Use of AKR1C3-activating compounds

Abstract

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)phosphoramidate, or a pharma- ceutically acceptable salt, isotopic variant or solvate thereof, as well as to compositions comprising said compound and at least one anti-cancer agent, and to the medical use thereof.

Description

[Technical 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)phosphoramidate, or a pharmaceutically acceptable salt, isotopic variant, or solvate thereof, as well as to compositions comprising said compound and at least one anticancer agent, and to the medical use thereof. [Background technology]

[0002] Cancer is one of the leading causes of human morbidity and mortality. Cancer treatment is challenging because it is difficult to kill cancer cells without harming or killing normal cells. Harming or killing normal cells during cancer treatment can cause side effects in patients and limit the amount of anticancer drugs that can be administered to cancer patients.

[0003] Aldo-ketoreductase 1C3 (AKR1C3) is also known as type 5, 17β-hydroxysteroid dehydrogenase (17β-HSD) and prostaglandin F synthase. AKR1C3 is one of the 15-gene family of aldo-ketoreductases (AKRs). AKR1C3 was originally cloned from cDNA libraries of human prostate (Non-Patent Document 1) and placenta (Non-Patent Document 2). AKR1C3 is a monomeric, cytosolic, NAD(P)(H)-dependent oxidoreductase with 323 amino acids and a molecular mass of 37 kDa (Non-Patent Document 1). AKR1C3 shares high sequence identity with related human AKR1C family members, including AKR1C1, AKR1C2, and AKR1C4. AKR1C3 catalyzes the metabolism of androgens, estrogens, progesterone, and prostaglandins (PGs), and subsequently regulates nuclear receptor activity (Non-Patent Documents 3 and 4). AKR1C3 is expressed in normal tissues, including steroid hormone-dependent and steroid hormone-independent cells, but at low average expression levels, except in the liver, kidney, and small intestine (Non-Patent Document 5). Numerous studies have shown that AKR1C3 is abnormally overexpressed in many malignant solid tumors and hematological tumors. Data show that over 50% of liver tumors, bladder cancer, kidney cancer, and gastric cancer are detected with high AKR1C3 expression, as detected by an immunohistochemistry score (IHC score) of 4 or higher on a scale of 0 to 6 (Non-Patent Document 6). AKR1C3 is highly expressed in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (Non-Patent Document 7).

[0004] Increased expression of AKR1C3 in cancer has been reported to be associated with metastasis of castration-resistant prostate cancer (CRPC) (Non-Patent Document 8) and colorectal cancer (CRC) (Non-Patent Document 9), as well as poor prognosis and reduced survival rates (Non-Patent Documents 10, 11). Many types of treatment resistance have also been attributed to overexpression of AKR1C3. Chemoresistance to doxorubicin (Non-Patent Documents 12, 13), enzalutamide (Non-Patent Document 14), abiraterone (Non-Patent Document 15), and methotrexate (Non-Patent Document 16) has been reported to be directly associated with high intracellular expression of AKR1C3. Radioresistance in esophageal cancer (Non-Patent Document 17), prostate cancer (Non-Patent Document 18), and non-small cell lung cancer cells (Non-Patent Document 19) has been associated with overexpression of AKR1C3. The main mechanism of action of AKR1C3 against ionizing radiation is the reduction of intracellular reactive oxygen species (ROS) and the increase of PGF2α, which subsequently activates MAP kinase and inhibits PPARγ, significantly reducing DNA damage (Non-Patent Document 18). High expression of AKR1C3 has also been shown to contribute to immunotherapy resistance. One study, based on whole-genome microarray and multiplexed quantitative (q) RT-PCR gene expression analysis, showed that high expression of AKR1C3 is associated with the failure of PD-1-targeted therapy in patients with advanced renal cell carcinoma (RCC) who are PD-L1-positive (Non-Patent Document 20). Because AKR1C3 is tumor-specifically overexpressed, the design of AKR1C3-activating prodrugs is an attractive approach for specifically targeting cancer. For example, the AKR1C3-activating prodrug PR104, originally designed as a hypoxia-activated prodrug (Non-Patent Documents 22, 24), has shown excellent antitumor activity in vitro and in vivo (Non-Patent Documents 6, 21).

[0005] The anticancer prodrug of the present invention, structural formula I-1 (referred to herein as 3424), is a chemically synthesized potent nitrogen mustard that is selectively cleaved by AKR1C3 in the presence of NADPH to a cytotoxic aziridine (referred to herein as 2660). The active molecule released by 3424, 2660, resembles the standard chemotherapy drugs thiotepa and mitomycin C, and alkylates and crosslinks DNA at the N7 (or O6) position of guanine. Prodrug 3424 is currently being developed for the treatment of malignant tumors by Ascentawits Pharmaceuticals, Ltd. in Asian countries and by OBI Pharma, Inc. in non-Asian countries (drug code: OBI-3424). The prodrug 3424 is currently being tested in multiple Phase 1 clinical trials in the United States (NCT04315324 & NCT03592264) and China (CXHL1900137 & CXHL2000263) for the treatment of over 14 types of human cancer, including solid tumors and hematological malignancies. Prodrug 3424 is designed to be specifically activated in tumors due to the high expression of AKR1C3 in tumors, while sparing normal cells, which have low levels of AKR1C3 expression, for tumor-specific targeting. Furthermore, the tumor-selective activation of 3424 distinguishes it from non-selective conventional alkylating agents such as cyclophosphamide and ifosfamide, suggesting its potential as a broad-spectrum and highly selective antitumor agent. Prodrug 3424 has been reported to show potent efficacy in preclinical models of T-ALL in vitro and in vivo (Non-Patent Documents 25, 26).

[0006] In the presence of NADPH, 3424 is reduced by AKR1C3, releasing the cytotoxic moiety 2660, an aziridine bisalkylating agent, which crosslinks DNA at the N7 (or O6) position of guanine, leading to subsequent cell death. [ka]

[0007] In recent years, cancer-targeting prodrugs have emerged as an attractive strategy for cancer treatment. However, due to the lack of effective biomarkers for patient selection, many prodrugs have failed in phase 3 clinical trials (Non-Patent Document 33). Because AKR1C3 expression can be assessed using RT-PCR and immunohistochemistry, selecting patients with high AKR1C3 expression and susceptibility to prodrugs can facilitate clinically efficient development of 3424. AKR1C3 has been shown to be overexpressed in association with chemotherapy resistance (Non-Patent Documents 13, 14), radioresistance (Non-Patent Document 19), and immunoresistance (Non-Patent Document 20). Furthermore, cancers with homologous recombination deficiency (HRD), such as ovarian, breast, and pancreatic cancers, are known to be highly sensitive to DNA-damaging agents (Non-Patent Document 34). 3424, a DNA alkylating agent, may be a promising candidate for treating HRD cancers expressing AKR1C3. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Lin HK, Jez JM, Schlegel BP, Peehl DM, Pachter JA, Penning TM.Expression and characterization of recombinant type 2 3 alpha-hydroxysteroid dehydrogenase(HSD) from human prostate:demonstration of bifunctional 3 alpha / 17 beta-HSD activity and cellular distribution.Mol Endocrinol 1997;11:1971-84 [Non-patent document 2] Dufort I, Rheault P, Huang XF, Soucy P, Luu-The V.Characteristics of a highly labile human type 5 17beta-hydroxysteroid dehydrogenase.Endocrinology 1999;140:568-74 [Non-patent document 3] Penning TM,Drury JE.Human aldo-keto reductases:Function, gene regulation, and single nucleotide polymorphisms.Arch Biochem Biophys 2007;464:241-50 [Non-patent document 4] Rizner TL,Penning TM.Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism.Steroids 2014;79:49-63 [Non-patent document 5] Chang TS,Lin HK,Rogers KA,Brame LS,Yeh MM,Yang Q,et al.Expression of aldo-keto reductase family 1 member C3(AKR1C3) in neuroendocrine tumors & adenocarcinomas of pancreas,gastrointestinal tract,and lung.Int J Clin Exp Pathol 2013;6:2419-29 [Non-patent document 6] [ PubMed ] Guise CP, Abbattista MR, Singleton RS, Holford SD, Connolly J, Dachs GU, et al

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[0009] There is a need for compounds that are selective AKR1C3 reductase-activated prodrugs and are suitable for treating cancer patients, and for novel, selective, and broad-spectrum anti-cancer agents. The present invention addresses this need. [Means for solving the problem]

[0010] The present invention is based on the compounds, or pharmaceutically acceptable salts, or solvates thereof, disclosed in PCT / US2016 / 021581 (WO2016 / 145092) and PCT / US2016 / 062114 (WO2017 / 087428), and provides medical uses of the compounds, and also provides compositions containing the compounds, or pharmaceutically acceptable salts, isotopic variants, or solvates thereof, and anti-cancer medical uses thereof.

[0011] In one aspect, the present invention provides the use of a compound having formula I, 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, or a pharmaceutically acceptable salt, isotopic variant, or solvate thereof, in the manufacture of a medicament for treating cancer in a patient. [ka]

[0012] Here, the AKR1C3 reductase level in cancer is represented by the AKR1C3 protein level or RNA level, which is equal to or greater than a predetermined value. The AKR1C3 level is measured according to conventional methods well known to those skilled in the art.

[0013] According to certain embodiments of the present invention, the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, having formula I-1 below, or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, having formula I-2 below (designated herein as 3423). [ka]

[0014] The preparation of compounds of Formula I, Formula I-1, or Formula I-2 is disclosed in PCT / US2016 / 021581 (WO2016 / 145092), and PCT / US2016 / 062114 (WO2017 / 087428), the disclosures of which are incorporated herein by reference in their entireties, wherein compound 2870 is a racemic mixture of R-enantiomer 3423 and S-enantiomer 3424 in a 1:1 ratio.

[0015] Here, the salt may be a basic salt, including a salt of the compound with an inorganic base (such as an alkali metal hydroxide or an alkaline earth metal hydroxide) or an organic base (such as monoethanolamine, diethanolamine, or triethanolamine). Alternatively, the salt may be an acid salt, including a salt of the compound with an inorganic acid (such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid, or phosphoric acid) or an organic acid (such as methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, or citric acid). The selection and preparation of salts, solvates, etc. of compounds are well known in the art.

[0016] According to a particular embodiment of the present invention, the compounds of formula I-1 or I-2 have an enantiomeric excess of 80% or less, preferably 90% or less, more preferably 95% or less.

[0017] According to certain embodiments of the present invention, the compound of formula I-1 or I-2 is substantially pure.

[0018] According to certain embodiments of the invention, the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma cancer or rectal cancer.

[0019] According to certain embodiments of the invention, the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma or pancreatic cancer.

[0020] The dosage of a pharmaceutical used in the treatment of cancer, or the dosage of a compound, its salt, isotope variant or solvate, or other anticancer agent contained in the pharmaceutical, generally depends on the specific compound being used, the patient, the specific disease or condition and its severity, the route and frequency of administration, etc., and must be determined by the attending physician in accordance with specific conditions. For example, when the composition or pharmaceutical provided by the present invention is orally administered, the dosage is 0.1 to 30 mg / 7 days, preferably 1 to 10 mg / 7 days, more preferably 5 mg / day, and may be administered once every 7 days or in two divided doses every 7 days, preferably once every 7 days.

[0021] The pharmaceutical may be in any dosage form for clinical administration, such as a tablet, suppository, powder, enteric-coated tablet, chewable tablet, orally disintegrating tablet, capsule, sugar-coated tablet, granule, dry powder, oral solution, small needle for injection, freeze-dried powder for injection, or infusion solution.

[0022] In another aspect, the present invention provides a method for treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, having formula I: [ka] Here, the AKR1C3 reductase level in cancer is expressed as an AKR1C3 protein level or RNA level and is equal to or greater than a predetermined value.

[0023] According to certain embodiments of the present invention, the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having the following formula I-1 or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having the following formula I-2: [ka]

[0024] According to certain embodiments of the invention, the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma cancer or rectal cancer.

[0025] According to certain embodiments of the invention, the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma or pancreatic cancer.

[0026] According to a particular embodiment of the present invention, the method further comprises a step of measuring the AKR1C3 reductase content in the patient's cancer cells using an AKR1C3 antibody, and if the AKR1C3 reductase content is measured to be equal to or greater than a predetermined value, the compound is administered to the patient.

[0027] In another aspect, the invention provides a method for inhibiting cell growth, comprising contacting the cell with an effective amount of a compound having Formula I, 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, or a pharmaceutically acceptable salt, isotopic variant, or solvate thereof, wherein the cellular AKR1C3 reductase level, expressed as the AKR1C3 protein level or RNA level, is equal to or greater than a predetermined value.

[0028] According to certain embodiments of the invention, the cells are cancer cells.

[0029] According to certain embodiments of the present invention, the method further comprises measuring the AKR1C3 reductase content of the cells using an AKR1C3 antibody, and if the AKR1C3 reductase content is measured to be equal to or greater than a predetermined value, contacting the cells with a compound.

[0030] In another aspect, the present invention provides use of the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having formula I, or a pharmaceutically acceptable salt, isotopic isomer, or solvate thereof, wherein the cellular AKR1C3 reductase level, expressed as the AKR1C3 protein level or RNA level, is equal to or greater than a predetermined value.

[0031] According to certain embodiments of the invention, the cells are cancer cells.

[0032] In another aspect, the present invention provides a composition comprising: 1) The compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate is represented by Formula I, or a pharmaceutically acceptable salt, isotopic variant, or solvate thereof, and 2) at least one other anticancer agent.

[0033] According to certain embodiments of the invention, the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having formula I-1 or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having formula I-2.

[0034] According to certain embodiments of the present invention, the anticancer drug is gemcitabine, 5-fluorouracil (5-FU), sunitinib, abiraterone acetate, prednisolone, erlotinib, meturedepa, uredepa, altretamine, imatinib, triethylenemelamine, trimethylmelamine, chlorambucil, chlornaphazine, estramustine, gefitinib, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, Fotemustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin, chromomycin, dactinomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-L-norleucine, mycophenolic acid, nogamicin, olivomycin, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin, streptomycin Leptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, tegafur, L-asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestravcil, bisantrene, defofamide, demecol tin, diazicon, eflornithine, ellipticine acetmethylate, etoglucide, flutamide, hydroxyurea, interferon α, interferon β, interferon γ, interleukin 2, lentinan, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamt, pirarubicin, podophyllic acid, 2-ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, erlotinib, teniposide, tenuazonic acid, triazicon, 2,2',Selected from the group consisting of 2''-trichlorotriethylamine, urethane, vinblastine, and vincristine.

[0035] According to certain embodiments of the present invention, the anti-cancer agent is selected from the group consisting of gemcitabine, abiraterone acetate, prednisolone, 5-FU, sunitinib, or a combination of abiraterone acetate and prednisolone.

[0036] According to certain embodiments of the present invention, when the cancer is renal cell carcinoma (RCC), the anticancer agent is selected from the group consisting of gemcitabine and sunitinib; when the cancer is gastric cancer, the anticancer agent is 5-FU; and when 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.

[0037] According to certain embodiments of the present invention, the composition further comprises a pharmaceutically acceptable excipient, preferably selected from inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants and oils.

[0038] In another aspect, the invention provides the use of a composition according to the invention in the manufacture of a medicament for treating cancer in a patient.

[0039] According to certain embodiments of the invention, the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma cancer or rectal cancer.

[0040] According to certain embodiments of the invention, the AKR1C3 reductase level in the cancer is equal to or greater than a predetermined value.

[0041] According to certain embodiments of the invention, the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma or pancreatic cancer.

[0042] In another aspect, the present invention provides a method for treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a composition according to the present invention.

[0043] According to certain embodiments of the invention, the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma cancer or rectal cancer.

[0044] According to certain embodiments of the invention, the AKR1C3 reductase level in the cancer is equal to or greater than a predetermined value.

[0045] According to certain embodiments of the invention, the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma or pancreatic cancer.

[0046] According to certain embodiments of the present invention, the method further comprises measuring the AKR1C3 reductase content in cancer cells in the patient using an AKR1C3 antibody, and if the AKR1C3 reductase content is measured to be equal to or greater than a predetermined value, the composition is administered to the patient. [Brief explanation of the drawings]

[0047] [Figure 1] Figure 1 depicts AKR1C3-dependent activation of 3424. (A) shows the decrease of 3424, and (B) shows the production of 2660. [Figure 2] FIG. 2 shows the detection of AKR1C3 protein expression in liver cancer cells by Western blot. [Figure 3]Figure 3 depicts the AKR1C3-dependent in vitro cytotoxicity of 3424. (A) Correlation between AKR1C3 protein expression and 3424 IC50 in liver cancer cells (left), correlation between AKR1C3 RNA expression and 3424 IC50 in liver cancer cells (middle), and correlation between AKR1C3 RNA expression and 3424 IC50 in NSCLC cancer cells (right). (B) The AKR1C3-specific inhibitor 3021 efficiently inhibited the cytotoxicity of 3424 (left), 3423 (middle), and the racemic mixture 2870. (C) Compound 2870 induced DNA cross-linking in a concentration-dependent manner. [Figure 4] Figure 4 shows 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 of care, and combinations thereof, as indicated in the legend. Here, "AA" represents abiraterone acetate, "P" represents prednisolone, "S" represents sunitinib, and "G" represents Gemzar. [Figure 5] FIG. 5 shows the antitumor activity of 3424 in the H460 CDX subcutaneous lung cancer model. [Figure 6] FIG. 6 shows the results of post-treatment serum prostate-specific antigen (PSA) measurements at the indicated times. [Figure 7] Figure 7 shows the antitumor activity of 3424 against a panel of PDXs: pancreatic cancer PA1280 (A), gastric cancer GA6201 (B), lung cancer LU250 with high AKR1C3 expression (C), and lung cancer LU2057 with low AKR1C3 expression (D). DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention will now be described with reference to specific examples. Those skilled in the art will understand that these examples are only used to illustrate the present invention and do not limit its scope in any way.

[0049] [Definition] The following definitions are provided to assist the reader. Unless otherwise defined, all technical terms, notations, and other scientific or medical terms or terminology used herein are intended to have the meaning commonly understood by one of ordinary skill in the chemical and medical arts. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or quick reference, and the inclusion of such definitions herein should not be construed as representing a substantive difference from the definition of the term as commonly understood in the art.

[0050] All numerical designations, such as pH, temperature, time, concentration, and weight (including their respective ranges), are typically approximations that may be varied (+) or (-) by increments of 0.1, 1.0, or 10.0, as appropriate. All numerical designations may be understood to be preceded by the term "about." Reagents described herein are exemplary, and equivalents thereof may be known in the art.

[0051] "A" and "said" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a compound refers to one or more compounds, or at least one compound. As such, the terms "a," "one or more," and "at least one" are used interchangeably herein.

[0052] The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" 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.

[0053] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements essential to the composition or method. Also, "consisting of" means, for the claimed compositions and substantial method steps, excluding more than trace elements of other components. Embodiments defined by each of these transition terms are within the scope of the present invention. Thus, the methods and compositions can include (comprise) additional steps and components, or alternatively, include (consist essentially of) insignificant steps and compositions, or alternatively, are meant to contemplate (consist of) only the recited method steps or compositions.

[0054] "Administering" or "dispensing" a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which is administration or self-administration by a healthcare professional to a patient, and indirect administration, which is the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and / or provides a patient with a prescription for a drug is administering the drug to the patient.

[0055] "Cancer" refers to leukemias, lymphomas, carcinomas, and other malignant tumors, including solid tumors, that can spread locally by infiltration and systemically by metastasis, potentially with unlimited growth potential. Examples of cancer include, but are not limited to, cancer of the adrenal gland, bone, brain, breast, bronchus, colon and / or rectum, gallbladder, head and neck, kidney, larynx, liver, lung, nervous tissue, pancreas, prostate, parathyroid gland, skin, stomach, and thyroid gland. Specific other examples of cancer include acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia, and intraepithelial neoplasia, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma multiforme, pilocytoma, intestinal ganglioneuroma, hyperplastic corneal neuropathy, pancreatic islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid, melanoma, malignant hypercalcemia, These include Marfanoid habitus tumor, medullary carcinoma, metastatic cutaneous cell tumor, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic sarcoma, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung tumor, ulcerative and papillary squamous cell carcinoma, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, localized skin lesions, reticulum cell sarcoma, and Wilm's tumor.

[0056] The terms "contacting" or "contacting" refer to bringing a therapeutic agent into contact with a cell or tissue, resulting in a physiological and / or chemical effect. Contacting may occur in vitro, ex vivo, or in vivo. In one embodiment, a therapeutic agent is contacted with cells in cell culture (in vitro) to determine the effect of the therapeutic agent on the cells. In another embodiment, contacting a therapeutic agent with a cell or tissue comprises administering the therapeutic agent to a subject having the cell or tissue to be contacted.

[0057] The term "optically active" refers to a collection of molecules having an enantiomeric 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 certain embodiments, the enantiomeric excess of optically active compounds is about 90% or more, about 95% or more, about 98% or more, or about 99% or more. The enantiomeric excess of a compound can be determined by any standard method used by those skilled in the art, including, but not limited to, chiroptical chromatography (gas chromatography, high performance liquid chromatography, and thin layer chromatography) using optically active stationary phases, isotope dilution, electrophoresis, calorimetry, polarimetry, NMR separation methods using chiral inducers, and NMR methods with chiral solvating agents or chiral conversion reagents.

[0058] In describing an optically active compound, the prefixes R and S are used to indicate the absolute configuration of the molecule about its chiral center(s).

[0059] "Substantially pure" means sufficiently homogeneous to be free of readily detectable impurities as determined by standard analytical methods employed by those of ordinary skill in the art, 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 of sufficient purity that further purification does not detectably alter the physical, chemical, biological, and / or pharmacological properties (such as enzymatic and biological activity) of the substance. In certain embodiments, "substantially pure" refers to a collection of molecules wherein 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% by weight of the molecules are a single stereoisomer of the compound as determined by standard analytical methods.

[0060] "Patient" and "subject" are used interchangeably to refer to a mammal in need of cancer treatment. Generally, a patient is a human. Generally, a patient is a human who has been diagnosed with cancer. In certain embodiments, a "patient" or "subject" may refer to a non-human mammal used in screening, characterizing, and evaluating drugs and treatments, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat.

[0061] A "prodrug" refers to a compound that is metabolized or converted into a biologically active or more active compound (or drug) with respect to at least one property after administration. A prodrug is chemically modified to render it less active or inactive relative to the drug, such that the corresponding drug is produced by metabolism or other biological processes after the prodrug is administered. A prodrug may have altered metabolic stability or transport properties, fewer side effects or lower toxicity, or improved flavor relative to the active drug. (See, e.g., Reference Nogrady, 1985, Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pp. 388-392, incorporated herein by reference.) Prodrugs may also be synthesized using reactants other than the corresponding drug.

[0062] "Solid tumor" refers to solid tumors, including, but not limited to, metastatic tumors of the bone, brain, liver, lung, lymph node, pancreas, prostate, skin, and soft tissue (sarcomas).

[0063] A "therapeutically effective amount" of a drug refers to the amount of drug that, when administered to a cancer patient, produces the intended therapeutic effect, e.g., relief, improvement, alleviation, or elimination of one or more symptoms of cancer in the patient. The therapeutic effect does not necessarily occur in a single administration, but may occur only after a series of administrations. Thus, a therapeutically effective amount can be administered in one or more administrations.

[0064] "Treating" a patient's condition refers to taking steps to achieve beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, relief or amelioration of one or more symptoms of cancer; reduction in the extent of disease; delay or slowing of disease progression; relief, palliation, or stabilization of the disease state; or other beneficial results. Treatment of cancer may in some cases result in a partial response or stable disease.

[0065] "Tumor cell" refers to a tumor cell of any appropriate species (eg, mammalian, such as murine, canine, feline, equine, or human).

[0066] The term "isotopic variant" refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound. In certain embodiments, an "isotopic variant" of a compound is an isotope of 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), iodine-131( 131 I) containing one or more unnatural proportions of isotopes, including but not limited to:

[0067] In certain embodiments, an "isotopic variant" of a compound is in a stable form, i.e., non-radioactive. In certain embodiments, an "isotopic variant" of a compound is in a stable form, i.e., non-radioactive. 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), Iodine-127( 127 I) containing one or more unnatural proportions of isotopes, including but not limited to:

[0068] In certain embodiments, an "isotopic variant" of a compound is in an unstable form, i.e., radioactive. In certain embodiments, an "isotopic variant" of a compound is in an unstable form, i.e., radioactive. 3 H), 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( 131I) containing one or more unnatural proportions of isotopes, including but not limited to:

[0069] In the compounds as provided herein, where practicable according to the judgment of one of skill in the art, any hydrogen may be, for example, 2 H, or any carbon can be, e.g. 13 C, or any nitrogen may be, e.g. 15 N, and any oxygen may be 18 It will be understood that the isotope may be O. In certain embodiments, an "isotopic variant" of a compound contains unnatural proportions of deuterium.

[0070] The term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute, such as a compound provided herein, and one or more molecules of a solvent present 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. 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, hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate.

[0071] The term "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, 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 of being compatible with the other components of the pharmaceutical formulation and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, or allergic response. [Example]

[0072] (material and method) (cell line) All human cancer cell lines were obtained from either the American Type Culture Collection (ATCC, Manassas, VA), the Japanese Collection of Research Biosources (JCRB, Osaka, Japan), or Cobioer Biosciences.

[0073] (Reagents and Chemicals) 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 obtained 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 according to a reported method (Non-Patent Document 27). Standard therapeutic drugs were purchased as follows: abiraterone (Bos Science, USA), prednisolone (Saen Chemical Technology, China), 5-FU (Shanhai Xudong Haipu Pharmaceutical Co., China), gemcitabine (Vianex SA, Greece), and sunitinib (Cayman, USA).

[0074] In the following examples, we investigated the 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. Additionally, we investigated the in vivo antitumor activity of 3424 in an extensive panel of CDX and PDX models.

[0075] Example 1: AKR1C3-dependent activation of 3424 (Enzyme activity assay) The assay mixture consisted of 10–50 μM 3424 or 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 in a total volume of 200 μL. Reactions were incubated at 25 °C and terminated at various time points by adding acetonitrile and methanol (9:1 ratio) and subjected to LC-MS / MS analysis. For enzyme kinetic analysis, the decrease in absorbance of NADPH at 340 nm (ε = 6270 M) was measured using a SpectraMax M2e spectrophotometer (Molecular Devices, San Jose, CA). -1 cm -1 Activity was determined by measuring the kinetics of the reaction. After initiation of the reaction by addition of substrate, the reaction was monitored at 30-second intervals at 25°C. The non-substrate reaction rate was also monitored as background, and its slope was used to determine the initial rate of the reaction. Kinetic data reported were the average of triplicate measurements.

[0076] (LC-MS / MS analysis) AKR1C3-mediated 3424 metabolism was analyzed by LC-MS / MS using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA) coupled to a Sciex API-4000 Qtrap (ABSciex, Framingham, MA) mass spectrometer. For 3424 analysis, reversed-phase liquid chromatography separation was performed using a Waters Xbridge C18 column (2.1 x 100 mm, 3.5 μm, Waters Corporation, Milford, MA) at a flow rate of 0.3 mL / min with a total run time of 12 min. Mobile phase A consisted of water (0.1% formic acid), and mobile phase B consisted of ACN (acetonitrile) (0.1% formic acid). The gradient consisted of 15% mobile phase B in 1.5 min, then a 3-min gradient to 50% mobile phase B, followed by a 6-min gradient to 95% mobile phase B with a 1-min hold, followed by a 0.1-min gradient back to 15% mobile phase B and a 4.9-min equilibration at 15% mobile phase B. The column oven temperature was 40 °C, and the sample injection volume was 2 μL. Mass spectra were acquired in positive MRM mode. In positive ion mode, the ion spray voltage was set to 4500 V, the declustering potential was set to 80 V, the collision energy was set to 20 V, the source temperature was set to 350 °C, the curtain gas was set to 10 psi, and source gases 1 and 2 were both set to 60 psi. The MRM pairs for 3424 and 3424-IS were m / z: 461 → 313 and m / z: 465 → 313, respectively. For the 2660 analysis, normal-phase liquid chromatography separation was performed using a Waters Atlantis HILIC Silica column (2.1 mm × 100 mm, 3 μm, Waters Corporation, Milford, MA) at a flow rate of 0.3 mL / min for a total run time of 9 min. Mobile phase A consisted of 1 mM ammonium formate in water, and mobile phase B was ACN.The gradient consisted of a 1-minute isocratic run at 89% mobile phase B, followed by a 1.5-minute gradient to 60% mobile phase B, followed by a 2.5-minute gradient to 40% mobile phase B with a 2-minute hold, and finally a 0.1-minute return to 89% mobile phase B and equilibration at 15% mobile phase B for 4.4 minutes. The column oven temperature was 40 °C, and the sample injection volume was 2 μL. Mass spectra were acquired in negative MRM mode. In negative ion mode, the ion spray voltage was set to -4500 V, the declustering potential was set to -60 V, the collision energy was set to -30 V, the source temperature was set to 350 °C, the curtain gas was set to 10 psi, and source gases 1 and 2 were both set to 60 psi. The MRM pairs for the 2660 and 2660-IS were m / z: 147→63 and m / z: 151→63, respectively. The peak area ratios (analyte peak area / analyte peak area - IS) of each MRM transition of the calibration standards and samples were used for quantitative analysis using Analyst 1.6 software (ABSciex, Framingham, MA).

[0077] Activation of 3424 by AKR1C3 was monitored by LC / MS-MS analysis, monitoring the reduction of 3424 and the generation of the activated form 2660. As shown in Figure 1, recombinant human AKR1C3 was able to activate 3424 to 2660 in 60 min (Figures 1A and 1B). However, 3424 was not metabolized by AKR1C1 or AKR1C4, two members of the AKR1C family (Table 1). Therefore, AKR1C3-dependent activation of the prodrug 3424 was demonstrated. AKR1C3 showed similar catalytic efficiency for 3424 (S-enantiomer) and its R-enantiomer 3423 (Table 2). Compared with the physiological substrates 4-androstenedione and 5-α-dihydrotestosterone (5α-DHT), 3424 was activated at a higher rate by AKR1C3.

[0078] [Table 1]

[0079] [Table 2]

[0080] Thus, the reduction of 3424 was confirmed to be AKR1C3-dependent. Human recombinant AKR1C3, but not AKR1C1 or AKR1C4, reduced 3424 to its active aziridine nitrogen mustard moiety, 2660.

[0081] Example 2: AKR1C3-dependent 3424 cytotoxicity (in vitro proliferation assay) Exponentially growing cells were seeded 24 hours before the addition of test compounds. After the addition of test compounds, plates were incubated at 37°C in a standard tissue incubator for the indicated time. After the experiment, viable cells were detected using either the CellTiter Glo (CTG) assay kit or AlamarBlue (Non-Patent Documents 28, 29). The drug concentration that resulted in 50% growth inhibition compared to untreated controls (IC 50 ) was calculated using either XLfit (IDBS, Boston, MA) or Prism 6 (GraphPad, San Diego, CA). For 3021 experiments, cells were pretreated with 3 μM 3021 for 2 hours, followed by compound treatment in air. IC 50 was calculated as above.

[0082] (Western blot) Human cell extracts were prepared and protein concentrations were measured. 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.

[0083] (Comet assay) After seeding for 24 hours, test substances were added at the indicated concentrations and incubated for 2 hours. Cells were washed twice to completely remove the compound. 20 μmol / L bleomycin was added and incubated under air for 1 hour. Cells were then allowed to rest for 2 hours before inducing DNA strand breaks. After washing twice with PBS, comet assays were performed using a single-cell electrophoresis system from Trevigen (Gaithersburg, MD). Data were analyzed using the Comet Assay IV software from Perceptive Instruments (Non-Patent Document 29).

[0084] The cytotoxicity of 3424 was evaluated using liver cancer cell lines and non-small cell lung cancer (NSCLC) cell lines. AKR1C3 protein expression in liver cancer cell lines was determined using Western blot, with tubulin used as a loading control (Figure 2). AKR1C3 RNA expression data was obtained from the CrownBio (Beijing, China) database. As shown in Table 3, after 96 hours of exposure to 3424, liver cancer cell lines with high AKR1C3 expression at both the protein and RNA levels exhibited IC 50 The IC value was in the low nM range, indicating high sensitivity to 3424. On the other hand, cells with low AKR1C3 expression were less sensitive to 3424, with IC 50The IC values ​​were above 1000 nM. Similarly, NSCLC cells also exhibited AKR1C3-dependent cytotoxicity characteristics after 72 h of exposure to 3424 (Table 4). The IC values ​​of 3424 in hepatocellular carcinoma and NSCLC cells were 50 There was a high correlation between AKR1C3 protein levels (R2 = 0.71, Figure 3A, top) and RNA expression levels (R2 = 0.87, Figure 3A, center) (R2 = 0.80, Figure 3A, bottom), respectively. These results demonstrate that 3424-mediated cytotoxicity is highly correlated with AKR1C3 expression levels in both liver cancer and NSCLC cell lines.

[0085] [Table 3]

[0086] [Table 4]

[0087] The specific activation of 3424 via AKR1C3 was confirmed in H460 cells using the AKR1C3 inhibitor 3021. H460 cells were pretreated with 3 μM 3021 for 2 hours, followed by co-treatment with 3424 for 2 hours. 3021, an AKR1C3-specific inhibitor, exhibited an IC of 6.3 μM. 50 value, and IC in the absence of 3021 50 While 3021 had an IC value of 4 nM (Figure 3B, top), it was able to effectively inhibit 3424 cytotoxicity in H460 cells, which was also reported by Evans et al. (Non-Patent Document 25). We also demonstrated the cytotoxicity of the R-enantiomer, 3423, and 2870, a racemic mixture of R- and S-enantiomers in a 1:1 ratio, in H460 cells in the absence or presence of 3021. As shown in Figure 3B, 3423 (center) and 2870 (bottom) had IC values ​​of 1:1 and 2:1, respectively. 50 The IC values ​​were 5 nM and 4 nM, respectively, demonstrating potent cytotoxicity comparable to that of 3424. Similar to 3424, the cytotoxicity of 3423 and 2870 was highly dependent on AKR1C3, and 3021 exhibited IC 50These cytotoxic effects were inhibited at concentrations of 6.3 μM and 5 μM, respectively, more than 1000-fold higher than in the absence of inhibitor. Inhibitor 3021 alone did not exert cytotoxicity up to 100 μM (data not shown).

[0088] To assess whether 3424 crosslinks DNA, a single-cell electrophoresis-based comet assay, a direct biochemical assay for DNA crosslinking, was employed using H460 cells. The racemic mixture 2870 was used in this assay. As shown in Figure 3C, in H460 cells treated with the solvent dimethyl sulfoxide, no detectable double-strand breaks were observed at a tail moment of 0.3. Bleomycin-induced DNA strand breaks were evident from an increase in tail moment to 35. 2870-induced DNA crosslinks were tested using three concentrations under the same conditions as those used for bleomycin. Compound 2870 induced concentration-dependent DNA crosslinks, evident from a decrease in tail moment from 36 to 1.6. Thus, 3424-mediated cytotoxicity was demonstrated to be highly AKR1C3-dependent.

[0089] (in vivo antitumor activity) All procedures related to the handling, care, and treatment of animals in this study were conducted in accordance with the guidelines of CrownBio (Beijing, China), WuXi AppTec (Shanghai, China), or Eurofins Pharmacology Discovery Services Taiwan (Taipei, Taiwan), as approved by the Institutional Animal Care and Use Committee (IACUC) in accordance with the International Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines. During regular monitoring, animals were checked for the effects of tumor growth on normal behavior, including mobility, food and water consumption, weight gain / loss, eye / hair matting, and other abnormal effects. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

[0090] In all animal studies, the efficacy of drugs was evaluated by their effect on tumor growth inhibition. Tumor volume (mm 3 ) was measured twice a week according to the following ellipsoid formula: Length (mm) x [Width (mm)] 2 ×0.5 The tumor growth inhibition rate (% TGI) was calculated twice a week during the administration period using the following formula. %TGI=(1-[(T-T) / (C-C)])×100, where T=mean tumor volume of the treatment group, T=mean tumor volume of the treatment group at the start of the study, C=mean tumor volume of the control group, and C=mean tumor volume of the control group at the start of treatment. Statistical significance of groups compared with vehicle controls was assessed using two-way analysis of variance and Bonferroni tests using SPSS Statistics 23 (IBM Corp., Armonk, NY) or R (version 3.3.1). P values ​​of less than 0.05 were considered statistically significant.

[0091] Example 3: Antitumor effect of 3424 in the CDX model Example 3-1: Antitumor effects of 3424 in HepG2 and H460 models The in vivo antitumor activity of 3424 was evaluated using a GFP-expressing cancer cell line in an orthotopic liver cancer model (HepG2) and a subcutaneous lung cancer model (H460) CDX model from AntiCancer Inc. (Beijing, China). Female athymic nude mice (6 weeks old: BALB / c-nu, Beijing HFK Bioscience Co., Ltd., Beijing, China) were used for the study. Each mouse received a HepG2-GFP tumor mass (~1 mm in diameter) in the right lobe of the liver to develop tumors. 3 ) or H460-GFP tumor masses (diameter ~1 mm 3) were subcutaneously inoculated. Approximately 3–10 days later, whole-body scans of the mice were performed using a FluorVivo Model-100 fluorescence imager (INDEC Biosystems, Inc., Los Altos, CA). Mice with similar fluorescent areas were selected and randomly assigned to groups. Both cell lines expressed high levels of AKR1C3. The prodrug 3424 was administered intravenously (IV) to the HepG2 orthotopic model at 1.25, 2.5 mg / kg, or 5 mg / kg Q7D (every 7 days) × 2, and to the H460 xenograft model at 0.625, 1.25, or 2.5 mg / kg Q7D (every 7 days) × 2, followed by a one-week rest period, and then Q7D (every 7 days) × 2. Sorafenib was used as a positive control in the HepG2 model and was orally administered at 30 mg / kg once daily for 5 × 7 cycles. In the H460 model, taxol was used as a positive control and administered intravenously at 15 mg / kg BIW (every other week) for four doses. Mice were observed daily and weighed twice weekly throughout the experiment. Tumor burden was monitored twice weekly by either caliper measurement (H460) or FluorVivo fluorescence imager (H460-GFP and HepG2-GFP).

[0092] The dose-dependent antitumor activity of 3424 was investigated using whole-body fluorescence imaging in a HepG2 orthotopic xenograft model. When 3424 was administered intravenously at doses of 1.25, 2.5, or 5 mg / kg once weekly for 2 weeks, tumor growth inhibition (TGI) at day 34 was 52.4%, 91.5%, and 101.2%, respectively (Table 5). The antitumor effect of 3424 was observed to be dose-dependent (Figure 4A). Compared with the vehicle group, the tumor inhibition induced by 2.5 mg / kg and 5 mg / kg 3424 was statistically significant, with complete regression rates of 80% and 100%, respectively. At the end of the experiment, tumor fluorescence images (Figure 4B) and tumor weight data were consistent with the fluorescence area measurements (Figure 4A). Oral administration of sorafenib, a first-line treatment for hepatocellular carcinoma (HCC), at 30 mg / kg reduced tumor growth by 52.1%, but this difference was not statistically significant. At this dosing regimen, sorafenib induced weight loss (-11%, data not shown). In contrast, no weight loss was observed in the 3424-treated groups at any dose tested in this study (Table 5).

[0093] In the H460 subcutaneous model, prodrug 3424 was administered twice weekly, followed by a one-week rest period, and then twice weekly at doses of 0.625, 1.25, and 2.5 mg / kg. As shown in Figure 5 and Table 5, prodrug 3424 demonstrated dose-dependent antitumor activity, with TGIs of 60.2%, 67.2%, and 88%, respectively. The antitumor efficacy of 3424 was comparable to that of paclitaxel, with paclitaxel at 15 mg / kg demonstrating a TGI of 64%. At the end of the study (day 35), 3424 administration resulted in minimal weight loss at the low and mid-dose levels and a statistically insignificant 14% loss at the high dose, whereas paclitaxel administration resulted in a significant weight loss of 11% (Table 5).

[0094] [Table 5]

[0095] Example 3-2: Antitumor effects of 3424 in VCaP, SNU-16, and A498 models 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 studies were 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 × 10 or 1 × 10 human cancer cells were injected subcutaneously into the right flank of the mice with 1:1 Matrigel. Tumor volumes of 150–200 mm were determined. 3 Once the 3-day follow-up time reached 100 mg / kg / day, the vehicle and test substance were administered (referred to as day 1 or day 0 in the SNU-16 model). Depending on the model, the vehicle or test substance was administered intravenously once a week for a total of four or five doses. Standard treatments, including abiraterone / prednisolone (for CRPC), 5-fluorouracil (for gastric cancer), sunitinib, and gemcitabine (both for RCC), were administered as recommended in the literature (Non-Patent Documents 15, 30-32). On the day of coadministration, 3424 was administered intravenously first, followed by the concomitant drug within one hour.

[0096] The in vivo antitumor efficacy of 3424 as monotherapy or in combination with standard of care 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 express high levels of AKR1C3 at both the protein and RNA levels. AKR1C3 protein expression in VCaP, SNU-16, and A498 was measured by Western blot using AKR1C3-to-tubulin ratios of 8.9, 1.9, and 1.6, respectively. AKR1C3 RNA expression levels (LOG2 FPKM) in VCaP, SNU-16, and A498 were 5.2, 8.0, and 10.0, respectively.

[0097] In these studies, animals were treated with various concentrations of 3424 as monotherapy, anti-cancer drugs as monotherapy or drugs as monotherapy (standard of care, control), or compositions of the present invention combining 3424 with at least one anti-cancer drug.

[0098] Example 3-2-1: 3424 monotherapy or combination therapy with abiraterone acetate + prednisolone In a CRPC model, castrated male BALB / c nude mice were treated with 3424 (five weekly intravenous injections), abiraterone acetate plus prednisolone (daily oral administration), or the combination (Figure 4C). At 5 mg / kg, 3424 demonstrated a significant TGI of 148%, which was further enhanced to 158% in combination with abiraterone acetate / prednisolone.

[0099] Furthermore, at the time of terminal euthanasia on day 32, animals receiving the combination treatment showed a decrease in serum prostate-specific antigen (PSA) (Figure 6).

[0100] Example 3-2-2: 3424 monotherapy or combination therapy with 5-FU In the SNU-16 gastric cancer model, female BALB / c mice were treated with 3424 (four weekly intravenous injections), 5-fluorouracil (5-FU) (twice weekly IP injections), or a combination of 3424 and 5-FU (Figure 4D). Animals treated with 2.5 or 5 mg / kg of 3424 demonstrated significant tumor growth inhibitor (TGI) of 87.8% or 96.2%, respectively, whereas 5-FU at 30 mg / kg did not demonstrate antitumor activity. Synergistic effects were observed when 3424 was combined with 5-FU.

[0101] Example 3-2-3: 3424 monotherapy or combination therapy with sunitinib In the A498 renal cell carcinoma model, female SCID mice were treated with 3424 (four weekly intravenous injections), sunitinib (25 mg / kg, daily oral administration), or the combination (Figure 4E). At 2.5 mg / kg, 3424 demonstrated a significant TGI of 73%, whereas sunitinib had a modest TGI of 52%. When animals were treated with the combination of 3424 and sunitinib, the TGI further increased to 88%.

[0102] Example 3-2-4: 3424 monotherapy or combination therapy with gemcitabine (Gemzar) When the efficacy of 3424 in combination with gemcitabine was tested in an A498 xenograft model, gemcitabine at 80 mg / kg (administered four times weekly via IP injection) showed a TGI of 19%, and in combination with 3424 at 2.5 mg / kg, the TGI increased to 87% (Figure 4F).

[0103] In all three models, 3424 was well tolerated by mice, and no significant weight loss was observed during treatment (data not shown). Figure 4 shows the antitumor activity of 3424 or compositions of the present invention in various CDX models. The inventors demonstrated that, in combination therapy, 3424 can enhance the efficacy of standard treatment in CDX models of CRPC, gastric cancer, and RCC.

[0104] Example 4: Antitumor activity of 3424 in PDX models The antitumor activity of 3424 in PDX models was evaluated at CrownBio Bioscience (Beijing, China) using female BALB / c nude mice (6-7 weeks old, Beijing Anikeeper Biotech Co., Ltd., Beijing, China). Tumor fragments (PA1280, GA6201, LU2057, and LU2505) were harvested from stock mice inoculated with selected primary human cancer tissues (pancreatic, gastric, and lung) and used to inoculate BALB / c nude mice. Tumors were developed subcutaneously in each mouse. Tumors were analyzed using StudyDirector™ Ver. 3.1.399.19 (Studylog Systems Inc., South San Francisco, CA, USA) until the mean tumor diameter was approximately 100 mm. 3 At the time of reaching 100 mg / kg, mice were randomly assigned to experimental groups. Prodrug 3424 was administered intravenously at the indicated doses on a Q7D (every 7 days) x 3 dosing schedule. Each group consisted of 5-6 mice. The day of group assignment was designated as day 0. Prodrug 3424 was administered to tumor-bearing mice from day 0 until the day indicated for each study.

[0105] Furthermore, the in vivo antitumor activity of 3424 was evaluated in patient-derived xenograft (PDX) models, including pancreatic cancer, gastric cancer, and lung cancer with high and low AKR1C3 expression. The prodrug 3424 was administered intravenously three times weekly. Patient information for the four PDX models is shown in Table 6. In the pancreatic PDX model (Figure 7A and Table 7), 3424 at 2.5 mg / kg demonstrated statistically significant antitumor activity with a TGI of 77.4%. In the gastric PDX model, 3424 at 5 mg / kg demonstrated significant antitumor inhibition with a TGI of 110% (Figure 7B and Table 7). Tumor volume continued to decrease and remained low or unmeasurable even one month after treatment cessation. At the end of the study, 60% of the mice were tumor-free. Two lung cancer PDX models with different levels of AKR1C3 expression were selected to examine whether the in vivo antitumor activity of 3424 was AKR1C3-dependent. The LU2505 PDX model expressed high levels of AKR1C3 RNA (log2 = 6.03), whereas the LU2057 model expressed low levels of AKR1C RNA (log2 = 2.08).

[0106] As shown in Figures 7 and 7D and Table 7, at the same dose of 2.5 mg / kg, 3424 exhibited excellent tumor growth inhibition in LU2505 tumors with high AKR1C3 expression (TGI: 105.2%), but not in LU2057 PDX tumors with low AKR1C3 expression (TGI: -20.9%), demonstrating the AKR1C3-dependent in vivo antitumor activity of 3424. Even at a dose as low as 1.25 mg / kg, 3424 statistically significantly inhibited LU2505 tumor growth (TGI: 105.0%). In all PDX models, the prodrug 3424 was well tolerated, with no weight loss observed at any dose tested (Table 7).

[0107] [Table 6]

[0108] [Table 7]

[0109] Using over 20 cell lines derived from either liver cancer or NSCLC, we found that 3424 exhibited enhanced cytotoxicity in all cell lines with high AKR1C3 expression compared with cells with low or undetectable AKR1C3 expression. 50 3424 exhibited low nM potency, characteristic of potent nitrogen mustards. Of particular note was the 5000-fold enhancement of cytotoxicity in the NCI-H2228 NSCLC line, which expresses high AKR1C3. This result is consistent with targeting tumors with high AKR1C3 expression while sparing areas of low AKR1C3 expression found in normal tissues.

[0110] The results described herein highlight that 3424 exhibits AKR1C3-dependent cytotoxicity in vitro and antitumor activity in vivo across a wide range of human cancer types. We demonstrate superior in vivo antitumor activity of 3424 at clinically achievable doses in a broad panel of CDX and PDX models highly expressing AKR1C3. In particular, 3424 demonstrates remarkable efficacy in vivo against liver, gastric, renal, lung, pancreatic, and castration-resistant prostate cancers. The AKR1C3-dependent activity of 3424 may serve as the basis for ongoing and future clinical trials specifically targeting cancer cells, or as a biomarker to characterize cancer patients and further guide patient selection for 3424 treatment.

[0111] It should be noted that the above description of the embodiments of the present invention does not limit the present invention. Those skilled in the art can make various modifications and changes according to the present invention, and modifications and changes within the spirit of the present invention are intended to be encompassed in the scope of the claims appended to the present invention.

[0112] All references cited herein are incorporated by reference to the full extent permitted by law. The discussion of these documents is intended merely to summarize the assertions made by their authors. No admission is made that any document (or portion of a document) is pertinent prior art. Applicant reserves the right to challenge the accuracy and pertinence of the cited documents.

Claims

1. 1. Use of the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having formula I, or a pharmaceutically acceptable salt, isotopic variant, or solvate thereof, in the manufacture of a medicament for treating cancer in a patient: Used in combination with at least one other anticancer drug, [Chemical Formula I] the cancer is associated with overexpression of AKR1C3; The other anticancer drug is at least one selected from the group consisting of gemcitabine, abiraterone acetate, prednisolone, 5-fluorouracil (5-FU), sunitinib, and a combination of abiraterone acetate and prednisolone.

2. The use according to claim 1, wherein the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having the following formula I-1, or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having the following formula I-2: 【Chemistry II】

3. 2. The use of claim 1, wherein the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma cancer or rectal cancer.

4. The use described in claim 3, wherein the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma or pancreatic cancer.

5. A method for treating cancer comprising administering to a patient a therapeutically effective amount of the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having the following formula I, or a pharmaceutically acceptable salt, isotopic variant, or solvate thereof, and at least one other anticancer agent: [Chemical Formula I] for use in a method of treatment for a subject in need of cancer treatment, the cancer is associated with overexpression of AKR1C3; The composition, wherein the other anticancer drug is at least one selected from the group consisting of gemcitabine, abiraterone acetate, prednisolone, 5-fluorouracil (5-FU), sunitinib, and a combination of abiraterone acetate and prednisolone.

6. The composition according to claim 5, wherein the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having the following formula I-1, or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having the following formula I-2: 【Chemistry II】

7. The composition of claim 5, wherein the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma cancer, or rectal cancer.

8. The composition described in claim 7, wherein the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell cancer or pancreatic cancer.

9. The composition described in claim 5, wherein the compound is administered to a subject when the content of AKR1C3 reductase in the subject's cancer cells is measured to be equal to or greater than a predetermined value by a process using an AKR1C3 antibody.

10. an effective amount of the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having formula I or a pharmaceutically acceptable salt, isotopic variant, or solvate thereof, and at least one other anticancer agent; [Chemical Formula I] for use in a method for inhibiting cell proliferation, the cancer is associated with overexpression of AKR1C3; The composition, wherein the other anticancer drug is at least one selected from the group consisting of gemcitabine, abiraterone acetate, prednisolone, 5-fluorouracil (5-FU), sunitinib, and a combination of abiraterone acetate and prednisolone.

11. The composition described in claim 10, wherein the cells are cancer cells.

12. The composition described in claim 10, wherein the compound is contacted with the cells when the content of AKR1C3 reductase in the cells is measured to be equal to or greater than a predetermined value by a step of measuring the content of AKR1C3 reductase in the cells using an AKR1C3 antibody.

13. 1. Use of the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having formula I, or a pharmaceutically acceptable salt, isotope or solvate thereof, in the manufacture of a medicament for inhibiting cell proliferation, Used in combination with at least one other anticancer drug, [Chemical Formula I] the cancer is associated with overexpression of AKR1C3; The other anticancer drug is at least one selected from the group consisting of gemcitabine, abiraterone acetate, prednisolone, 5-fluorouracil (5-FU), sunitinib, and a combination of abiraterone acetate and prednisolone.

14. The use according to claim 13, wherein the cells are cancer cells.

15. A composition comprising the following 1) and 2): 1) the compound 1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having formula I, or a pharmaceutically acceptable salt, isotopic variant, or solvate thereof; and 2) at least one other anticancer agent selected from the group consisting of gemcitabine, abiraterone acetate, prednisolone, 5-fluorouracil (5-FU), sunitinib, and a combination of abiraterone acetate and prednisolone. [Chemical Formula I]

16. The composition of claim 15, wherein the compound is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having formula I-1, or (R)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate having formula I-2. 【Chemistry II】 17. The composition of claim 15, wherein the composition further comprises a pharmaceutically acceptable excipient.

18. When the cancer to be treated is renal cell carcinoma (RCC), the anticancer agent is selected from the group consisting of gemcitabine and sunitinib; when the cancer is gastric cancer, the anticancer drug is 5-fluorouracil (5-FU); 16. The composition of claim 15, wherein when 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.

19. 20. Use of a composition according to any one of claims 15 to 18 in the manufacture of a medicament for treating cancer in a patient.

20. 20. The use of the composition of claim 19, wherein the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma cancer or rectal cancer.

21. The use described in claim 20, wherein the AKR1C3 reductase level of the cancer is equal to or greater than a predetermined value.

22. The use described in claim 20, wherein the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma or pancreatic cancer.

23. 19. A medicament comprising the composition of any one of claims 15 to 18 for use in a method of treating cancer in a patient in need thereof, The method comprises administering to the patient an effective amount of the medicament.

24. The pharmaceutical composition of claim 23, wherein the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma cancer, or rectal cancer.

25. The pharmaceutical described in claim 24, wherein the AKR1C3 reductase level of the cancer is equal to or greater than a predetermined value.

26. The pharmaceutical described in claim 24, wherein the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell cancer or pancreatic cancer.

27. The pharmaceutical composition of claim 23, wherein the composition is administered to a patient when the content of AKR1C3 reductase in the patient's cancer cells is measured to be equal to or greater than a predetermined value using an AKR1C3 antibody.

28. 1. A composition for use in treating cancer in a patient, comprising: The composition comprises at least one compound selected from the group consisting of a compound having the following formula I-1, a compound having the following formula I-2, and a racemic mixture of the compound having the following formula I-1 and the compound having the following formula I-2 in a 1:1 ratio, and at least one anticancer agent, The composition, wherein the anticancer agent is selected from the group consisting of gemcitabine, 5-fluorouracil (5-FU), sunitinib, or a combination of abiraterone acetate and prednisolone. 【Chemistry II】

29. 29. The composition of claim 28, wherein the cancer is liver cancer, hepatocellular carcinoma (HCC), lung cancer, melanoma, prostate cancer, breast cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcoma cancer, or rectal cancer.

30. The composition described in claim 29, wherein the AKR1C3 reductase level of the cancer is equal to or greater than a predetermined value.

31. The composition described in claim 29, wherein the cancer is liver cancer, non-small cell lung cancer, castration-resistant prostate cancer, gastric cancer, renal cell carcinoma or pancreatic cancer.

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