into the bloodstream. Improvements in the overall stability of the compound and increased in vivo circulation time are also desired. Some examples of such moiety include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, PVP, and polyproline. (Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts,” In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367–383 (1981); Newmark et al., J ApplBiochem 4:185–189 (1982).) Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. For pharmaceutical applications, as indicated above, the polyethylene glycol portion is suitable.

[0188] The site of release of the compounds described herein may be the stomach, small intestine (e.g., duodenum, jejunum, or ileum), or large intestine. Those skilled in the art can obtain formulations of substances that do not dissolve in the stomach but are released in the duodenum or other parts of the intestine. Release can avoid the harmful effects of the gastric environment by protecting the compound or by releasing the compound outside the gastric environment (e.g., in the intestine).

[0189] To ensure complete gastric resistance, an impermeable coating at at least pH 5.0 is necessary. Some examples of common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings can be used as blended membranes.

[0190] Coatings or coating mixtures can also be used on tablets, and are not intended to resist the stomach. This can include sugar coatings or coatings that make the tablet easier to swallow. Capsules may consist of a hard shell (e.g., gelatin) for delivering dry therapeutic agents (e.g., powders); for liquid forms, a soft gelatin shell may be used. The shell material for caches may be thick starch paper or other edible paper. For pills, lozenges, molded tablets, or tablet triturates, a wet massing technique may be used.

[0191] The compound can be contained in formulations as finely broken particles in the form of granules or pellets with a particle size of about 1 mm. Formulations of materials used for capsule administration can also be in the form of powders, lightly compressed plugs, or even tablets. Therapeutic agents can be prepared by compression.

[0192] Both colorants and flavoring agents can be included. For example, compounds can be formulated (e.g., encapsulated via liposomes or microspheres) and subsequently included in edible products, such as chilled beverages containing colorants and flavoring agents.

[0193] Inert materials can be used to dilute or increase the volume of compounds. These diluents may include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran, and starch. Certain inorganic salts can also be used as fillers, including calcium triphosphate, magnesium carbonate, and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

[0194] Disintegrants can be included in formulations that conjugate therapeutic agents into solid dosage forms. Materials used as disintegrants include, but are not limited to, starch, including the commercially available starch-based disintegrant Explotab. Sodium glycolate starch, amberlite, sodium carboxymethyl cellulose, ultrabranched starch, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponges, and bentonite are also possible. Another form of disintegrant is an insoluble cation exchange resin. Powdered gum can be used as both a disintegrant and a binder, and these may include powdered gums such as agar, ark tarragon gum, or tragacanth gum. Alginic acid and its sodium salts can also be used as disintegrants.

[0195] Binders can be used to hold compounds together to form hard tablets and include materials derived from natural products such as gum arabic, tragacanth gum, starch, and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC), and carboxymethyl cellulose (CMC). Both PVP and hydroxypropyl methylcellulose (HPMC) can be used in alcoholic solutions to granulate therapeutic agents.

[0196] Anti-friction agents may be included in the formulation of the therapeutic agent to prevent sticking during formulation. Lubricants may be used as a layer between the therapeutic agent and the mold wall, and these may include, but are not limited to: stearic acid (including its magnesium and calcium salts), polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, and waxes. Soluble lubricants may also be used, such as sodium dodecyl sulfate, magnesium dodecyl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000, and 6000.

[0197] A flow aid may be added to improve drug flow properties during formulation and to aid rearrangement during compression. Flow aids may include starch, talc, thermally induced silica, and hydrated aluminosilicates.

[0198] To aid in the dissolution of therapeutic agents in an aqueous environment, surfactants may be added as wetting agents. Surfactants may include anionic detergents such as sodium dodecyl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents that may be used include benzalkonium chloride and benzyl chloride. Potential nonionic detergents that may be included as surfactants in formulations include polidocanol 400; polyoxyethylene 40 stearate; polyoxyethylene hydrogenated castor oil 10, 50, and 60; glyceryl monostearate; polysorbates 40, 60, 65, and 80; sucrose fatty acid esters; methylcellulose; and carboxymethylcellulose. These surfactants may be present alone or in mixtures in varying proportions in formulations of compounds or their derivatives.

[0199] Orally administered pharmaceutical formulations include push-fit capsules made of gelatin and soft-sealable capsules made of gelatin and plasticizers (such as glycerin or sorbitol). Push-fit capsules may contain the active ingredient mixed with a filler (such as lactose), a binder (such as starch), and / or a lubricant (such as talc or magnesium stearate), and optionally a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Additionally, a stabilizer may be added. Microspheres formulated for oral administration may also be used. Such microspheres are clearly defined in the art. All formulations intended for oral administration should be at a dosage suitable for such administration.

[0200] For buccal administration, the composition may be in the form of tablets or lozenges formulated in a conventional manner.

[0201] For surface application, compounds can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is known in the art. Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection), as well as those designed for transdermal, transmucosal, oral, or transpulmonary administration.

[0202] For inhalation administration, when using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas), the compound can be conveniently delivered as an aerosol from a pressurized package or nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver the measured amount. Capsules and cartridges (e.g., gelatin) for inhalers or blowpipes can be formulated as a powder mixture containing the compound with a suitable powder matrix (e.g., lactose or starch).

[0203] Lung delivery of the compound (or its salt) was also considered. The compound is delivered to the lungs of a mammal upon inhalation and enters the bloodstream through the epithelial lining of the lung. Other reports on inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(Supplement 5):143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989) (α1-antitrypsin); Smith et al., 1989, J Clin Invest 84:1145-1146 (α-1-protease); Oswein et al., 1990, “Aerosolization of Proteins,” Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant hepatocyte growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (Interferon-γ and Tumor Necrosis Factor α) and Platz et al., U.S. Patent No. 5,284,656 (Granocyte Colony-Stimulating Factor; incorporated herein by reference). Methods and compositions for pulmonary delivery of drugs for systemic action are described in U.S. Patent No. 5,451,569, issued to Wong et al., September 19, 1995 (the disclosure thereof is specifically incorporated herein by reference).

[0204] Consider using a variety of mechanical devices designed for delivering therapeutic products to the lungs, including but not limited to nebulizers, metered inhalers, and powder inhalers, all of which are well known to those skilled in the art.

[0205] Nasal delivery pharmaceutical compositions were also considered. Nasal delivery allows the pharmaceutical composition to enter the bloodstream directly after the therapeutic product is applied to the nose, without requiring deposition in the lungs. Formulations for nasal delivery include those containing dextran or cyclodextrin.

[0206] When systemic delivery of the compound is desired, the compound may be formulated for parenteral administration by injection (e.g., by bolus or continuous infusion). Formulations for injection may be present in unit dosage forms (e.g., in ampoules or in multi-dose containers) with the addition of preservatives. The composition may be in the form of a suspension, solution, or emulsion, for example, in an oily or aqueous carrier, and may contain formulations such as suspending agents, stabilizers, and / or dispersants.

[0207] Pharmaceutical formulations intended for parenteral administration include aqueous solutions of the active compound in its water-soluble form. Alternatively, suspensions of the active compound can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or carriers include fatty oils (e.g., sesame oil), synthetic fatty acid esters (e.g., ethyl oleate or triglycerides), or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or substances that increase the solubility of the compound to allow for the preparation of highly concentrated solutions.

[0208] Alternatively, the active compound may be in powder form for use with a suitable carrier (e.g., sterile, pyrogen-free water) prior to use.

[0209] The compound can also be formulated into rectal or vaginal compositions, such as suppositories or retention enemas, for example, containing a conventional suppository base, such as cocoa butter or other glycerides.

[0210] In addition to the formulations described above, compounds can also be formulated into depot preparations. Such long-acting preparations can be formulated using suitable polymers or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives (e.g., sparingly soluble salts).

[0211] The pharmaceutical composition may also contain a suitable solid or gel phase carrier or excipient. Some examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

[0212] Suitable liquid or solid pharmaceutical formulations include, for example, aqueous or saline solutions for inhalation; microencapsulated, encochleated, coated with microscopic gold particles, contained in liposomes, nebulized, aerosols, dried on sharp objects for skin penetration, or pellets for implantation. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or formulations with prolonged release of the active compound, typically prepared using excipients and additives and / or excipients, such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweeteners, or solubilizers, as described above. Pharmaceutical compositions are suitable for a variety of drug delivery systems. For a brief overview of drug delivery methods, see Langer R, Science 249:1527-1533 (1990).

[0213] The compound and optionally one or more other therapeutic agents may be administered either in their pure form or as a pharmaceutically acceptable salt. When used in a medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutical salts may be conveniently used to prepare their pharmaceutically acceptable form. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Similarly, such salts may be prepared as alkali metal or alkaline earth salts, such as sodium, potassium, or calcium salts of a carboxylic acid group.

[0214] Suitable buffers include: acetic acid and its salt (1% to 2% w / v); citric acid and its salt (1% to 3% w / v); boric acid and its salt (0.5% to 2.5% w / v); and phosphoric acid and its salt (0.8% to 2% w / v). Suitable preservatives include benzalkonium chloride (0.003% to 0.03% w / v); chlorobutanol (0.3% to 0.9% w / v); parabens (0.01% to 0.25% w / v); and thimerosal (0.004% to 0.02% w / v).

[0215] The pharmaceutical composition contains an effective amount of the compound as described herein and optionally one or more other therapeutic agents contained in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic organic or inorganic component combined with the active ingredient to facilitate application. Components of the pharmaceutical composition may also be mixed with the compound and with each other in a manner that prevents interactions that would significantly impair the desired pharmaceutical efficacy.

[0216] Therapeutic agents (specifically, including but not limited to compounds) may be provided in the form of particles. As used herein, “particles” means nanoparticles or microparticles (or, in some cases, larger particles) that may consist wholly or partially of compounds or other therapeutic agents as described herein. Particles may contain a therapeutic agent in a core surrounded by a coating (including, but not limited to, enteric coating). Therapeutic agents may also be dispersed throughout the particle. Therapeutic agents may also be adsorbed into the particle. Particles may have release kinetics of any order, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof. In addition to therapeutic agents, particles may also contain any of those materials conventionally used in the pharmaceutical and medical fields, including but not limited to erosive, non-erosive, biodegradable, or non-biodegradable materials, or combinations thereof. Particles may be microcapsules containing compounds in a solution or semi-solid state. Particles may be virtually any shape.

[0217] Both non-biodegradable and biodegradable polymeric materials can be used to prepare particles for delivering therapeutic agents. Such polymers can be natural or synthetic. The polymer is selected based on the desired release time period. Of particular interest are bioadhesive polymers, including bio-erosive hydrogels described in Sawhney et al., Macromolecules 26:5823-2787 (1993), the teachings of which are specifically incorporated herein by reference. These include polyhyaluronic acid, casein, gelatin, gelatin protein, polyanhydride, polyacrylic acid, alginate, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(laurate methacrylate), poly(phenyl methacrylate), poly(methyl methacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0218] Therapeutic agents may be included in controlled-release systems. The term "controlled release" refers to any formulation containing a drug in which the manner and spectrum of drug release from the formulation are controlled. This includes both immediately released and non-immediately released formulations, with non-immediately released formulations including, but not limited to, sustained-release and delayed-release formulations. The term "sustained-release" (also known as "extended-release") refers to a pharmaceutical formulation that provides a gradual release of the drug over an extended period of time and may result in a substantially constant blood level of the drug over the extended period of time. The term "delayed-release" refers to a pharmaceutical formulation in which there is a time delay between the administration of the formulation and the release of the drug therefrom. "Delayed-release" may or may not involve a gradual release of the drug over an extended period of time, and therefore may or may not be "sustained-release".

[0219] The use of long-term sustained-release implants is particularly suitable for the treatment of chronic conditions. As used herein, “long-term” release means that the implant is constructed and positioned to deliver therapeutic levels of active ingredient for at least 7 days and up to 30 to 60 days. Long-term sustained-release implants are well known to those skilled in the art and include some of the aforementioned release systems.

[0220] As used herein, the terms “salt” and “medicinal salt” refer to derivatives of the disclosed compounds, wherein the parent compound is modified by preparing its acid or base salt. Some examples of medicinal salts include, but are not limited to, inorganic or organic acid salts of basic groups (e.g., amines); and basic or organic salts of acidic groups (e.g., carboxylic acids). Medicinal salts include conventional nontoxic salts or quaternary ammonium salts of parent compounds formed from, for example, nontoxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids: such as hydrochlorides, hydrobroms, sulfates, aminosulfonates, phosphates, and nitrates; and salts prepared from organic acids, such as acetates, propionates, succinates, glycolates, stearates, lactates, malates, tartrates, citrates, ascorbic acid salts, pyrates, maleates, hydroxymaleates, phenylacetates, glutamates, benzoates, salicylates, sulfanilic acids, 2-acetoxybenzoates, fumarates, toluenesulfonates, methanesulfonates, ethanedisulfonates, oxalates, and hydroxyethylsulfonates, etc.

[0221] Pharmaceutically usable salts can be synthesized from parent compounds containing basic or acidic moieties using conventional chemical methods. In some cases, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington's PharmaceuticalSciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990, the disclosure of which is incorporated herein by reference.

[0222] The term "solvent" refers to a compound or its salt that also contains stoichiometric or non-stoichiometric amounts of a solvent bound together by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0223] The term "prodrug" refers to a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound, particularly the compounds of the present invention. Examples of prodrugs include, but are not limited to, derivatives and metabolites of the compounds of the present invention that contain a biohydrolyzable moiety, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable acylureas, and biohydrolyzable phosphate ester analogs. Specific prodrugs of compounds having a carboxyl functional group are lower alkyl esters of carboxylic acids. Carboxylic acid esters are conveniently formed by esterifying any carboxylic acid moiety present on the molecule. Prodrugs are generally prepared using well-known methods, such as those described in: Burger's Medicinal Chemistry and Drug Discovery, 6th edition (edited by Donald J. Abraham, 2001, Wiley) and Design and Application of Prodrugs (edited by H. Bundgaard, 1985, Harwood Academic Publishers GmbH).

[0224] Furthermore, in each of the foregoing and following embodiments, it should be understood that the formula includes and represents not only all pharmaceutically acceptable salts of the compound, but also any and all hydrates and / or solvates of the compound formula or its salts. It should be understood that in the various physical forms of the compound, certain functional groups, such as hydroxyl, amino, etc., form complexes and / or coordination compounds with water and / or various solvents. Therefore, the above formula should be understood to include and represent those various hydrates and / or solvates. In each of the foregoing and following embodiments, it should also be understood that the formula, whether alone or in any and all possible mixtures, includes and represents every possible isomer, such as stereoisomers and geometric isomers. In each of the foregoing and following embodiments, it should also be understood that the formula includes and represents any and all crystalline, partially crystalline, and amorphous and / or non-crystalline forms of the compound.

[0225] The term “medicatable carrier” is recognized in the art and refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting any subject composition or its components. Each carrier must be “acceptable” in the sense that it is compatible with the subject composition and its components and is harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, red sesame oil, etc. Flower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotropic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic and compatible substances used in pharmaceutical preparations.

[0226] The term "administration" includes all methods of introducing the compounds and compositions described herein into a patient, including but not limited to oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), percutaneous, inhalation, buccal, ocular, sublingual, vaginal, and rectal administration. The compounds and compositions may be administered in unit dosage forms and / or formulations containing conventional nontoxic pharmaceutically acceptable carriers, excipients, and loading agents.

[0227] Illustrative forms for oral administration include tablets, capsules, elixirs, syrups, etc. Illustrative routes for parenteral administration include intravenous, intra-arterial, intraperitoneal, epidural, intraurethral, ​​intrasternal, intramuscular, and subcutaneous administration, as well as any other parenteral administration route recognized in the art.

[0228] Illustrative methods of parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques, as well as any other parenteral administration method recognized in the art. Parenteral preparations are typically aqueous solutions that may contain excipients (e.g., salts, carbohydrates, and buffers, preferably at a pH of about 3 to about 9), but for some applications, they may be more suitable as sterile, non-aqueous solutions or as dry forms for use in combination with a suitable carrier (e.g., sterile, pyrogen-free water). The preparation of parenteral preparations under sterile conditions (e.g., by lyophilization) can be readily performed using standard pharmaceutical techniques known to those skilled in the art. Parenteral administration of compounds is illustratively carried out in the form of salt solutions or by incorporating the compound into liposomes. In cases where a compound is to be dissolved that is not sufficiently soluble on its own, a solubilizer such as ethanol may be used.

[0229] The dosage of each compound in the claimed combination depends on several factors, including: the method of administration, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health status of the patient. Additionally, pharmacogenomics information (the influence of genotype on pharmacokinetics, pharmacodynamics, or therapeutic efficacy characteristics) of a particular patient can influence the dosing regimen used.

[0230] In the method described, individual components that are co-administered or combined may be administered concurrently, simultaneously, sequentially, separately, or as a single pharmaceutical formulation in any suitable manner. When co-administered compounds or compositions are administered in separate dosage forms, the daily doses of 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, separately or as a single form, at the same or different times during the course of treatment, depending on a simultaneous or alternating regimen.

[0231] The term "therapeutic effective dose" refers to the amount of an active compound or agent that elicits a biological or pharmaceutical response in an tissue system, animal, or human, as sought by an investigator, veterinarian, physician, or other clinician, including the reduction of symptoms of the treated disease or condition. In one aspect, a therapeutic effective dose is an amount that can treat or reduce a disease or disease symptoms at a reasonable benefit / risk ratio applicable to any medical treatment. However, it should be understood that the total daily dose of the compounds and compositions described herein may be determined by the attending physician within the bounds of reasonable medical judgment. The specific therapeutic effective dose level for any particular patient will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the time of administration, route of administration, and rate of excretion of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors known to investigators, veterinarians, physicians, or other clinicians of common skill.

[0232] Depending on the route of administration, a wide range of permissible doses are considered, including doses ranging from approximately 1 μg / kg to approximately 1 g / kg. Dosage can be a single dose or divided doses and can be administered according to various regimens, including qd (once a day), bid (twice a day), tid (three times a day), or even every other day, once a week, once a month, once a quarter, etc. In each of these cases, the therapeutically effective dose corresponds to the administration method, or alternatively to the total daily, weekly, monthly, or quarterly dose, as determined by the dosing regimen.

[0233] The effective amount of any one or mixture of compounds described herein may be determined by the attending physician or doctor using known techniques and / or by observing results obtained under similar circumstances. In determining the effective amount or dosage, the attending physician or doctor considers a number of factors, including but not limited to the species of mammal (including humans), its size, age and general health condition, the specific disease or condition involved, the extent or severity of the disease or condition, the individual patient's response, the specific compound administered, the route of administration, the bioavailability characteristics of the administered formulation, the chosen dosing regimen, the use of concomitant medications, and other relevant circumstances.

[0234] The term "patient" includes both humans and non-human animals, such as companion animals (dogs and cats) and livestock. Livestock are animals raised for food production. Patients seeking treatment are preferably mammals, especially humans.

[0235] The following terms and phrases shall have the meanings indicated below. Unless otherwise specified, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art.

[0236] The term “about” allows for a degree of variability in a value or range, such as within 20%, 10%, 5%, or 1% of a specified value, or within 20%, 10%, 5%, or 1% of a specified limit of the range.

[0237] Unless the context clearly specifies otherwise, nouns without quantifiers are used to include one or more than one. Unless otherwise stated, the term "or" is used to indicate a non-exclusive "or". Furthermore, it should be understood that any wording or terminology used herein without further qualification is for descriptive purposes only and not for limiting purposes. The use of any section headings is intended to aid reading the document and should not be construed as restrictive. Additionally, information relating to section headings may appear within or outside that particular section. Moreover, all publications, patents, and patent documents mentioned in this document are incorporated herein by reference in their entirety, as if incorporated individually by reference. In the event of any inconsistency between the usage in this document and those documents incorporated by reference, the usage in the incorporated references shall be considered supplementary to the usage in this document; in the case of contradictory inconsistencies, the usage in this document shall prevail.

[0238] The term “substantially” means the majority or most, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0239] The term “substantially not” as used in this article means less than about 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.001%, or less than about 0.0005% or less, or about 0% or 0%.

[0240] In this method, steps can be performed in any order without departing from the principles of the invention, except when the timing or sequence of operations is explicitly stated. Furthermore, unless the explicit language of the claims specifies that a particular step is performed individually, specific steps can be performed simultaneously. For example, claimed step X and claimed step Y can be performed simultaneously in a single operation, and the resulting method will fall within the scope of the claimed method.

[0241] Those skilled in the art will understand that many modifications to the described embodiments are possible without departing from the spirit and scope of this disclosure. Therefore, the description is not intended and should not be construed as limiting to the examples given, but should be granted the full scope of protection provided by the appended claims and their equivalents. Furthermore, some features of this disclosure may be used without correspondingly using others. Therefore, the purpose of providing the above description or illustrative embodiments is to illustrate the principles of this disclosure and not to limit it, and modifications and arrangements thereof may be included.

[0242] This disclosure also relates to the subject matter of the following provisions, which are not listed in any particular order of importance:

[0243] 1. A conjugate of formula TLA, wherein:

[0244] T is a ligand that targets prostate-specific membrane antigen (PSMA).

[0245] L is the connector, and

[0246] A contains a DNA repair enzyme inhibitor, wherein the DNA repair enzyme inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor, an ataxia-telangiectasia mutation (ATM) inhibitor, or a DNA protein kinase (DNA-PK) inhibitor.

[0247] 2. The conjugate described in Clause 1, wherein T is:

[0248] .

[0249] 3. The conjugate described in Clause 1, wherein T is:

[0250] , where R 1 and R 2 Independently selected from hydrogen, optionally substituted carboxylic acids, malonic acid, succinic acid, glutamic acid, and adipic acid.

[0251] 4. The conjugate described in Clause 3, wherein the substituted carboxylic acid is thioacetic acid or thiopropionic acid.

[0252] 5. The conjugate described in Clause 1, wherein T is (a) an aminodicarboxylic acid or a derivative thereof and (b) an aminodicarboxylic acid or a derivative thereof, wherein (a) and (b) may be the same or different.

[0253] 6. The conjugate of any one of clauses 1 to 5, wherein L comprises an atomic chain of about 3 atoms to about 30 atoms in length.

[0254] 7. The conjugate of any one of clauses 1 to 6, wherein L comprises an atomic chain of about 5 Å to about 45 Å in length.

[0255] 8. The conjugate of any one of clauses 1 to 7, wherein L comprises a peptide.

[0256] 9. The conjugate of any one of clauses 1 to 8, wherein L comprises one or more phenylalanine residues, each of which is optionally substituted independently.

[0257] 10. The conjugate of any one of clauses 1 to 9, wherein L comprises at least one phenylalanyl-phenylalanyl group, wherein at least one phenyl group is optionally substituted independently.

[0258] 11. The conjugate of any one of clauses 1 to 10, wherein L comprises polyoligoethylene glycol n (POEGn), polyethylene glycol n (PEGn), or a mixture thereof, wherein n = 1 to 36.

[0259] 12. The conjugate of any one of clauses 1 to 11, wherein L comprises a disulfide.

[0260] 13. The conjugate described in Clause 1, wherein the PRMT5 inhibitor comprises a group of JNJ-64619178 or a group of GSK3326595.

[0261] 14. The conjugate described in Clause 1, wherein the ATM inhibitor comprises a group of AZD0156.

[0262] 15. The conjugate described in Clause 1, wherein the DNA-PK inhibitor comprises the following groups: Nedisertib, AZD7648, KU57748, Torin 2, PI-103, NU7026, Samotolisib, PIK 90, CC-115, PIK-75 hydrochloride, VX-984, KU-0060648, BAY-8400, DNA-PK-IN-2, DNA-PK-IN-1, DNA-PK-IN-4, DNA-PK-IN-5, DNA-PK-IN-6, XRD-0394, NU5455, STL127705, PI-103 hydrochloride, LTURM34, ZL-2201 free base, DNA-PK-IN-14, DNA-PK-IN-10, DNA-PK-IN-11, or DNA-PK-IN-13.

[0263] 16. The conjugate of any one of clauses 1 to 15, further comprising an albumin-binding group.

[0264] 17. The conjugate of Clause 16, wherein the albumin-binding group has the following formula:

[0265] .

[0266] 18. The conjugate of Clause 16, wherein the albumin-binding group is linked to L via a substituent or group present on L.

[0267] 19. The conjugate of Clause 16, wherein the albumin-binding group is wherein R 4 It is H or alkyl -NR 4 - or -O- group is attached to L.

[0268] 20. The following conjugate:

[0269] ,

[0270] ,

[0271] .

[0272] 21. A conjugate of formula TL-(X)A, wherein:

[0273] T is a ligand that targets prostate-specific membrane antigen (PSMA).

[0274] L stands for connector.

[0275] A contains a DNA repair enzyme inhibitor, which is a protein arginine methyltransferase 5 (PRMT5) inhibitor, an ataxia-telangiectasia mutation (ATM) inhibitor, or a DNA protein kinase (DNA-PK) inhibitor; and

[0276] X is a radiotherapy agent or a radioimaging agent.

[0277] 22. The conjugate described in Clause 21, wherein A and X are connected to L.

[0278] 23. The conjugate described in Clause 21 or 22, further comprising an albumin-binding group.

[0279] 24. The conjugate described in Clause 23, wherein the albumin-binding group has the following formula:

[0280] .

[0281] 25. The conjugate described in Clause 21 or 22, wherein the albumin-binding group is linked to L by a substituent or group present on L, and wherein the albumin-binding group is optionally linked to L according to Clause 19 or 20.

[0282] 26. The conjugate of any one of Clauses 21 to 25, wherein T is defined as in Clauses 2 to 5.

[0283] 27. The conjugate of any one of Clauses 21 to 26, wherein L is defined as in Clauses 6 to 12.

[0284] 28. The conjugate of any one of Clauses 21 to 27, wherein A is defined in Clauses 13 to 15.

[0285] 29. A pharmaceutical composition comprising any one of the conjugates or pharmaceutically acceptable salts thereof, as well as a pharmaceutically acceptable carrier, according to any one of the provisions 1 to 20.

[0286] 30. A pharmaceutical composition comprising any one of the conjugates or pharmaceutically acceptable salts thereof, as well as a pharmaceutically acceptable carrier, according to any one of clauses 21 to 28.

[0287] 31. A method of treating a patient with prostate cancer or metastases using radiation therapy, the method comprising administering to the patient any of the conjugates or pharmaceutically acceptable salts thereof, as described in any one of Clauses 1 to 20, or a pharmaceutical composition as described in Clause 29, thereby treating the patient with prostate cancer or metastases thereof.

[0288] 32. The method of Clause 31, further comprising administering, to the patient, the TLX conjugate or its pharmaceutically acceptable salt before or after administration of the conjugate or pharmaceutical composition of any one of Clauses 1 to 20, wherein:

[0289] T is a ligand that targets prostate-specific membrane antigen (PSMA).

[0290] L is the connector, and

[0291] X is a radiotherapy agent or a radioimaging agent.

[0292] 33. The method described in Clause 32, wherein T is:

[0293] .

[0294] 34. The method described in Clause 32, wherein T is:

[0295] , where R 1 and R 2 Independently selected from hydrogen, optionally substituted carboxylic acids, malonic acid, succinic acid, glutamic acid, and adipic acid.

[0296] 35. The method described in Clause 32, wherein the substituted carboxylic acid is thioacetic acid or thiopropionic acid.

[0297] 36. The method described in Clause 32, wherein T is (a) an aminodicarboxylic acid or a derivative thereof and (b) an aminodicarboxylic acid or a derivative thereof, wherein (a) and (b) may be the same or different.

[0298] 37. The method described in Clause 32, wherein L comprises an atomic chain of about 3 atoms to about 30 atoms in length.

[0299] 38. The method described in Clause 32, wherein L comprises a chain of atoms of about 5 Å to about 45 Å in length.

[0300] 39. The method described in Clause 32, wherein L comprises a peptide.

[0301] 40. The method of Clause 32, wherein L comprises one or more phenylalanine residues, each of which is optionally substituted independently.

[0302] 41. The method of Clause 32, wherein L comprises at least one phenylalanyl-phenylalanyl group, wherein at least one phenyl group is optionally substituted independently.

[0303] 42. The method of Clause 32, wherein L comprises polyethylene oligoethylene glycol n (POEGn), polyethylene glycol n (PEGn), or a mixture thereof, wherein n = 1 to 36.

[0304] 43. The method of Clause 32, wherein X comprises a radioactive isotope bound to a chelating agent, said chelating agent being selected from:

[0305] • DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or its derivatives;

[0306] • TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) or its derivatives;

[0307] • SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine or its derivatives;

[0308] • NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) or its derivatives;

[0309] • NETA (4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazacyclononane-1-yl)acetic acid or a derivative thereof

[0310] • TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphonic acid) or its derivatives;

[0311] • HBED (N,NO-bis(2-hydroxybenzyl)-ethylenediamine-N,NO-diacetic acid) or its derivatives;

[0312] • 2,3-HOPO (3-hydroxypyridin-2-one) or its derivatives;

[0313] • PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadecane-1(15),11,13-triene-3,6,9-triacetic acid) or its derivatives;

[0314] • DFO (deferriphosphate) or its derivatives;

[0315] • DTPA (diethylenetriaminepentaacetic acid) or its derivatives;

[0316] • OCTAPA (N,NO-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,NO-diacetic acid) or its derivatives; or H2-MACROPA (N,N'-bis[(6-carboxy-2-pyridylmethyl]-4,13-diaza-18-crown-6) or its derivatives;

[0317] • H2dedpa (1,2-[[carboxyl)-pyridin-2-yl]-methylamino]ethane or a derivative thereof; and

[0318] • EC20-head, which contains β-l-diaminopropionic acid, aspartic acid and cysteine.

[0319] 44. The method of Clause 32, wherein X comprises a radioactive isotope selected from: 18 F, 44 Sc、 47 Sc、 52 Mn, 55 Co、 64 Cu、 6 7Cu, 67 Ga、 68 Ga、 86 Y、 89 Zr、 90 Y、 99m Tc, 111 In、 114m In、 117m Sn、124 I, 125 I, 131 I, 149 Tb, 153 Sm、 152 Tb, 155 Tb, 161 Tb, 177 Lu、 186 Re、 188 Re、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 224 Ra、 225 Ab、 225 Ac and 227 Th.

[0320] 45. The method described in Clause 32, further comprising administering an immune checkpoint inhibitor to the patient.

[0321] 46. ​​The method of Clause 45, wherein the immune checkpoint inhibitor is selected from cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein 1 (PD-1) inhibitors, and programmed cell death ligand 1 (PD-L1) inhibitors.

[0322] 47. The method described in Clause 45, wherein the immune checkpoint inhibitor is selected from nivolumab (Opdivo), pembrolizumab (Keytruda), ipilimumab (Yervoy), atezolizumab, avelumab, and durvalumab.

[0323] 48. A method of treating a patient with prostate cancer or metastases using radiation therapy, the method comprising administering to the patient any of the conjugates or pharmaceutically acceptable salts thereof, as described in any one of clauses 21 to 28, or a pharmaceutical composition as described in clause 30, thereby treating the patient with prostate cancer or metastases thereof.

[0324] Example

[0325] This disclosure can be better understood by referring to the following embodiments provided by way of example. This disclosure is not limited to the embodiments given herein.

[0326] Example 1: Synthesis of ATM Inhibitors

[0327] Synthesis of activated DNAPKcs 3 (Protocol 1 below). Heterofunctional adapter 2 (0.078 mmol, 0.027 g) was added to compound 1 (0.052 mmol, 0.025 g) and DMAP (0.052 mmol, 0.006 g) in DCM at room temperature under a nitrogen atmosphere. The reaction mixture was stirred at reflux temperature for 3 hours. LCMS was monitored to observe the reaction progress. After 3 hours, the reaction was quenched by adding DCM. The product was purified by column chromatography using a 5% DCM-methanol mixture as the mobile phase to obtain pure product 3 (20 mg, 55% yield, t) as a green solid. R = 4.337 minutes). Compound 3 LCMS (+ESI) for [M+H] + (C 32 H 28 ClFN6O5S2) + The calculated value is 695.1, and the measured value is 695.1.

[0328]

[0329] Synthesis of conjugate 6 (Protocol 2 below). Compound 4 (1 equivalent) was dissolved in DMSO (1 mL). DMAP (1 equivalent) was added to the reaction mixture. The reaction mixture was continuously degassed with argon. Compound 5 (1 equivalent) was dissolved in DMSO and added to the reaction mixture in 3 portions at 20-minute intervals. LCMS was monitored after 1 hour. After the reaction was complete, product 6 was purified by reversed-phase HPLC using 10 mmol ammonium acetate (pH = 5) and acetonitrile. The pure fraction of 6 was collected using an automated fractionating collector, the acetonitrile was evaporated under reduced pressure, and the purified bioconjugate 6 was obtained after lyophilization.

[0330] Conjugate 6 LCMS (+ESI) for [M+H] + (C 83 H 98 ClFIN 13 O 20 S2) + The calculated value was 1841.5, and the measured value was 1841.5. The product was separated as a yellow solid. (Yield 60%; t) R = 3.748 minutes).

[0331]

[0332] Synthesis of Compound 8 (Scheme 3 below). MC-Val-Cit-PAB-OH,7 (0.087 mmol, 50 mg) was suspended in DMF (500 μL) and heated to 80 °C to dissolve all solids. After cooling to 0 °C, thionyl chloride (50 μL) was added in portions. After the addition, the reaction was maintained at 0 °C for 2 to 5 hours. The reaction progress was monitored by LCMS. The crude reaction mixture was purified by column chromatography using a 5% DCM-methanol mixture as the mobile phase to obtain pure product 8 as a yellow solid. (31 mg, yield 60%, t) R = 3.980 minutes). Compound 8 LCMS (+ESI) for [M+H] + (C 28 H 39 ClN6O6) + The calculated value is 591.3, and the measured value is 591.3.

[0333]

[0334] Synthesis of Compound 11 (Activation of the ATM inhibitor; Scheme 5 below). In a vial, tertiary amine 9 (1 equivalent) and 10 N-[(1S)-1-[[(1S)-1-[[4-(chloromethyl)phenyl]carbamoyl]-4-ureo-butyl]carbamoyl]-2-methyl-propyl]-6-(2,5-dioxopyrrole-1-yl)hexamethylenediamine (1.1 equivalent) were mixed in DMF at room temperature. 0.5 equivalents of tetrabutylammonium iodide were added to the solution, followed by N,N-diisopropylethylamine (2.5 equivalents), and the mixture was stirred until all the starting amines were consumed, or until decomposition was observed by LCMS (3 to 48 hours). The mixture was diluted with 1.5 mL of DMF and purified directly by HPLC, eluting with 20% to 60% acetonitrile:0.1% formic acid aqueous solution to give the quaternary salt product. Pure product 11 was obtained as a white solid (yield 45%, t). R = 3.980 minutes). Compound 11 LCMS (+ESI) for [M+H] + (C 54 H 70 N 11 O9) + The calculated value is 1016.5, and the measured value is 1016.5.

[0335]

[0336] Synthesis of conjugate 12 (Protocol 5 below). Compound 11 (1 equivalent) was dissolved in DMSO (1 mL). The reaction mixture was continuously degassed with argon. Compound 5 (1 equivalent) was dissolved in DMSO and added to the reaction mixture. LCMS was monitored after 1 hour. After the reaction was complete, product 12 was purified by reversed-phase HPLC using 10 mmol ammonium acetate (pH=5) and acetonitrile. The pure fraction of 12 was collected using an automated fractionating collector, the acetonitrile was evaporated under reduced pressure, and the fraction was lyophilized to obtain pure bioconjugate 12.

[0337] Conjugate 12 LCMS (+ESI) for (M+H / 2) + [(C 110 H 145 IN 19 O 24 S) + The calculated value of [ / 2] is 1138.2, and the measured value is 1138.2. The product was separated as a yellow solid. (Yield 60%; t) R = 3.678 minutes).

[0338]

[0339] Example 2

[0340] Synthesis of competitive ligand 20 (Scheme 6 below). In the first step, isocyanate 13 of the glutamyl moiety was generated in situ by adding a mixture of bis(tert-butyl)-L-glutamic acid hydrochloride 14 (2.1 mmol, 0.62 g, 3 equivalents) and 1 mL of DIPEA in 5 mL of dry DCM to a solution of triphosgene (0.7 mmol, 0.207 g, 1 equivalent) in 5 mL of dry DCM at -78 °C. The reaction mixture was stirred at -78 °C for 1 h and then further stirred at room temperature for 1.5 h. The generated isocyanate 13 was transferred to a peptide vessel containing HL Lys(Alloc)-2-Cl-triphenylmethyl resin 15 (0.35 mmol, 500 mg, resin loading 0.7 mmol / g). The reaction was bubbled with argon at room temperature for 16 h to form urea moiety 16. The resin beads were washed with DCM (3 × 4 mL). The completion of the reaction was confirmed by a Kaiser test. Subsequently, the allyloxy protecting group was removed by stirring the resin beads with 100 mg tetra(triphenylphosphine)palladium (0) and 500 μL morpholine and 6 mL of dry DCM for 3 hours. After deprotection, the resin beads were washed with DCM (3 × 4 mL), dried under vacuum, and the completion of deprotection was confirmed by a Kaiser test. The resin was further washed with sodium diethyldithiocarbamate (0.03 M in DMF) to remove excess palladium complex, thus forming 17. Subsequently, coupling of Fmoc-naphthylalanine (0.7 mmol, 0.306 g) and Fmoc-tranexamic acid (0.7 mmol, 0.266 g) was performed using PyBOP (0.7 mmol, 0.364 g), DIPEA (0.5 mL), and DMF (6 mL). After each peptide coupling, the Fmoc group was deprotected for 30 minutes using 20% ​​piperidine in DMF to form 18. Finally, DOTA-tBu ester NHS ester (0.428 g, 0.525 mmol) carboxylic acid was coupled with compound 18 to form conjugate 19.

[0341] The general procedure for cleaving peptides from resin beads is as follows: Prepare a mixture of 14.25 mL trifluoroacetic acid (TFA), 0.375 mL triisopropylsilane (TIPS), and 0.375 mL H₂O. Add 7.5 mL of this mixture to the resin beads and bubble the solution through argon for 30 minutes. Repeat the same procedure once more using the remaining 7.5 mL (30 minutes) of the mixture. Evaporate the mother liquor collected from the cleavage under reduced pressure and precipitate the concentrated viscous liquid in ice-cold diethyl ether to form crude compound 20.

[0342] Analytical HPLC method. Product formation was further confirmed by LCMS. The purity of bioconjugate 20 was analyzed using an Agilent 1260 Infinity II system. Typically, a solution of bioconjugate 20 in DMF was injected via an autosampler and analyzed using XBridge. Elution was performed using a Shield RP18 3.5 μm, 3.0 × 50 mm column at a flow rate of 0.750 mL / min (mobile phase, A = 10 mmol ammonium bicarbonate at pH 7 and B = acetonitrile). The initial mobile phase was 95% buffer and 5% acetonitrile. The percentage of acetonitrile was gradually increased from 5% to 95% over a 4.5 min time interval and returned to its initial composition over another 2.5 min interval. Chromatograms of 20 were recorded at 220 nm on a variable wavelength detector, where t R = 2.6 minutes.

[0343] The RP-Combi rinsing method was used for purification. Crude conjugate 20 was purified using a 15.5 g C18Aq high-performance column at a flow rate of 30 mL / min (A = 20 mmol ammonium acetate, pH = 7, and B = acetonitrile). The acetonitrile percentage was gradually increased from 0% to 50% over a 25-minute period to obtain the pure compound. Chromatograms were recorded at 220 nm. The pure fraction of 20 was collected using an automated fraction collector, the acetonitrile was evaporated under reduced pressure, and the fraction was lyophilized to obtain pure bioconjugate 20.

[0344]

[0345] Example 3

[0346] Radiolabeling. PSMA-617 20 was dissolved in NH4OAc buffer (10 mmol, pH 6.0) and […]. 177 Lu]Lu +3 (National Isotope Development Center) mark.

[0347] Animal husbandry. Mice were provided with normal rodent food and water, and a standard 12-hour light-dark cycle was maintained. All animal procedures were approved by the Purdue Animal Care and Use Committee.

[0348] The tumor model used 150 μL PBS and Matrigel (2:1) to prepare 5 × 10⁶ cells / mL of PBS. 5 LNCaP cells were inoculated into the shoulder area of ​​athymic nude mice.

[0349] Radiation therapy. Mice bearing LNCaP tumors were randomly assigned to control and treatment groups to ensure identical initial tumor volumes. Each group received either a separate radiotherapy agent or a combined radiotherapy agent on day 0. 177 A single intravenous injection of Lu-radiolabeled PSMA-617 20 conjugate was administered. Mice bearing LNCaP tumors were distributed into five different groups, with five mice in each group. The groups are as follows:

[0350] • Untreated group or control group;

[0351] • Using 9.25 MBq 177 Lu PSMA-617 20 treatment; using 9.25 MBq 177 Lu PSMA-617 and 10 nanomoles of compound 21 were administered daily for 28 days; and

[0352] • Using 9.25 MBq 177 Lu PSMA 617 and 10 nanomoles of compound 22 were administered (daily for 28 days).

[0353] Except for the untreated group, each group received 9.25 MBq. 177 Lu PSMA-617 20.

[0354] For ease of reference, the structure of each compound is as follows:

[0355]

[0356]

[0357] Tumor growth was measured in two perpendicular directions using calipers. Mice were euthanized upon reaching one of the predefined endpoint criteria, in accordance with the agency's Animal Care and Use Committee guidelines. Data showed that, compared to the control, [the tumor growth was significantly reduced]. 177 Combination therapy with LuPSMA-617 20 and compound 22 showed 81% tumor growth inhibition. Figure 9 No significant weight loss was observed during the treatment study. For those derived from... 177 Data on combination therapy with Lu PSMA-617 20 and compound 22 can be found in [link to relevant documentation]. Figure 10 Survival curves showed that mice treated with the combination therapy survived approximately 30 days longer than the control group. For mice treated with... 177 Combination therapy of Lu PSMA-617 20 and compound 22, see [link to relevant information]. Figure 11 ; and for 177 Combination therapy of Lu PSMA-617 29 and compound 34, see [link to relevant information]. Figure 14 .

[0358] Example 4

[0359] γH2AX staining and fluorescence imaging. LNCaP (PSMA) + DU145 (PSMA) + PC3 (PSMA) - Cells were grown at 37°C in T-75 flasks in RPMI 1640 medium supplemented with 1% penicillin-streptavidin and 10% FBS. Cells were incubated in a humidified incubator with 5% CO2. For this experiment, cells were seeded into 6 cm culture dishes (4 × 10⁻⁶) with coverslips. 5 1 cell / culture dish), and add 2 mL of culture medium to each well. Add 2 μl of fresh working solution of the following three different compounds (PSMA-617 20, JNJ64619178 21, PSMA-617-JNJ 23) to each well at three different concentrations (10 nM, 1 nM, 0.1 nM):

[0360] .

[0361] Cells were incubated for 1 hour. One hour after adding the compound, cells were treated with IR: 2 Gy. The culture medium was replaced immediately after IR. After irradiation, cells were incubated for 6 hours to restore damaged DNA before harvest.

[0362] Cells were washed once with 1×PBS and fixed with 3.7% formaldehyde. Cells were incubated at room temperature for 20 minutes. Cells were washed three times with 1×PBS, then 0.2% Triton X-100 solution was added to each well, incubated at room temperature for 5 minutes, and washed three times again with 1×PBS. Cells were blocked at room temperature with 5% emulsion in 1×PBS for 1 hour. Cells were incubated overnight at 40°C with the primary antibody [phosphohistone H2A.X (ser139) (20E3)] solution (1:400 in 5% emulsion PBS) on coverslips (100 μL). The next day, cells were washed three times with 1×PBS. Then, secondary antibody containing DAPI solution (anti-rabbit-Alexa Fluors: 1:2000, final concentration of 1 mg / mL DAPI in 5% emulsion PBS) was added to the cells and incubated at room temperature for 1 hour. Cells were washed three times with 1×PBS and then blocked with ProLong. The Antifade kit (Invitrogen) mounted cells from coverslips onto slides. Images were captured using an inverted fluorescence microscope under oil immersion (60 × objective) (Nikon Instruments Melville, NY, USA) and via NIS Elements software. LNCaP, DU145, and PC-3 cells were incubated with different compounds for 1 hour, and results showed that for LNCaP (PSMA) cells... + DU145 (PSMA) + Both showed significantly lower double-strand DNA break repair (more focal sites) at 1 nM of compound 23. See also Figures 12 to 14 The PC-3 cell line does not express PSMA, therefore its effect was not significant, further demonstrating the specificity of compound 23. See also Figure 15 .

[0363] Figures 12 to 14 The results shown indicate that, compared to the carrier (i.e., DMSO), the PRMT5 inhibitor alone (21) and the carrier... 177 Lu, but not containing a PRMT5 inhibitor, PSMA-617 (20), and compound 23 containing a PRMT5 inhibitor significantly reduced DSB repair.

[0364] Example 5

[0365] Tumor model and treatment studies. Five-week-old male nu / nu mice were subcutaneously inoculated with LNCaP cells (5.0 × 10⁻⁶ cells in 100 μL PBS and 50 μL matrix gel) in the shoulder area. 6 Tumor growth was measured every 2 days using calipers in two vertical directions (while simultaneously monitoring body weight), and tumor volume was calculated as 0.5 × L × W² (L being the longest axis and W being the axis perpendicular to L, in millimeters). Once the tumor volume reached 150 to 200 mm², tumor growth was recorded. 3 The animals were then treated. For xenograft tumors, FIR (5 Gy / session) was performed, and 12 mice were divided into four different groups:

[0366] • Group 1 (untreated): 3 mice

[0367] • Group 2 tumor-bearing mice treated with external beam radiation (IR): 3 mice

[0368] • Group 3: Tumor-bearing mice treated with compound 23 (10 nmol) only: 3 mice.

[0369] • Group 4 tumor-bearing mice treated with external beam radiation (IR) + compound 23 (10 nmol): 3 mice.

[0370] For group 4, compound 23 was applied 1 hour before external beam irradiation.

[0371] Data showed that, compared to the control, combination therapy with compound 23 exhibited tumor growth inhibition ( Figure 16 No significant weight loss was observed during the treatment study. See also Figure 17 .

Claims

1. A conjugate of formula TLA, wherein: T is a ligand that targets prostate-specific membrane antigen (PSMA). L is the connector, and A contains a DNA repair enzyme inhibitor, wherein the DNA repair enzyme inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor, an ataxia-telangiectasia mutation (ATM) inhibitor, or a DNA protein kinase (DNA-PK) inhibitor.

2. The conjugate according to claim 1, wherein T is: 。 3. The conjugate according to claim 1, wherein T is: , where R 1 and R 2 Independently selected from hydrogen, optionally substituted carboxylic acids, malonic acid, succinic acid, glutamic acid, and adipic acid.

4. The conjugate of claim 3, wherein the substituted carboxylic acid is thioacetic acid or thiopropionic acid.

5. The conjugate of claim 1, wherein T is (a) an aminodicarboxylic acid or a derivative thereof and (b) an aminodicarboxylic acid or a derivative thereof, wherein (a) and (b) may be the same or different.

6. The conjugate according to any one of claims 1 to 5, wherein L comprises an atomic chain of about 3 atoms to about 30 atoms in length.

7. The conjugate according to any one of claims 1 to 6, wherein L comprises an atomic chain of about 5 Å to about 45 Å in length.

8. The conjugate of any one of claims 1 to 7, wherein L comprises a peptide.

9. The conjugate of any one of claims 1 to 8, wherein L comprises one or more phenylalanine residues, each of which is optionally substituted independently.

10. The conjugate of any one of claims 1 to 9, wherein L comprises at least one phenylalanyl-phenylalanyl group, wherein at least one phenyl group is optionally substituted independently.

11. The conjugate of any one of claims 1 to 10, wherein L comprises polyoligoethylene glycol n (POEGn), polyethylene glycol n (PEGn), or a mixture thereof, wherein n = 1 to 36.

12. The conjugate of any one of claims 1 to 11, wherein L comprises a disulfide.

13. The conjugate of claim 1, wherein the PRMT5 inhibitor comprises a group of JNJ-64619178 or a group of GSK3326595.

14. The conjugate of claim 1, wherein the ATM inhibitor comprises a group of AZD0156.

15. The conjugate of claim 1, wherein the DNA-PK inhibitor comprises the following groups: Nedisertib, AZD7648, KU57748, Torin 2, PI-103, NU7026, Samotolisib, PIK 90, CC-115, PIK-75 hydrochloride, VX-984, KU-0060648, BAY-8400, DNA-PK-IN-2, DNA-PK-IN-1, DNA-PK-IN-4, DNA-PK-IN-5, DNA-PK-IN-6, XRD-0394, NU5455, STL127705, PI-103 hydrochloride, LTURM34, ZL-2201 free base, DNA-PK-IN-14, DNA-PK-IN-10, DNA-PK-IN-11, or DNA-PK-IN-13.

16. The conjugate according to any one of claims 1 to 15, further comprising an albumin-binding group.

17. The conjugate of claim 16, wherein the albumin-binding group has the following formula: 。 18. The conjugate of claim 16, wherein the albumin-binding group is linked to L via a substituent or group present on L.

19. The conjugate of claim 16, wherein the albumin-binding group is wherein R 4 It is H or alkyl -NR 4 - or -O- group connected to L.

20. The following conjugate: 。 21. A conjugate of formula TL-(X)A, wherein: T is a ligand that targets prostate-specific membrane antigen (PSMA). L stands for connector. A contains a DNA repair enzyme inhibitor, wherein the DNA repair enzyme inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor, an ataxia-telangiectasia mutation (ATM) inhibitor, or a DNA protein kinase (DNA-PK) inhibitor; and X is a radiotherapy agent or a radioimaging agent.

22. The conjugate of claim 21, wherein A and X are connected to L.

23. The conjugate of claim 21 or 22, further comprising an albumin-binding group.

24. The conjugate of claim 23, wherein the albumin-binding group has the following formula: 。 25. The conjugate of claim 21 or 22, wherein the albumin-binding group is connected to L via a substituent or group present on L, and wherein the albumin-binding group is optionally connected to L as described in claim 19 or 20.

26. The conjugate of any one of claims 21 to 25, wherein T is defined in claims 2 to 5.

27. The conjugate of any one of claims 21 to 26, wherein L is defined in claims 6 to 12.

28. The conjugate of any one of claims 21 to 27, wherein A is defined in claims 13 to 15.

29. A pharmaceutical composition comprising the conjugate or pharmaceutically acceptable salt thereof as described in any one of claims 1 to 20, and a pharmaceutically acceptable carrier.

30. A pharmaceutical composition comprising the conjugate or pharmaceutically acceptable salt thereof as described in any one of claims 21 to 28, and a pharmaceutically acceptable carrier.

31. A method of treating a patient with prostate cancer or metastases using radiation therapy, the method comprising administering to the patient the conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 20, or the pharmaceutical composition of claim 29, thereby treating the patient with prostate cancer or metastases.

32. The method of claim 31, further comprising administering to the patient, before or after administration of the conjugate or pharmaceutical composition of any one of claims 1 to 20, a conjugate of formula TLX or a pharmaceutically acceptable salt thereof, wherein: T is a ligand that targets prostate-specific membrane antigen (PSMA). L is the connector, and X is a radiotherapy agent or a radioimaging agent.

33. The method of claim 32, wherein T is: 。 34. The method of claim 32, wherein T is: , where R 1 and R 2 Independently selected from hydrogen, optionally substituted carboxylic acids, malonic acid, succinic acid, glutamic acid, and adipic acid.

35. The method of claim 32, wherein the substituted carboxylic acid is thioacetic acid or thiopropionic acid.

36. The method of claim 32, wherein T is (a) an aminodicarboxylic acid or a derivative thereof and (b) an aminodicarboxylic acid or a derivative thereof, wherein (a) and (b) may be the same or different.

37. The method of claim 32, wherein L comprises an atomic chain of about 3 atoms to about 30 atoms in length.

38. The method of claim 32, wherein L comprises an atomic chain of about 5 Å to about 45 Å in length.

39. The method of claim 32, wherein L comprises a peptide.

40. The method of claim 32, wherein L comprises one or more phenylalanine residues, each of which is optionally substituted independently.

41. The method of claim 32, wherein L comprises at least one phenylalanyl-phenylalanyl group, wherein at least one phenyl group is optionally substituted independently.

42. The method of claim 32, wherein L comprises polyoligoethylene glycol n (POEGn), polyethylene glycol n (PEGn), or a mixture thereof, wherein n = 1 to 36.

43. The method of claim 32, wherein X comprises a radioactive isotope bound to a chelating agent, the chelating agent being selected from: • DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or its derivatives; • TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) or its derivatives; • SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) or its derivatives; • NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) or its derivatives; • NETA (4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazacyclononane-1-yl)acetic acid or a derivative thereof; • TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphonic acid) or its derivatives; • HBED (N,NO-bis(2-hydroxybenzyl)-ethylenediamine-N,NO-diacetic acid) or its derivatives; • 2,3-HOPO (3-hydroxypyridin-2-one) or its derivatives; • PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadecane-1(15),11,13-triene-3,6,9-triacetic acid) or its derivatives; • DFO (deferriphosphate) or its derivatives; • DTPA (diethylenetriaminepentaacetic acid) or its derivatives; • OCTAPA (N,NO-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,NO-diacetic acid) or its derivatives; or H2-MACROPA (N,N'-bis[(6-carboxy-2-pyridylmethyl]-4,13-diaza-18-crown-6) or its derivatives; • H2dedpa (1,2-[[carboxyl)-pyridin-2-yl]-methylamino]ethane or a derivative thereof; and • EC20-head, which contains β-l-diaminopropionic acid, aspartic acid and cysteine.

44. The method of claim 32, wherein X comprises a radioactive isotope selected from: 18 F, 44 Sc、 47 Sc、 52 Mn, 55 Co、 64 Cu、 6 7Cu, 67 Ga、 68 Ga、 86 Y、 89 Zr、 90 Y、 99m Tc, 111 In、 114m In、 117m Sn、 124 I, 125 I, 131 I, 149 Tb, 153 Sm、 152 Tb, 155 Tb, 161 Tb, 177 Lu、 186 Re、 188 Re、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 224 Ra、 225 Ab、 225 Ac and 227 Th.

45. The method of claim 32, further comprising administering an immune checkpoint inhibitor to the patient.

46. ​​The method of claim 45, wherein the immune checkpoint inhibitor is selected from cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein 1 (PD-1) inhibitors, and programmed cell death ligand 1 (PD-L1) inhibitors.

47. The method of claim 45, wherein the immune checkpoint inhibitor is selected from nivolumab (Opdivo), pembrolizumab (Keytruda), ipilimumab (Yervoy), atezolizumab, avelumab, and durvalumab.

48. A method of treating a patient with prostate cancer or metastases using radiation therapy, the method comprising administering to the patient the conjugate or pharmaceutically acceptable salt of any one of claims 21 to 28, or the pharmaceutical composition of claim 30, thereby treating the patient with prostate cancer or metastases.

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