Uses of AKR1C3-activated compounds

Abstract

In the manufacture of medicines for the treatment of cancer, use is provided for a compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate or its pharmaceutically acceptable salt, isotope variant, or solvate, and a composition comprising said compound and at least one anticancer agent.

Description

Technology Field

[0001] The present invention relates to the medical use of the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate or its pharmaceutically acceptable salts, isotope variants, or solvates, and to a composition comprising said compound and at least one anticancer agent and its medical use. Background Technology

[0002] Cancer is one of the major causes of human morbidity and mortality. Cancer treatment is difficult because it is difficult to kill cancer cells without damaging or killing normal cells. Damage or death of normal cells during cancer treatment causes side effects in patients and may limit the amount of anticancer drugs administered to cancer patients.

[0003] Aldo-keto reductase 1C3 (AKR1C3) is also known as type 5, 17β-hydroxysteroid dehydrogenase (17β-HSD) and prostaglandin F synthase. AKR1C3 is a member of the 15 gene families of aldoketo reductases (AKR). AKR1C3 was originally [indicated] in the human prostate (1) and placenta (2) It was replicated from a cDNA library. AKR1C3 has 323 amino acids and a molecular weight of 37 kDa (1) It is a monomeric cytoplasmic NAD(P)(H)-dependent oxidoreductase. AKR1C3 shares high sequence homology with the related human AKR1C family, including AKR1C1, AKR1C2, and AKR1C4. AKR1C3 catalyzes the metabolism of androgens, estrogens, progesterone, and prostaglandins (PGs) and subsequently participates in the regulation of nuclear receptor activity. (3,4) AKR1C3 is expressed in normal tissues, including steroid hormone-dependent and steroid hormone-independent cells, and the average expression level is low except in the liver, kidney, and small intestine. (5)Numerous studies have demonstrated that AKR1C3 is abnormally overexpressed in many malignant solid and hematological tumors. Data indicate that high AKR1C3 expression was detected in over 50% of liver tumors, bladder cancers, kidney cancers, and gastric cancers, with immunohistochemical scores (IHC scores) ranging from 0 to 6 and found to be 4 or higher. (6) AKR1C3 is highly expressed in non-small cell lung cancer (NSCLC) but is not expressed in small cell lung cancer. (7) .

[0004] AKR1C3 upregulation in cancer is castration-resistant prostate cancer (CRPC (8) ) and colorectal cancer (CRC (9) It has been reported to be associated with metastasis, and is also associated with a poor prognosis and low survival rate. (10,11) In addition, many types of treatment resistance are attributed to AKR1C3 overexpression. Doxorubicin (12,13) , enzalutamide (14) , abirateron (15) and methotrexate (16) Chemotherapy resistance to [it] has been reported to be directly associated with high AKR1C3 expression in cells. Esophageal cancer (17) , prostate cancer (18) and NSCL cancer cells (19) In [the context], resistance to radiotherapy is associated with AKR1C3 overexpression. The primary mechanism of action of AKR1C3 against ionizing radiation is to reduce ROS (reactive oxygen species) and increase PGF2α in cells, leading to MAP kinase activation and PPARγ inhibition, which consequently significantly reduces DNA damage. (18) Furthermore, immunotherapy resistance is attributed to high expression of AKR1C3. A study based on whole-genome microarray and multiplex quantitative (q)RT-PCR gene expression analysis showed that high expression of AKR1C3 was associated with the failure of PD-1 targeted therapy performed on PD-L1-positive patients with advanced renal cell carcinoma (RCC). (20)Due to the tumor-specific overexpression of AKR1C3, the design of AKR1C3-activated prodrugs has become an attractive approach for specifically targeting cancer. One such example is the AKR1C3-activated prodrug PR104, which was originally designed as a hypoxia-activated prodrug, but (22-24) It demonstrated excellent antitumor activity in vitro and in vivo. (6, 21) .

[0005] The anticancer prodrug having chemical formula I-1 (referred to herein as 3424) is a chemically synthesized potent nitrogen mustard, which is selectively cleaved by AKR1C3 in the presence of NADPH to become cytotoxic aziridine (referred to herein as 2660). The active molecule 2660 released by 3424 is similar to the standard chemotherapy drugs thiotepa and mitomycin C, resulting in alkylation and cross-linking of DNA at the N7 (or O6) position of guanine. The prodrug 3424 is currently being developed by Ascentawits Pharmaceuticals, LTD in Asian countries and by OBI Pharma, Inc. in countries outside of Asia for the treatment of malignant tumors (drug code OBI-3424). Prodrug 3424 is currently being studied in multiple Phase 1 clinical trials in the United States (NCT04315324 and NCT03592264) and China (CXHL1900137 and CXHL2000263) for the treatment of over 14 types of human cancer, including solid tumors and hematological malignancies. Prodrug 3424 is designed to achieve tumor-specific targeting because it is specifically activated in tumors due to the high expression of AKR1C3 in tumors, but remains inactive (spared) in normal cells with low AKR1C3 expression levels. Furthermore, the tumor-selective activation of 3424 can be differentiated from existing non-selective alkylating agents such as cyclophosphamide and ifosfamide, indicating that 3424 has the potential to be a broad-spectrum, highly selective antitumor drug. Prodrug 3424 has been reported to demonstrate potent efficacy against preclinical models of T-ALL in vitro and in vivo. (25,26) .

[0006] In the presence of NADPH, 3424 is reduced by AKR1C3, releasing the cytotoxic moiety 2660, an aziridine bis-alkylating agent, which leads to DNA cross-linking at the N7 (or O6) position of guanine and subsequent cell death.

[0007]

[0008] 3424 Overview of the Reduction-Activation Pathway

[0009] Prodrugs designed to target cancer cells have attracted attention and emerged as a strategy for cancer treatment in recent years; however, many prodrugs failed in Phase 3 clinical trials due to the lack of effective biomarkers for patient selection. (33) If AKR1C3 expression can be evaluated using RT-PCR or immunohistochemistry, 3424 can be developed in a clinically efficient manner by selecting patients with high AKR1C3 expression who are most likely to respond to the prodrug. AKR1C3 is chemoresistant (13,14) , radiation resistance (19) and immune resistance (20) It has been proven to be overexpressed upon acquisition. In addition, homologous recombination deficiency (HRD) cancers, such as ovarian, breast, and pancreatic cancers, are known to be sensitive to DNA damaging agents. (34) 3424 as a DNA alkylating agent may be a good candidate drug for treating HRD cancers in which AKR1C3 is expressed.

[0010] There is still a need for a compound suitable for treating cancer patients that is a selective AKR1C3 reductase-activated prodrug, novel, selective, and broad-spectrum anticancer agent. The present invention meets this need.

[0011] Summary of the Invention

[0012] The present invention is based on a compound or its pharmaceutically acceptable salt or solvate as disclosed in patent application PCT / US2016 / 021581 (WO2016 / 145092) and patent application PCT / US2016 / 062114 (WO2017 / 087428), provides a medical use of said compound, and provides a composition comprising said compound or its pharmaceutically acceptable salt, isotope variant, or solvate and its anticancer medical use.

[0013] In one aspect, the present invention provides a use of 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate, a compound having the following formula I, or a pharmaceutically acceptable salt, isotope variant, or solvate thereof, in the manufacture of a medicine for treating cancer in a patient, wherein the AKR1C3 reductase level of said cancer is expressed as an AKR1C3 protein level or RNA level and is equal to or greater than a predetermined value:

[0014]

[0015] Chemical formula I (compound 2870)

[0016] The AKR1C3 level is measured according to conventional methods well known to those skilled in the art.

[0017] According to a specific embodiment of the present invention, the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the following formula I-1 or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the following formula I-2 (referred to herein as 3423):

[0018]

[0019] Chemical formula I-1 Chemical formula I-2

[0020] (Compound 3424) (Compound 3423)

[0021] The preparation of compounds of Formula I, Formula I-1 or Formula I-2 is disclosed in PCT application PCT / US2016 / 021581 (WO2016 / 145092) and patent application PCT / US2016 / 062114 (WO2017 / 087428), the entire contents of which are incorporated herein by reference. Herein, compound 2870 is a racemic mixture of R-enantiomer 3423 and S-enantiomer 3424 in a 1:1 ratio.

[0022] In the above, the salt may be a basic salt comprising a salt of the compound with an inorganic base (e.g., alkali metal hydroxide and alkaline earth metal hydroxide) or a salt with an organic base (e.g., monoethanolamine, diethanolamine, or triethanolamine). Alternatively, the salt may be an acid salt comprising a salt of the compound with an inorganic acid (e.g., hydrochloric acid, hydrobromide, hydroiodide, nitric acid, perchloric acid, sulfuric acid, or phosphoric acid) or a salt with an organic acid (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, and citric acid). The preparation of acceptable salts, solvates, etc. of the compound is a technique well known in the art.

[0023] According to a specific embodiment of the present invention, a compound of Formula I-1 or Formula I-2 has an enantiomer excess of 80% or more. Preferably, the compound has an enantiomer excess of 90% or more, more preferably 95% or more.

[0024] According to a specific embodiment of the present invention, the compound of formula I-1 or formula I-2 is substantially pure.

[0025] According to a specific embodiment of the present invention, the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colorectal cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma, or rectal cancer.

[0026] According to a specific embodiment of the present invention, the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma, or pancreatic cancer.

[0027] The dosage of a drug used for cancer treatment, or the dosage of the compound or its salt, isotope variant, or solvate, or the dosage of other anticancer agents contained in the drug, generally depends on the specific compound, patient, specific disease or pathological condition and its severity, the route and frequency of administration, etc., and needs to be determined by the attending physician according to the specific pathological condition. For example, when the composition or drug provided in the present invention is administered via an oral route, the dosage may be 0.1 to 30 mg / 7 days, preferably 1 to 10 mg / 7 days, more preferably 5 mg / day; the dosage may be administered once every 7 days or divided into two doses to be administered twice every 7 days; preferably, the dosage is administered once every 7 days.

[0028] The medicine may be any dosage form for clinical administration, such as tablets, suppositories, disperse tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated preparations, granules, dry powder, oral solution, injection needle, injection lyophilized powder, or infusion solution.

[0029] In another aspect, the present invention provides a method for treating cancer in a patient requiring cancer treatment, comprising the step of administering an effective amount of a compound having the following formula I, 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate, or a pharmaceutically acceptable salt, isotope variant, or solvate thereof, to said patient, wherein the AKR1C3 reductase level of said cancer is expressed as an AKR1C3 protein level or RNA level and is equal to or greater than a predetermined value:

[0030]

[0031] Chemical Formula I

[0032] According to a specific embodiment of the present invention, the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the following formula I-1 or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the following formula I-2:

[0033]

[0034] Chemical formula I-1 Chemical formula I-2.

[0035] According to a specific embodiment of the present invention, the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colorectal cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma, or rectal cancer.

[0036] According to a specific embodiment of the present invention, the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma, or pancreatic cancer.

[0037] According to a specific embodiment of the present invention, the method further comprises the step of measuring the content of AKR1C3 reductase in a patient's cancer cells using an AKR1C3 antibody, and when the content of AKR1C3 reductase is measured to be equal to or greater than a predetermined value, the compound is administered to the patient.

[0038] In another aspect, the present invention provides a method for inhibiting cell growth, comprising the step of contacting said cells with an effective amount of 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the formula I or a pharmaceutically acceptable salt, isotope variant, or solvate thereof; wherein the AKR1C3 reductase level of said cells is expressed as an AKR1C3 protein level or RNA level and is equal to or greater than a predetermined value.

[0039] According to a specific embodiment of the present invention, the cell is a cancer cell.

[0040] According to a specific embodiment of the present invention, the method further comprises the step of measuring the content of AKR1C3 reductase in a cell using an AKR1C3 antibody, and when the content of AKR1C3 reductase is measured to be equal to or greater than a predetermined value, the compound is brought into contact with the cell.

[0041] In another aspect, the present invention provides for the use of a compound having the formula I, 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate, or a pharmaceutically acceptable salt, isotopic variant, or solvate thereof, in the manufacture of a drug for inhibiting cell growth, wherein the AKR1C3 reductase level of said cell is expressed as an AKR1C3 protein level or RNA level and is equal to or greater than a predetermined value.

[0042] According to a specific embodiment of the present invention, the cell is a cancer cell.

[0043] In another aspect, the present invention provides a composition comprising the following:

[0044] 1) a compound having the chemical formula I 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate, or its pharmaceutically acceptable salts, isotopic variants, or solvates; and

[0045] 2) At least one other anticancer drug.

[0046] According to a specific embodiment of the present invention, the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the formula I-1 or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the formula I-2.

[0047] According to a specific embodiment of the present invention, the anticancer agent comprises gemcitabine, 5-fluorourasie (5-FU), sunitinib, abiraterone acetate, prednisolone, erlotinib, meturedepa, uredepa, altretamine, imatinib, triethylenemelamine, trimethylmelamine, chlorambucil, chlornafazine, estramustine, gefitinib, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novelichine, phenesterin, prednimustine, trophosphamide, uracil mustard, carmustine, chlorozotocin, potemustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipovroman, aclaxinomycin, actinomycin, antramycin, azacerin, bleomycin, Cactinomycin, Carubicin, Casinophylline, Cromomycin, Dactinomycin, Daunorubicin, Daunomycin, 6-Diazoth-5-oxo-l-norleucine, Mycophenolic acid, Nogalamycin, Olivomycin, Peflomycin, Plicamycin, Porphyromycin, Puromycin, Streptonigreen, Streptozosin, Tubercidin, Uvenimex, Genostatin, Zorubicin, Denopterin, Pteropterin, Trimetrexate, Fludarabine, 6-Mercaptopurine, Thiamiphrine, Thioguanine, Ancitabine, Azacitidine, 6-Azauridine, Camofur, Cytarabine, Dideoxyuridine, Doxyfluridine, Enositabine, Fluxuridine, Tegafur, L-Asparaginase, Pulmozyme, Aceglatone, Aldophosphamide Glycoside, Aminolevulinic Acid, Amsacrin, Bestabucil, Biesantren, Depopamide, Demecolsin, Diaziquone, L-Fornitin, Elliptinium Acetate, Etogluside, Flutamide, Hydroxyurea, Interferon-Alpha, Interferon-Beta, Interferon-Gamma, Interleukin-2, Lentinan, Mitoguazone, Mithoxantrone, Morpidamol, Nitracrine, Pentostatin, Fenamet, Pirarubicin, Podophyllinic Acid, 2-Ethylhydrazide, Procarbazine, Razoxic Acid, Sizopyran, Spirogermanium, Paclitaxel, Tamoxifen, Erlotonib, Tenifoside, Tenuazonic Acid, Triaziquone, 2,2',2"-trichlorotriethylamine, urethane,It is selected from the group consisting of vinblastine and vincristine.

[0048] According to a specific embodiment of the present invention, the anticancer agent is selected from the group consisting of gemcitabine, abiraterone acetate, prednisolone, 5-FU, sunitinib, or a combination of abiraterone acetate and prednisolone.

[0049] According to a specific embodiment of the present invention, if the cancer is renal cell carcinoma (RCC), the anticancer agent is selected from the group consisting of gemcitabine and sunitinib; if the cancer is gastric cancer, the anticancer agent is 5-FU; and if the cancer is castration-resistant prostate cancer (CRPC), the anticancer agent is selected from the group consisting of abiraterone acetate and prednisolone or a combination thereof.

[0050] According to a specific embodiment of the present invention, the composition further comprises a pharmaceutically acceptable excipient. Preferably, the excipient is selected from inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and oils.

[0051] In another aspect, the present invention provides a use of the composition according to the present invention in the manufacture of a medicine for treating cancer in a patient.

[0052] According to a specific embodiment of the present invention, the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colorectal cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma, or rectal cancer.

[0053] According to a specific embodiment of the present invention, the level of AKR1C3 reductase in the cancer is equal to or greater than a predetermined value.

[0054] According to a specific embodiment of the present invention, the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma, or pancreatic cancer.

[0055] In another aspect, the present invention provides a method for treating cancer in a patient requiring cancer treatment, comprising the step of administering an effective amount of a composition according to the present invention to the patient.

[0056] According to a specific embodiment of the present invention, the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colorectal cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma, or rectal cancer.

[0057] According to a specific embodiment of the present invention, the level of AKR1C3 reductase in the cancer is equal to or greater than a predetermined value.

[0058] According to a specific embodiment of the present invention, the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma, or pancreatic cancer.

[0059] According to a specific embodiment of the present invention, the method further comprises the step of measuring the content of AKR1C3 reductase in a patient's cancer cells using an AKR1C3 antibody, and when the content of AKR1C3 reductase is measured to be equal to or greater than a predetermined value, the composition is administered to the patient. Brief explanation of the drawing

[0060] Figure 1 illustrates AKR1C3-dependent 3424 activation. (a) 3424 decrease; (b) 2660 increase. Figure 2 illustrates the detection of AKR1C3 protein expression in liver cancer cells by Western blot. Figure 3 illustrates the AKR1C3-dependent in vitro cytotoxicity of 3424. (a) AKR1C3 protein expression and 3424 IC in hepatocellular carcinoma cells. 50 Correlation between (left); AKR1C3 RNA expression and 3424 IC in liver cancer cells 50Correlation between (median); AKR1C3 RNA expression and 3424 IC in NSCLC cancer cells 50 (b) Correlation between (right); (b) AKR1C3-specific inhibitor 3021 effectively inhibited the cytotoxicity of 3424 (left), 3423 (middle) and racemic mixture 2870; (c) Compound 2870 induced concentration-dependent DNA cross-linking. Figure 4 illustrates the antitumor activity of 3424 in various human cell line-derived xenograft (CDX) models. HepG2 liver orthotopic model (a and b). VCap castration-resistant prostate cancer in castrated male BALB / c nude mice (c), SNU-16 gastric cancer in female BALB / c nude mice (d), and A498 renal cell carcinoma in female SCID mice (e and f). Animals were treated with various concentrations of 3424, standard therapy regimens, and combinations indicated in the legend; where "AA" represents abiraterone acetate, "P" represents prednisolone, "S" represents sunitinib, and "G" represents Gemza. Figure 5 shows the anti-tumor activity of 3424 in the subcutaneous lung cancer model H460 CDX model. Figure 6 shows the measurement of serum prostate-specific antigen (PSA) after treatment at a specified time. Figure 7 illustrates the antitumor activity of 3424 against PDX panels. Pancreatic cancer PA1280 (a), gastric cancer GA6201 (b), lung cancer LU2505 (c) with high AKR1C3 expression, and lung cancer LU2057 (d) with low AKR1C3 expression. Specific details for implementing the invention

[0061] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are used merely to illustrate the invention and do not limit the scope of the invention in any way.

[0062] definition

[0063] The following definitions are provided to assist readers of this specification. Unless otherwise defined, all technical terms, notations, and other scientific or medical terms used herein are intended to have the meanings generally understood by those skilled in the art of chemistry and medicine. In some cases, terms having a generally understood meaning are defined in this specification for clarity and / or ease of reference, and the inclusion of such definitions in this specification should not be interpreted as representing a substantial difference from the definitions of terms generally understood in the art.

[0064] All numerical designations, including their respective ranges, e.g., pH, temperature, time, concentration, and weight, are generally approximations that can be appropriately changed (+) or (-) in increments of 0.1, 1.0, or 10.0. All numerical designations may be understood to be preceded by the term "approx." The reagents described herein are exemplary, and equivalents may be known in the art.

[0065] "A(a)," "an," and "the" include multiple referents unless otherwise specified in the context. Thus, for example, a reference to a compound means one or more compounds or at least one compound. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this specification.

[0066] The term “approximately” or “roughly” means an acceptable error for a specific value determined by a person skilled in the art, depending in part on the manner in which said value is measured or determined. In certain embodiments, the term “approximately” or “roughly” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “approximately” or “roughly” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0067] As used herein, the term “comprising” is intended to mean that the composition and method include the cited elements but do not exclude other elements. When used to define the composition and method, “essentially comprising” means excluding other elements that are essentially significant to the composition or method. “Comprising” means excluding elements that exceed the amount of other components in the claimed composition and substantial method steps. Embodiments defined by each of these transition terms are within the scope of the invention. Accordingly, the method and composition may include additional steps and components, otherwise, insignificant steps and compositions, otherwise, the method or composition of the mentioned steps only.

[0068] "Administering" or "administering" a drug to a patient (and its grammatical equivalents) refers to direct administration, which may be administered to the patient by a medical professional or self-administration, and / or indirect administration, which may be the act of prescribing the drug. For example, a physician who instructs a patient to self-administer a drug and / or provides a prescription for the drug to the patient is administering the drug to the patient.

[0069] "Cancer" means leukemia, lymphoma, carcinoma, and other malignant tumors, and includes solid tumors of potentially unlimited growth that may spread locally by invasion and throughout the body by metastasis. Examples of cancer include, but are not limited to, adrenal cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colorectal and / or rectal cancer, gallbladder cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, nervous tissue cancer, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, gastric cancer, and thyroid cancer. Other specific examples of cancer include acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma pleomorpha, hairy-cell tumor, intestinal ganglion neuroma, hyperplastic corneal neuroma, islet cell carcinoma, Kaposi sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, marphanoid habitual tumor, medullary carcinoma, metastatic cutaneous carcinoma, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, myeloma sarcoma, osteogenesis and other sarcomas, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung tumor, squamous cell carcinoma of both ulcerative and papillary types, hyperplastic, seminoma, soft tissue sarcoma. Retinoblastoma, rhabdomyosarcoma, renal cell tumor, focal skin lesions, bullous cell sarcoma, and Wilms tumor are included.

[0070] The term “contacting” or “contact” means bringing a therapeutic agent and a cell or tissue together so that physiological and / or chemical effects occur as a result of such contact. Contact may occur in vitro, in vitro, or in vivo. In one embodiment, the therapeutic agent is brought into contact with cells in a cell culture (in vitro) to determine the therapeutic effect on the cells. In another embodiment, contact between the therapeutic agent and the cell or tissue involves administering the therapeutic agent to a subject having the cell or tissue to be contacted.

[0071] The term “optical activity” means a series of molecules having an enantiomer excess of about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, about 99.8% or more, or about 99.9% or more. In a specific embodiment, the enantiomer excess for the optically active compound is about 90% or more, about 95% or more, about 98% or more, or about 99% or more. The excess of enantiomers of a compound can be determined by a person skilled in the art using any standard method including, but not limited to, chiroptical chromatography using an optically active stationary phase (gas chromatography, high-performance liquid chromatography and thin-layer chromatography), isotope dilution, electrophoresis, calorimetry, polarimetry, NMR decomposition methods using chiral derivatization, and NMR methods using a chiral solvator or chiral transfer reagent.

[0072] When describing optically active compounds, the prefixes R and S are used to indicate the absolute molecular form with respect to the chiral center(s).

[0073] The term “substantially pure” means homogeneity sufficient to appear free of impurities that are easily detectable when determined by a person skilled in the art using standard analytical methods, including but not limited to thin-layer chromatography (TLC), gel electrophoresis, high-performance liquid chromatography (HPLC), gas chromatography (GC), nuclear magnetic resonance (NMR), and mass spectrometry (MS); or means purity sufficient to ensure that physical, chemical, biological, and / or pharmacological properties, such as the enzymatic and biological activity of the substance, are not detectably altered by further purification. In certain embodiments, “substantially pure” means a series of molecules that are monostereoisomers of the compound when, by weight of the molecules, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% is determined by standard analytical methods.

[0074] "Patient" and "subject" are used interchangeably to refer to mammals requiring cancer treatment. Generally, the patient is a human. Generally, the patient is a human diagnosed with cancer. In certain embodiments, "patient" or "subject" may refer to non-human mammals, such as non-human mammals, dogs, cats, rabbits, pigs, mice, or rats, used to screen, characterize, and evaluate drugs and therapies.

[0075] "Prodrug" means a compound that is metabolized or otherwise converted into a compound (or drug) that is biologically active or more active in at least one characteristic after administration. Prodrugs are less active or inactive compared to drugs, but are chemically modified relative to drugs such that chemical modifications occur after administration of the prodrug through metabolism or other biological processes to produce the corresponding drug. Prodrugs may have modified metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor compared to the active drug (e.g., see Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, referenced herein). Prodrugs may be synthesized using reactants other than the corresponding drug.

[0076] "Solid tumor" refers to a solid tumor including, but not limited to, metastatic tumors of bone, brain, liver, lungs, lymph nodes, pancreas, prostate, skin, and soft tissues (sarcoma).

[0077] The "therapeutic effective dose" of a drug refers to the amount of drug that, when administered to a cancer patient, produces the intended therapeutic effect, for example, the reduction, improvement, alleviation, or elimination of one or more signs of the patient's cancer. The therapeutic effect does not necessarily occur with a single dose administration but may occur only after a series of doses have been administered. Therefore, the therapeutic effective dose may be administered in one or more doses.

[0078] "Treatment" for a condition or patient refers to steps taken to obtain beneficial or desired results, including clinical outcomes. Beneficial or desired clinical outcomes for the purposes of the present invention include, but are not limited to, alleviation or improvement of one or more symptoms of cancer; reduction in the severity of the disease; delay or slowing of disease progression; alleviation, remission, or stabilization of the disease state; or other beneficial outcomes. Cancer treatment may result in a partial response or stable disease in some cases.

[0079] "Tumor cells" means tumor cells of any suitable species, for example, mammals such as murine, dog, feline, horse, or human.

[0080] The term "isotope variant" means a compound containing an abnormal proportion of isotopes in one or more atoms constituting the compound. In certain embodiments, the "isotope variant" of the compound is hydrogen ( 1 H), deuterium ( 2 H), tritium ( 3 H), carbon-11( 11 C), carbon -12( 12 C), carbon-13( 13 C), carbon-14( 14 C), nitrogen-13( 13 N), Nitrogen-14( 14 N), Nitrogen-15( 15 N), Oxygen-14( 14 O), Oxygen- 15( 15 O), Oxygen-16 ( 16 O), Oxygen-17( 17 O), Oxygen-18( 18 O), Fluorine-17( 17 F), Fluorine-18 ( 18 F), Phosphorus-31( 31 P), Phosphorus-32( 32 P), Phosphorus-33( 33 P), sulfur-32( 32 S), Sulfur-33( 33 S), Sulfur-34( 34 S), Sulfur-35(35 S), Sulfur-36( 36 S), Chlorine-35( 35 Cl), Chlorine-36( 36 Cl), Chlorine-37( 37 Cl), Bromine-79( 79 Br), Bromine-81( 81 Br), iodine-123( 123 I), iodine-125( 125 I), iodine-127( 127 I), iodine-129( 129 I) and iodine-131( 131 It contains an anomalous proportion of one or more isotopes, including but not limited to I). In certain embodiments, the “isotope variant” of the compound is a stable form, i.e., non-radioactive. In certain embodiments, the “isotope variant” of the compound is hydrogen ( 1 H), deuterium ( 2 H), carbon-12( 12 C), carbon-13( 13 C), nitrogen-14( 14 N), Nitrogen-15( 15 N), oxygen-16( 16 O), Oxygen-17( 17 O), Oxygen-18( 18 O), Fluorine-17( 17 F), Phosphorus-31( 31 P), sulfur-32( 32 S), Sulfur-33( 33 S), Sulfur-34( 34 S), Sulfur-36( 36 S), Chlorine-35( 35 Cl), Chlorine-37( 37 Cl), Bromine-79( 79 Br), Bromine-81( 81 Br) and iodine-127( 127 It contains an anomalous proportion of one or more isotopes, including but not limited to I). In certain embodiments, the "isotope variant" of the compound is an unstable form, i.e., radioactive. In certain embodiments, the "isotope variant" of the compound is tritium ( 3H), carbon-11( 11 C), carbon-14( 14 C), nitrogen-13( 13 N), Oxygen-14( 14 O), Oxygen-15( 15 O), Fluorine-18( 18 F), Phosphorus-32( 32 P), Phosphorus-33( 33 P), Sulfur-35( 35 S), Chlorine-36( 36 Cl), iodine-123( 123 I), iodine-125( 125 I), iodine-129( 129 I) and iodine-131( 131 It contains an anomalous proportion of one or more isotopes, including but not limited to I). In compounds such as those provided herein, any hydrogen is, for example, 2 It can be H, or any carbon, for example 13 It could be C, or any nitrogen, for example 15 It may be N, and any oxygen may be appropriately determined by a person skilled in the art. 18 You will understand that it may be O. In certain embodiments, the "isotopic variant" of the compound contains an abnormal deuterium ratio.

[0081] The term “solvent” means a complex or aggregate formed by the presence of one or more solute molecules, for example, one or more solvent molecules of the compounds and solvents provided herein in stoichiometric or non-stoichiometric amounts. Suitable solvents include, but are not limited to, water, methanol, ethanol, n-propanol, isopropanol, and acetic acid. In certain embodiments, the solvent is pharmaceutically acceptable.

[0082] In one embodiment, the complex or aggregate is in a crystalline form. In another embodiment, the complex or aggregate is in an amorphous form. When the solvent is water, the solvate is a hydrate. Examples of hydrates include, but are not limited to, hemihydrates, monohydrates, dihydrates, trihydrates, tetrahydrates, and pentahydrates.

[0083] The term "pharmaceuticalally acceptable excipient" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense that it is compatible with other components of the pharmaceutical formulation and is suitable for use in contact with tissues or organs of humans and animals without excessive toxicity, irritation, or allergic reaction.

[0084] Examples

[0085] Materials and Methods

[0086] cell line

[0087] All human cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA), the Japanese Collection of Research Biosources (JCRB, Osaka, Japan), or Cobioer Biosciences (Nanjing, China).

[0088] Reagents and Chemicals

[0089] Anti-human AKR1C3 monoclonal antibody, bleomycin, NADPH, lyophilized bovine serum albumin (BSA), and positive control substrates for AKR1C1 / AKR1C3 (progesterone, androstenedione, and dihydrotestosterone) were purchased from Sigma (St. Louis, MO). Recombinant human AKR1C3 was purchased from Abcam (Cambridge, MA), and AKR1C1 and AKR1C4 were purchased from Sigma. The Comet assay kit was purchased from Trevigen (Gaithersburg, MD). The CellTiter Glo (CTG) assay kit was purchased from Promega (Madison, WI). Racemic 2870 was synthesized by Threshold Pharmaceuticals (South San Francisco, CA). Prodrugs 3423 and 3424 were synthesized by Ascentawits Pharmaceuticals, LTD (Shenzhen, China). 3021 was synthesized based on the reported method. (27) Standard treatment regimens were purchased from abiraterone (Bos Science, USA), prednisolone (Saen Chemical Technology, China), 5-FU (Shanghai Xudong Haipu Pharmaceutical Co., China), gemcitabine (Vianex SA, Greece), and sunitinib (Cayman, USA).

[0090] In the following examples, AKR1C3-dependent activation, in vitro cytotoxicity of 3424 in a wide range of human cancer cell lines, and concentration-dependent DNA cross-linking of 3424 were studied. Additionally, the in vivo antitumor activity of 3424 was studied in a wide range of CDX and PDX model panels.

[0091] Example 1: AKR1C3-dependent activation of 3424

[0092] Enzyme activity test

[0093] The assay mixture consisted of 10 to 50 μM 3424 or a positive control (androstenedione or dihydrotestosterone), 100 mM phosphate buffer, pH 7.0, 300 μM NADPH, 4% ethanol, and 8 M recombinant human AKR1C3, AKR1C1, or AKR1C4, providing a total volume of 200 μL. The reaction mixture was incubated at 25°C, terminated at various time points by adding acetonitrile and methanol (9:1 ratio), and analyzed by LC-MS / MS. SpectraMax M2 was used for enzyme kinetics analysis. e Decrease in NADPH absorbance at 340 nm using a spectrophotometer (Molecular Devices, LLC, San Jose, CA) (ε = 6270 M -1 cm -1 Activity was determined by measuring ). After initiating the reaction by adding the substrate, the reaction was monitored at 25°C at 30-second intervals. The non-substrate reaction rate was also monitored in the background, and a gradient was used to determine the initial reaction rate. The reported kinetic data were the average of three (triplicate) measurements.

[0094] LC-MS / MS analysis

[0095] For AKR1C3-mediated 3424 metabolism, LC / MS / MS was performed using a Sciex API-4000 Qtrap (ABSciex, LLC, Framingham, MA) mass spectrometer coupled to an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA). For 3424 analysis, reverse-phase liquid chromatography separation was performed using a Waters Xbridge C18 column (2.1 × 100 mm, 3.5 μm, Waters Corp., Milford, MA) at a flow rate of 0.3 mL / min for a total run time of 12 minutes. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in ACN (acetonitrile). The gradient was performed by running an isolytic run at 15% B for 1.5 minutes, then to 50% B at 3 minutes, followed by a gradient to 95% B at 6 minutes, holding for 1 minute, finally returning to the 15% B gradient within 0.1 minutes, and equilibrating at 15% B for 4.9 minutes. The column oven temperature was 40°C and the sample injection volume was 2 μL. Mass spectra were obtained in positive MRM mode. In cation mode, the ion spray voltage was set to 4500 V, the declustering potential to 80 V, the collision energy to 20 V, the source temperature to 350°C, the curtain gas to 10 psi, and both source gases 1 and 2 to 60 psi. The MRM pairs for 3424 and 3424-IS were m / z 461 → 313 and m / z 465 → 313, respectively. For the analysis of 2660, normal liquid chromatography separation was performed using a Waters Atlantis HILIC Silica column (2.1 mm x 100 mm, 3 μm, Waters Corp., Milford, MA) at a flow rate of 0.3 mL / min for a total run time of 9 minutes. Mobile phase A consisted of 1 mM ammonium formate in water, and mobile phase B used ACN. The gradient consisted of an isolytic run at 89% B for 1 minute, to 60% B at 1.5 minutes, and then 2.The process was performed by applying a gradient to 40% B in 5 minutes, holding for 2 minutes, finally returning to an 89% B gradient within 0.1 minutes, and equilibrating at 15% B for 4.4 minutes. The column oven temperature was 40°C and the sample injection volume was 2 μL. Mass spectra were obtained in negative MRM mode. In negative mode, the ion spray voltage was set to -4500 V, the declustering potential to -60 V, the collision energy to -30 V, the source temperature to 350°C, the curtain gas to 10 psi, and both source gases 1 and 2 to 60 psi. The MRM pairs for 2660 and 2660-IS were m / z 147 → 63 and m / z 151 → 63, respectively. For quantitative analysis using Analyst 1.6 software (ABSciex, Framingham, MA), the peak area ratio (peak area of ​​analyte / peak area of ​​analyte-IS) for each MRM transition of the calibration standard and samples were used.

[0096] 3424 activation by AKR1C3 was monitored using LC / MS-MS for the reduction of 3424 and the generation of the active form from 2660. As shown in Figure 1, recombinant human AKR1C3 was able to activate 3424 to 2660 within 60 minutes (Figures 1a and 1b). In contrast, 3424 was not metabolized by AKR1C1 or AKR1C4, the two members of the AKR1C family (Table 1). Thus, the AKR1C3-dependent activation of the prodrug 3424 was evident. AKR1C3 exhibited similar catalytic efficiency for 3424 (S-enantiomer) and its R-enantiomer 3423 (Table 2). Physiological substrates 4-androstenedione and 5-α-dihydrotestosterone (5 α Compared to -DHT, 3424 was activated by AKR1C3 at a higher rate.

[0097] Table 1. 3424 (prodrug) incubated with various AKR1C enzymes at a concentration of 2660 (active form).

[0098]

[0099] n=3, data is expressed as mean ± SD

[0100] BQL: Less than the quantification limit (0.25 ng / mL)

[0101] * 3424 Tested only at maximum concentration (50μM)

[0102] Table 2. Dynamic analysis of AKR1C3 on various substrates

[0103]

[0104] Therefore, it was confirmed that the reduction of 3424 is AKR1C3-dependent. Recombinant human AKR1C3, excluding AKR1C1 or AKR1C4, reduced 3424 to the active aziridine nitrogen mustard moiety 2660.

[0105] Example 2: AKR1C3-dependent 3424 cytotoxicity

[0106] In vitro proliferation test

[0107] Exponentially growing cells were seeded 24 hours prior to the addition of the test compound. After adding the test compound, the plates were incubated in a standard tissue culture incubator at 37°C for the indicated times. At the end of the experiment, viable cells were detected using the CellTiter Glo (CTG) assay kit or AlamarBlue. (28,29) . Drug concentration (IC) that causes 50% growth inhibition compared to the untreated control group using XLfit (IDBS, Boston, MA) or Prism 6 (GraphPad, San Diego, CA) 50 ) was calculated. For the 3021 experiment, cells were pretreated with 3 μM 3021 for 2 hours in air before treatment with the compound. IC 50 It was calculated as described above.

[0108] Western Blot

[0109] Human cell extracts were prepared and protein concentrations were determined. Proteins were detected using antibodies that recognize human AKR1C3 and tubulin or β-actin. Band densities of AKR1C3 and tubulin or actin were scanned and quantified using an Odyssey laser imaging system and software (LI-COR Biosciences, Lincoln, NE) or a UVP ChemStudio imaging system and VisionWorks software (Analytik Jena AG), and the ratio of AKR1C3 to tubulin or actin was calculated.

[0110] Comet Black

[0111] After seeding cells for 24 hours, the test substance was added at the indicated concentration and incubated for 2 hours. Cells were washed twice to completely remove the compound. 20 μmol / L of bleomycin was added, and DNA strand breaks were induced after 2 hours of cell rest by incubating in air for 1 hour. After washing twice with PBS, a Comet assay was performed using a single-cell electrophoresis system from Trevigen (Gaithersburg, MD). Data were analyzed using Perceptive Instruments' Comet Assay IV software. (29) .

[0112] The cytotoxicity of 3424 was evaluated in a panel of hepatocellular carcinoma and non-small cell lung cancer (NSCLC) cell lines. AKR1C3 protein expression in hepatocellular carcinoma cell lines was determined by Western blot, with tubulin used as a loading control (Fig. 2). AKR1C3 RNA expression data were obtained from the CrownBio (Beijing, China) database. As shown in Table 3, after 96 hours of exposure to 3424, hepatocellular carcinoma cell lines with high AKR1C3 expression at both protein and RNA levels were more sensitive to 3424 and had a low nanomolar IC50 range. 50They had values. Meanwhile, cells with low AKR1C3 expression were less sensitive to 3424 and had an IC50 higher than 1000 nM. 50 It had a value. Similarly, NSCLC cells also exhibited an AKR1C3-dependent cytotoxicity profile after 72 hours of exposure to 3424 (Table 4). 3424 IC 50 is liver cancer cells and NSCLC cells (R 2 = 0.80, Fig. 3a, right) AKR1C3 protein (R 2 = 0.71, Fig. 3a, left) and RNA expression (R 2 = 0.87, Fig. 3a, middle) There was a high correlation with the level, respectively. These results demonstrated that 3424-mediated cytotoxicity is highly correlated with AKR1C3 expression levels in both liver cancer and NSCLC cell lines.

[0113] Table 3 . Cytotoxicity of 3424 on a panel of human liver cancer cell lines 96 hours after drug exposure

[0114]

[0115] Table 4 . 3424 cytotoxicity on a human non-small cell lung cancer (NSCLC) cell line panel 72 hours after drug exposure

[0116]

[0117] The AKR1C3-mediated specific activation of 3424 was confirmed in H460 cells using the AKR1C3 inhibitor 3021. After pre-treating H460 cells with 3 μM 3021 for 2 hours followed by co-treatment with 3424 for 2 hours, the AKR1C3-specific inhibitor 3021 [indicated] IC 50 At 6.3 μM, it was able to effectively inhibit 3424 cytotoxicity in H460, whereas Evans et al. (25) As reported here, in the absence of 3021, IC 50It had 4 nM. Here, the inventors also determined the cytotoxicity profiles of 3423, an R-enantiomer, and 2870, a racemic mixture of the R-enantiomer and S-enantiomer in a 1:1 ratio, in H460 cells in the absence or presence of 3021. As shown in Fig. 3b, 3423 (center) and 2870 (right) are IC10, respectively. 50 With values ​​of 5 nM and 4 nM, they exhibited cytotoxicity potent as 3424. Similar to 3424, the cytotoxicity of 3423 and 2870 was highly AKR1C3-dependent, and 3021 inhibited cytotoxicity at concentrations more than 1000 times higher than in the absence of an inhibitor, respectively, at IC50s. 50 It had values ​​of 6.3 μM and >5 μM. Inhibitor 3021 alone did not show any cytotoxicity at up to 100 μM (not shown in data).

[0118] To evaluate DNA cross-linked DNA, a single-cell electrophoresis-based Comet assay, a direct biochemical assay for DNA cross-linking, was used with H460 cells. The racemic mixture 2870 was used in this assay. As shown in Figure 3c, the vehicle had a tail moment of 0.3 in DMSO-treated H460 cells, and there were no detectable double-strand breaks. Bleomycin was used to induce DNA strand breaks, which was evident by a tail moment increased to 35. 2870-induced DNA cross-linking was tested using three concentrations under the same conditions as used for bleomycin. Concentration-dependent DNA cross-linking induced by compound 2870 was evident by a tail moment decreased from 36 to 1.6.

[0119] As such, it has been proven that 3424-mediated cytotoxicity is highly dependent on AKR1C3.

[0120] In vivo antitumor activity

[0121] In this study, all procedures related to animal handling, care, and treatment were performed in compliance with the International Association for the Accreditation of Animal Care (AAALAC) guidelines, based on guidelines approved by the Institutional Animal Care and Use Committees (IACUC) of CrownBio (Beijing, China), WuXi AppTec (Shanghai, China), or Eurofins Pharmacology Discovery Services Taiwan (Taipai, Taiwan). At the time of routine monitoring, the impact of tumor growth and other abnormal effects on normal behaviors, such as animal mobility, food and water intake, body weight gain / loss, and eye / hair tangles, were assessed. Mortality and observed clinical signs were recorded based on the number of animals within each subgroup.

[0122] In all animal studies, drug efficacy was evaluated by tumor growth inhibition. Tumor volume (mm³) was measured twice weekly according to the following long ellipsoid formula: Length (mm) x [Width (mm)] 2 x 0.5. The percentage of tumor growth inhibition (%TGI) was determined twice weekly during the administration period using the following formula: %TGI = (1-[(T-T0) / (C-C0)])×100, where T = mean tumor volume of the treatment group, T0 = mean tumor volume of the treatment group at the start of the study, C = mean tumor volume of the control group, and C0 = mean tumor volume of the control group at the start of the study. Statistical significance of each group was evaluated using two-way ANOVA and Bonferroni tests, while for each vehicle control group, SPSS Statistics 23 (IBM, Armonk, NY) or R (version 3.3.1) was used. A P-value of <0.05 was considered statistically significant.

[0123] Example 3: Antitumor efficacy of 3424 in a CDX model

[0124] Example 3-1: Antitumor efficacy of 3424 in HepG2 and H460 models

[0125] The in vivo antitumor activity of 3424 was evaluated using GFP-expressing cancer cell lines in an orthotopic liver cancer model (HepG2) and a subcutaneous lung cancer model (H460) CDX model at AntiCancer, Inc (Beijing, China). Female athymic nude mice (6 weeks; BALB / c-nu, Beijing HFK Bioscience Co., Ltd., Beijing, China) were used in the study. For tumorigenesis, HepG2-GFP tumor masses (approx. 1 mm³ in diameter) were implanted into the right lobe of the liver or H460-GFP tumor masses (approx. 1 mm³ in diameter) were inoculated subcutaneously into each mouse. After approximately 3 to 10 days, whole-body scans of the mice were performed using a FluorVivo Model-100 fluorescence imaging machine (INDEC Biosystems, Inc., Los Altos, CA). Mice with similar fluorescence areas were selected and randomly divided into groups. Both cell lines expressed high levels of AKR1C3. Prodrug 3424 was administered intravenously (IV) at doses of 1.25, 2.5 mg / kg, or 5 mg / kg in the HepG2 orthotopic model via a Q7D x 2 regimen, and at doses of 0.625, 1.25, or 2.5 mg / kg in the H460 xenograft model via a Q7D x 2 regimen followed by a 1-week rest period and then a Q7D x 2 regimen. Sorafenib was used as a positive control in the HepG2 model and was administered orally at a dose of 30 mg / kg via a QD x 5 x 7 cycle regimen. Taxol was used as a positive control in the H460 model and was administered IV at a dose of 15 mg / kg via a BIW x 4 regimen. Mice were observed daily during the experiment, and body weight was measured twice a week. Tumor burden was monitored twice a week using caliper measurements (H460) or FluorVivo fluorescence imaging machines (H460-GFP and HepG2-GFP).

[0126] The dose-dependent antitumor activity of 3424 was investigated using whole-body fluorescence imaging with a HepG2 orthotopic xenograft model. When 3424 was administered intravenously once weekly for 2 weeks at doses of 1.25, 2.5, or 5 mg / kg, the tumor growth inhibition (TGI) on day 34 was 52.4%, 91.5%, and 101.2%, respectively (Table 5). The antitumor efficacy of 3424 was observed in a dose-dependent manner (Fig. 4a). Compared to the vehicle group, the tumor inhibition induced by 3424 at 2.5 mg / kg and 5 mg / kg was statistically significant, with complete regression rates of 80% and 100%, respectively. At the end of the experiment, the tumor fluorescence imaging (Fig. 4b) and tumor weight data were consistent with the fluorescence area measurements (Fig. 4a). Sorafenib, a first-line treatment for hepatocellular carcinoma (HCC), was administered orally at 30 mg / kg and reduced tumor growth by up to 52.1%, which was not statistically significant. In this regimen, sorafenib resulted in weight loss (-11%, not shown in data). In contrast, no weight loss was observed in the 3424 treatment group at any of the test doses in the current study (Table 5).

[0127] In the H460 subcutaneous model, the prodrug 3424 was administered weekly in two doses at 0.625, 1.25, and 2.5 mg / kg, followed by a one-week rest period, and an additional two doses were administered weekly. As shown in Figure 5 and Table 5, the prodrug 3424 exhibited dose-dependent antitumor activity with TGI values ​​of 60.2%, 67.2%, and 88%, respectively. The antitumor efficacy of 3424 was similar to that of paclitaxel, which showed a TGI of 64% at 15 mg / kg. At the end of the study (Day 35), treatment with 3424 resulted in minimal weight loss at low and intermediate doses and a statistically insignificant 14% weight loss at high dose, whereas treatment with paclitaxel resulted in a significant 11% weight loss (Table 5).

[0128] Table 5 Summary of 3424 In vivo efficacy in HepG2 and H460 CDX models

[0129]

[0130] Example 3-2: Antitumor efficacy of 3424 in VCaP, SNU-16, and A498 models

[0131] The antitumor activity of 3424 and compositions containing it was evaluated in castration-resistant prostate cancer (CRPC) VCaP, gastric cancer SNU-16, and renal cell carcinoma (RCC) A498 xenograft models. The study was conducted at WuXi AppTec (VCaP and SNU-16 models) and Eurofins Pharmacology Discovery Services Taiwan (A498 model). Male BALB / c nude, female BALB / c nude, and female SCID mice were used in the CDX models of CRPC, gastric cancer, and RCC, respectively. 5 x 10⁶ 6 or 1 x 10 7 Human cancer cells and 1:1 Matrigel were subcutaneously injected into the right flank of mice. The vehicle and test substance were administered when the tumor volume reached 150 to 200 mm³ (indicated as Day 1 or Day 0 in the SNU-16 model). The vehicle or test substance was administered intravenously once a week for a total of 4 or 5 administrations, depending on the model. Standard treatment regimens, including abiraterone / prednisolone (for CRPC), 5-fluorouracil (for gastric cancer), and sunitinib and gemcitabine (both for RCC), were performed as recommended in the literature. (15,30-32) On the day of concomitant administration, 3424 was injected IV first, and the concomitant agent was injected within 1 hour.

[0132] The in vivo antitumor efficacy of 3424 as monotherapy or in combination with standard therapy was evaluated in castration-resistant prostate cancer (CRPC) VCaP, gastric cancer SNU-16, and renal cell carcinoma A498 xenograft models. These three human cancer cell lines expressed high levels of AKR1C3 at both protein and RNA levels. In VCap, SNU-16, and A498, AKR1C3 protein expression was determined by Western blot to be AKR1C3-to-tubulin ratios of 8.9, 1.9, and 1.6, respectively. AKR1C3 RNA expression (LOG2 FPKM) in VCap, SNU-16, and A498 was 5.2, 8.0, and 10.0, respectively.

[0133] In these studies, animals were treated with various concentrations of 3424 as monotherapy, as a monotherapy anticancer agent or anticancer agents (standard management therapy, control group), or as a composition of the present invention in combination with 3424 and at least one anticancer agent.

[0134] Example 3-2-1: As monotherapy or in combination with abiraterone acetate + prednisolone 3424

[0135] In a CRPC model, castrated male BALB / c nude mice were treated with 3424 (weekly IV injection during 5 administrations), abiraterone acetate + prednisolone (daily oral gastric feeding), or in combination (Fig. 4c). 3424 showed a significant TGI of 148% at 5 mg / kg, which increased to 158% when combined with abiraterone acetate / prednisolone.

[0136] In addition, at the final sacrifice on day 32, animals treated with combination therapy showed a corresponding decrease in serum prostate-specific antigen (PSA) (Fig. 6).

[0137] Example 3-2-2: 3424 as monotherapy or in combination with 5-FU

[0138] Female BALB / c mice in a gastric cancer SNU-16 model were treated with 3424 (weekly IV injection during 4 administrations), 5-fluorouraci (5-FU) (IP injection, twice a week), or a combination of 3424 and 5-FU (Fig. 4d). Animals treated with 2.5 mg / kg or 5 mg / kg of 3424 showed significant TGI of 87.8% and 96.2%, respectively, whereas 5-FU showed no antitumor activity at the 30 mg / kg level. A synergistic effect was noted when 3424 was combined with 5-FU.

[0139] Example 3-2-3: As monotherapy or in combination with sunitinib 3424

[0140] In a renal cell carcinoma A498 model, female SCID mice were administered 3424 (weekly IV injection during 4 administrations), sunitinib (25 mg / kg, daily oral gastric feeding), or in combination (Fig. 4e). 3424 at 2.5 mg / kg showed a significant TGI of 73%, compared to a moderate TGI of 52% with sunitinib. When animals were treated with a combination of 3424 and sunitinib, the TGI increased further to 88%.

[0141] Example 3-2-4: As monotherapy or in combination with gemcitabine (Gemzar) 3424

[0142] The efficacy of 3424 in combination with gemcitabine was also tested in an A498 xenograft model, where gemcitabine at 80 mg / kg (weekly IP injection during 4 administrations) provided only 19% TGI, but when combined with 2.5 mg / kg of 3424, the TGI increased to 87% (Fig. 4f).

[0143] In all three models, 3424 was well tolerated in mice and there was no significant weight loss during treatment (not shown in data).

[0144] Figure 4 illustrates the antitumor activity of 3424 or the composition of the present invention in various CDX models. In combination therapy, the inventors have demonstrated that 3424 can enhance the efficacy of standard treatment in CDX models of CRPC, gastric cancer, and RCC.

[0145] Example 4: Antitumor activity of 3424 in a PDX model

[0146] The antitumor activity of 3424 in a PDX model was evaluated using female BALB / c nude mice (6 to 7 weeks old, Beijing Anikeeper Biotech Co., Ltd, Beijing, China) at CrownBio Bioscience (Beijing, China) Inc. Tumor fragments (PA1280, GA6201, LU2057, and LU2505) harvested from stock mice inoculated with selected primary human cancer tissues (pancreatic, gastric, and lung cancer) were used for inoculation of BALB / c nude mice. They were subcutaneously inoculated into each mouse for tumorigenesis. The mice were StudyDirector TM Using Ver 3.1.399.19 (Studylog Systems, Inc., S. San Francisco, CA, USA), mice were randomized into the experimental group when the average tumor size reached approximately 100 mm³. Prodrug 3424 was administered IV at the dose indicated by the Q7D x 3 regimen. Each group consisted of 5 to 6 mice. The date of group assignment was designated as Day 0. Prodrug 3424 was administered to tumor-bearing mice throughout the period from Day 0 to the date indicated in each study.

[0147] The in vivo antitumor activity of 3424 was further evaluated in a panel of patient-derived xenograft (PDX) models, including pancreatic cancer, gastric cancer, and two lung cancers: one with high AKR1C3 expression and one with low AKR1C3 expression. The prodrug 3424 was administered weekly via IV injection during three administrations. Patient information for the four PDX models is presented in Table 6. According to the pancreatic PDX model (Figure 7a and Table 7), 3424 demonstrated statistically significant antitumor activity of 77.4% TGI at 2.5 mg / kg. In the gastric PDX model, 3424 demonstrated significant antitumor inhibition of 110% TGI at 5 mg / kg (Figure 7b and Table 7). Tumor volume continued to decrease and remained low or was undetectable one month after discontinuation of therapy. At the end of the study, 60% of the mice remained tumor-free. Two lung cancer PDX models with different AKR1C3 expression levels were selected to investigate whether the in vivo antitumor activity of 3424 was AKR1C3-dependent. The LU2505 PDX model expressed higher AKR1C3 RNA (log2=6.03), while the LU2057 model expressed lower AKR1C3 RNA (log2=2.08). As shown in Figures 7c and 7d and Table 7, 3424 demonstrated superior tumor growth inhibition in LU2505, which has high AKR1C3 expression, at the same dose of 2.5 mg / kg (TGI 105.2%), but no inhibition was observed in LU2057 PDX tumors, which have low AKR1C3 expression (TGI -20.9%), indicating that the in vivo antitumor activity of 3424 is AKR1C3-dependent. 3424 still demonstrated statistically significant inhibition of LU2505 tumor growth even at a lower dose of 1.25 mg / kg (TGI 105.0%). In all PDX models, the prodrug 3424 was well tolerated at all tested doses, and no weight loss was observed (Table 7).

[0148] Table 6. PDX Patient Information

[0149]

[0150] Table 7. Summary of 3424 In vivo efficacy in the PDX model

[0151]

[0152] Using more than 20 cell lines from liver cancer or NSCLC, it was found that 3424 exhibited improved cytotoxicity in all cell lines with high AKR1C3 expression compared to cells with low or no detectable AKR1C3 expression. The IC50 of 3424 in cell lines with high AKR1C3 expression 50 The values ​​were in the low nanomolar range, indicating high potency characteristic of potent nitrogen mustard. Particularly noteworthy was the finding that cytotoxicity was improved by up to 5,000-fold in the NCI-H2228 NSCLC cell line, which has high AKR1C3 expression. This result is consistent with the finding that it targets tumors with high AKR1C3 expression while remaining inactive in regions of normal tissues with low AKR1C3 expression.

[0153] The results described herein highlight that 3424 exhibits in vitro AKR1C3-dependent cytotoxicity and in vivo antitumor activity across a wide range of human cancer types. The inventors demonstrate that 3424 possesses excellent in vivo antitumor activity at clinically achievable doses in a broad panel of CDX and PDX models with high AKR1C3 expression. Notably, 3424 demonstrates significant in vivo efficacy against liver, gastric, renal, lung, pancreatic, and castration-resistant prostate cancers. The AKR1C3-dependent activity of 3424 serves as the basis for current and future clinical studies specifically targeting cancer cells, and has acted as a biomarker for profiling cancer patients and guiding patient selection for regimens using 3424.

[0154] The above detailed description of embodiments of the present invention does not limit the invention. Those skilled in the art may make various modifications and changes according to the present invention, and all modifications and changes within the spirit of the present invention are included in the claims appended to this invention.

[0155] All references cited herein are incorporated herein by reference to the maximum extent permitted by law. Discussion of such references is merely intended to summarize the claims of the respective authors. It is not an acknowledgment that any reference (or part thereof) constitutes relevant prior art. The applicant has the right to object to the accuracy and appropriateness of the cited references.

[0156] References

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Claims

Claim 1 A method for treating cancer in subjects other than humans who require cancer treatment, comprising the step of administering to said subject an effective amount of a compound having the following chemical formula I, 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate, or a pharmaceutically acceptable salt, isotope variant, or solvate thereof, in combination with at least one other anticancer agent selected from the group consisting of gemcitabine, abiraterone acetate, prednisolone, 5-FU, sunitinib, and combinations of abiraterone acetate and prednisolone, wherein the level of aldo-keto reductase family 1 member C3 (AKR1C3) reductase in said cancer is expressed at an AKR1C3 protein level or RNA level, said AKR1C3 reductase is expressed higher than a predetermined value, and said cancer is liver cancer, non-small cell lung cancer, castration-resistant For prostate cancer, stomach cancer, renal cell carcinoma, or pancreatic cancer, method: Chemical formula I. Claim 2 Method of claim 1, wherein the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the following chemical formula I-1 or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the following chemical formula I-2: Chemical formula I-1 Chemical formula I-2. Claim 3 The method of claim 1 further comprises the step of measuring the content of AKR1C3 reductase in the cancer cells of a subject using an AKR1C3 antibody, and administering the compound to the subject when the content of AKR1C3 reductase is expressed at a higher level than a predetermined value. Claim 4 A method for inhibiting cell growth in vitro, comprising the step of contacting said cells with an effective amount of 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having Formula I or a pharmaceutically acceptable salt, isotope variant, or solvate thereof; wherein the level of aldo-keto reductase family 1 member C3 (AKR1C3) reductase in said cells is expressed at an AKR1C3 protein level or RNA level, said AKR1C3 reductase is expressed at a higher level than a predetermined value; and said cells are cancer cells. Chemical formula I. Claim 5 In claim 4, the method further comprises the step of measuring the content of AKR1C3 reductase in a cell using an AKR1C3 antibody, and the method wherein the compound is brought into contact with the cell when the content of AKR1C3 reductase is expressed higher in the cell than a predetermined value. Claim 6 1) a compound having the chemical formula I, 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate, or a pharmaceutically acceptable salt, isotope variant, or solvate thereof; and 2) a pharmaceutical composition for treating cancer comprising at least one other anticancer agent selected from the group consisting of gemcitabine, abiraterone acetate, prednisolone, 5-FU, sunitinib, and combinations of abiraterone acetate and prednisolone: Chemical formula I. Claim 7 A pharmaceutical composition according to claim 6, wherein the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the formula I-1 or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoamidate having the formula I-2: Chemical formula I-1 Chemical formula I-2. Claim 8 In claim 7, the above composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient. Claim 9 A pharmaceutical composition according to claim 6, wherein, if the cancer is renal cell carcinoma (RCC), the anticancer agent is selected from the group consisting of gemcitabine and sunitinib; if the cancer is gastric cancer, the anticancer agent is 5-FU; and if the cancer is castration-resistant prostate cancer (CRPC), the anticancer agent is selected from the group consisting of abiraterone acetate, prednisolone, or a combination thereof. Claim 10 A pharmaceutical composition according to any one of claims 6 to 9, wherein the level of aldo-keto reductase family 1 member C3 (AKR1C3) reductase of the cancer is expressed at a higher level than a predetermined value. Claim 11 A pharmaceutical composition according to claim 10, wherein the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma, or pancreatic cancer. Claim 12 A pharmaceutical composition for treating a patient's cancer, comprising compound 3424, compound 3423, or compound 2870 in combination with at least one other anticancer agent, Compound 3424, A pharmaceutical composition comprising compound 3423, wherein compound 2870 is a racemic mixture in a 1:1 ratio of compound 3423, which is an R-enantiomer, and compound 3424, which is an S-enantiomer, and the anticancer agent is selected from the group consisting of gemcitabine, 5-FU, sunitinib, and combinations of abiraterone acetate and prednisolone. Claim 13 A pharmaceutical composition according to claim 12, wherein the aldoketo-keto reductase family 1 member C3 (AKR1C3) reductase of the cancer is expressed at a higher level than a predetermined value, and the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma, or pancreatic cancer. Claim 14 A method for treating cancer in a subject other than a human, comprising the step of administering an effective amount of a pharmaceutical composition according to any one of claims 6 to 9 to the subject. Claim 15 In claim 14, the level of aldoketo-keto reductase family 1 member C3 (AKR1C3) reductase in the cancer is expressed at a higher level than a predetermined value, and the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma, or pancreatic cancer, method. Claim 16 In claim 15, the method further comprises the step of measuring the content of AKR1C3 reductase in a patient's cancer cells using an AKR1C3 antibody, and if the content of AKR1C3 reductase is expressed higher than a predetermined value, the composition is administered to the patient. Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete

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