AST-3424 metabolite and use

Abstract

An AST-3424 metabolite I-1 / I-2, a use, and a preparation method. The AST-3424 metabolite I-1 / I-2 can be used in the preparation of a drug for treating tumors and cancer, and can serve as a standard substance or a reference substance for the quality control of a formulation / active pharmaceutical ingredient containing AST-3424.

Description

AST-3424 metabolites and uses Technical Field

[0001] This invention relates to the further development of compound TH3424 (AST-3424 / OBI-3424) of the S-configuration in patent application PCT application number PCT / US2016 / 062114, publication number WO2017087428A1, corresponding to Chinese application number 2016800446081, publication number CN108290911A, and belongs to the field of cancer therapeutic compound development. Background Technology

[0002] Our company has developed AST-3424, a DNA alkylation cancer therapeutic drug targeting overexpression of aldehyde-ketone reductase 1C3 (AKR1C3). (See patent application: DNA alkylating agent, corresponding PCT application number PCT / US2016 / 021581, publication number WO2016 / 145092, corresponding Chinese application number 2016800150788, publication number CN107530556A, which discloses compound TH2870; (R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethyl)aminophosphate). The composition, its use, and preparation method, corresponding to PCT application number PCT / US2016 / 062114, publication number WO2017087428A1, and Chinese application number 2016800446081, publication number CN108290911A (S-configuration compound), with the Chinese name (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)aminophosphate, also known as the S-configuration compound of OBI-3424 and TH-2870, CAS number 2097713-69-2, has the following structure:

[0003] There are authoritative industry documents (Kathryn Evans, Jian Xin Duan, Tara Pritchard, et al. OBI-3424, a novel AKR1C3-activated prodrug, exhibits potent efficacy against preclinical models of T-ALL [J], Clinical Cancer Research, 2019, DOI: 10.1158 / 1078-0432.CCR-19-0551; Richard B. Lock, Kathr yn Evans, Raymond Yung, Tara Pritchard, Beverly A. Teicher, Jian Xin Duan, Yue long Guo, Stephen W. Erickson and Malcolm A. Smith, Abstract LB-B16: The AKR1C3-Activated Prodru g OBI-3424Exerts Profound In Vivo EfficacyAgainst Preclinical Models of T-Cell Acute Lymphoblastic Leukemia (T-ALL); a Pediatric Preclinical Testing Consortium Study [C], AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; October 26-30, 2017; Philadelphia, PA, DOI: 10.1158 / 1535-7163.) confirmed that this compound is a broad-spectrum small molecule anticancer prodrug with efficacy against various solid tumors and hematological malignancies.

[0004] The drug has currently completed Phase I clinical trials in China and the United States (Shukui Qin et al., Safety, tolerability, pharmacokinetics and clinical activity of AST-3424, an AKR1C3-activated bis-alkylating moiety prodrug, in subjects with advanced solid tumors in China: A phase I dose-escalation study..JCO 42,3121-3121(2024).DOI:10.1200 / JCO.2024.42.16_suppl.3121;Apostolia Maria Tsimberidou et al.,Safety,pharmacokinetics,and clinical activity of OBI-3424,an AKR1C3-activated prodrug,in patients with advanced or metastatic solid tumors:A phase 1dose-escalation study..JCO 40,3030-3030(2022).DOI:10.1200 / JCO.2022.40.16_suppl.3030;Tsimberidou,AM,Verschraegen,CF,Wesolowski,R.et al.Phase 1dose-escalation study evaluating the safety, pharmacokinetics, and clinical activity of OBI-3424 in patients with advanced or metastatic solid tumors.Br J Cancer 129,266-274(2023).https: / / doi.org / 10.1038 / s41416-023-02280-4), the trial results support the safety and preliminary efficacy of the drug, and further phase II and III clinical trials will be conducted. Summary of the Invention

[0005] Subsequent studies on the metabolism of AST-3424 in humans and animals revealed that, apart from the AKR1C3 enzyme-specific metabolism that produces AST-2660, AST-3424 exhibits completely different metabolic pathways in different species. Furthermore, its metabolites I-1 and I-2, obtained by removing monomethyl and dimethyl groups, were found to have AKR1C3 enzyme-dependent cell proliferation effects similar to AST-3424. Moreover, the monomethyl-removed metabolite I-1 showed higher AKR1C3 selectivity in vitro, suggesting that the monomethyl-removed metabolite I-1 of AST-3424 has the potential to be further developed into an AST-3424 analogue.

[0006] On the other hand, since the metabolites I-1 and I-2, after the removal of monomethyl and dimethyl groups, are both cytotoxic, excessive amounts of these metabolites could lead to unexpected toxic side effects if they are present in the AST-3424 active pharmaceutical ingredient (API) or formulation. Therefore, it is necessary to control the content of I-1 and I-2 in the AST-3424 API or formulation. Thus, these two metabolites, I-1 and I-2, can be used as standard references in the quality control of the AST-3424 API or formulation. Uses of I-1 and I-2 compounds as standards or references:

[0007] Uses of compounds I-1 and I-2 in quality control of formulations / active pharmaceutical ingredients containing AST-3424:

[0008] As mentioned above, since the metabolites I-1 and I-2 after the removal of monomethyl and dimethyl groups are both cytotoxic, it is necessary to control the content of metabolites I-1 and I-2 in AST-3424 active pharmaceutical ingredient or formulation. Therefore, these two metabolites I-1 and I-2 can be used as standard references in the quality control of AST-3424 active pharmaceutical ingredient or formulation.

[0009] For pharmaceutical technicians, compounds of higher purity can be used as "reference substances" or "standard substances." Reference substances generally refer to standard materials used for identification, testing, content determination, and calibration of instrument performance. Together, they are called standard reference substances, namely, high-purity metabolites I-1 and I-2, which are typically used as reference substances for substance identification and testing, and as standard substances for content determination and instrument calibration.

[0010] In some embodiments, the formulation containing AST-3424 is a concentrated solution of AST-3424 for injection, and the active pharmaceutical ingredient containing AST-3424 is an AST-3424 ethanol solution with a mass percentage of 30-40%.

[0011] The concentrated AST-3424 injection solution was manufactured by a pharmaceutical company commissioned by Shenzhen Aixindawei Pharmaceutical Technology Co., Ltd. The specification is 1 mL: 10 mg; it contains 0.75 mL of ethanol, 0.25 mL of propylene glycol, and 10 mg of AST-3424. This formulation has been used in a Phase II clinical trial of AST-3424 in China, with clinical trial registration number CTR20191399. For the specific preparation process and method, please refer to patent application PCT / CN2020 / 101870, publication number WO20210085201.

[0012] The AST-3424 active pharmaceutical ingredient is an ethanol solution of 30-40% by weight of AST-3424. This ethanol solution is used to prepare the above-mentioned concentrated AST-3424 injection solution. The 30-40% by weight ethanol solution of AST-3424 was prepared by a CDMO in accordance with PCT / CN2022 / 129548, publication number WO2024092614, and is an ethanol solution of 30-40% by weight.

[0013] In order to explain the necessity of using the above metabolites I-1 and I-2 for quality control, it is necessary to clarify the production process of metabolites I-1 and I-2 other than the generation of AST-3424 through cellular metabolism.

[0014] Regardless of the method used in the synthesis of AST-3424 (see patent publications WO2020172506 and WO2016145092), 3-hydroxy-N,N-dimethylbenzamide is required to construct the corresponding chemical structure. This compound may contain impurities of 3-hydroxy-N-methylbenzamide / 3-hydroxybenzamide. Therefore, the final AST-3424 will inevitably contain metabolites I-1 and I-2. Furthermore, the possibility of AST-3424 degrading under high temperature and light conditions, generating metabolites I-1 and I-2, cannot be ruled out. Therefore, metabolites I-1 and I-2 may be generated during the production and storage of AST-3424. To control the quality of the AST-3424 formulation / active drug, i.e., the content of metabolites I-1 and I-2 in the active drug / formulation, metabolites I-1 and I-2 can be used as standards for impurity identification in the active drug / formulation and as reference standards for impurity HPLC content determination.

[0015] The demethylated metabolite of AST-3424 of formula I-2,

[0016] Application of I-2 compounds in the preparation of drugs for treating tumors and cancer.

[0017] The pharmaceutical composition of compound I-2 also contains pharmaceutically acceptable excipients.

[0018] A pharmaceutical composition for the treatment of tumors and cancer.

[0019] Preferably, the tumor or cancer is selected from liver cancer or leukemia.

[0020] Liver cancer includes hepatocellular carcinoma and intrahepatic bile duct carcinoma, and leukemia includes acute lymphoblastic leukemia (such as acute T-lymphoblastic leukemia T-ALL and acute B-lymphoblastic leukemia B-ALL), especially relapsed or refractory B-ALL and T-ALL.

[0021] The above metabolites I-1 and I-2 can be prepared either through cellular metabolic processes or through direct chemical synthesis.

[0022] In some embodiments, compounds I-1 and I-2 are prepared by reacting AST-3424 with human cells:

[0023] The specific steps can be:

[0024] For hepatocytes, AST-3424 solution was directly mixed with human, monkey, rat and mouse hepatocytes, and incubated at an appropriate temperature for a period of time before post-processing and separation.

[0025] For blood cells, the AST-3424 solution was directly mixed with human whole blood and incubated at an appropriate temperature for a period of time before post-processing and separation.

[0026] As an alternative, compounds I-1 and I-2 are prepared by reacting AST-3424 with human liver microsomes. The preparation of liver microsomes is similar to that of hepatocytes described above.

[0027] Post-processing separation operation: After adding acetonitrile, the cells were broken by high-speed mixing and shaking (using a high-speed vortex shaker), and the proteins were separated by high-speed centrifugation. The supernatant was taken and extracted with organic solvents (DCM, EA). The organic phase was collected, concentrated, and then purified by chromatography.

[0028] In some embodiments, compounds I-1 and I-2 are prepared by the following chemical synthesis: compound II reacts with the corresponding 3-hydroxy-N-methylbenzamide / 3-hydroxybenzamide in acetonitrile solvent under the action of Cs2CO3 to prepare compounds I-1 and I-2 of the above formula:

[0029] To ensure a more efficient reaction, the molar ratio of 3-hydroxy-N-methylbenzamide / 3-hydroxybenzamide to raw material II is greater than 1, preferably greater than or equal to 1.5; the molar ratio of Cs2CO3 to raw material II is greater than or equal to 2.

[0030] The specific steps are as follows:

[0031] Add starter II, acetonitrile, 3-hydroxy-N-methylbenzamide / 3-hydroxybenzamide and cesium carbonate to the reaction vessel. After addition, react the reaction mixture overnight at room temperature. LC-MS or TLC is used to detect that starter II has reacted completely. Filter the mixture and wash the residue with a small amount of acetonitrile. Combine the filtrate and washings and concentrate under reduced pressure to dryness to obtain the crude product. The crude product is then purified by column chromatography.

[0032] Clearly, the metabolites I-1 and I-2 prepared by the above chemical synthesis are larger in scale, lower in cost, and easier to prepare high-purity compounds, making them more suitable as standard references for the quality control of AST-3424. Attached Figure Description

[0033] Figure 1 shows the UPLC-UV chromatogram of the AST-3424 standard solution.

[0034] Figure 2 shows the UPLC-UV chromatogram of AST-3424 after incubation in human hepatocytes. The horizontal axis is in minutes. A is the sample after hepatocyte incubation for 120 min, and B is the control sample after hepatocyte incubation for 0 min.

[0035] Figure 3 shows the UPLC-UV chromatogram of AST-3424 after incubation in monkey hepatocytes. The horizontal axis is in minutes. A is the sample after hepatocyte incubation for 120 min, and B is the control sample after hepatocyte incubation for 0 min.

[0036] Figure 4 shows the UPLC-UV chromatogram of AST-3424 after incubation in rat hepatocytes. The horizontal axis is in minutes. A is the sample after hepatocyte incubation for 120 min, and B is the control sample after hepatocyte incubation for 0 min.

[0037] Figure 5 shows the UPLC-UV chromatogram of AST-3424 after incubation in mouse hepatocytes. The horizontal axis is in minutes. A is the sample after hepatocyte incubation for 120 min, and B is the control sample after hepatocyte incubation for 0 min.

[0038] Figure 6 shows a simulated UPLC-MS chromatogram of AST-3424 metabolites in human hepatocytes.

[0039] Figure 7 shows a simulated UPLC-MS chromatogram of AST-3424 metabolites in monkey hepatocytes.

[0040] Figure 8 shows a simulated UPLC-MS chromatogram of AST-3424 metabolites in rat hepatocytes.

[0041] Figure 9 shows a simulated UPLC-MS chromatogram of AST-3424 metabolites in mouse hepatocytes.

[0042] Figure 10 shows the HPLC test results of the synthesized I-1.

[0043] Figure 11 shows the HPLC test results of the synthesized I-2.

[0044] Figure 12 shows the proliferation inhibition curve of compound I-1 (AST-A096 in the figure) on NCI-H460 cells.

[0045] Figure 13 shows the proliferation inhibition curve of compound I-2 (AST-A098 in the figure) on NCI-H460 cells.

[0046] Figure 14 shows the proliferation inhibition curve of compound AST-3424 on H460 cells. Detailed Implementation

[0047] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0049] The terms "patient" and "individual" are used interchangeably and refer to a mammal in need of cancer treatment. Typically, a patient is a human being diagnosed with cancer. In some embodiments, "patient" or "individual" may refer to a non-human mammal used to screen, characterize, and evaluate drugs and therapies, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat.

[0050] "Tumor" refers to a solid tumor, including (but not limited to) metastatic tumors in the bone, brain, liver, lungs, lymph nodes, pancreas, prostate, skin, and soft tissue (sarcoma).

[0051] Leukemia is a malignant clonal disease originating from hematopoietic stem cells. Based on cell differentiation and maturation and natural course, it can be divided into acute leukemia (AL) and chronic leukemia (CL). According to the primary cell lineage affected, it can be divided into lymphocytic leukemia and non-lymphocytic (myeloid) leukemia. Therefore, AL can be further divided into acute lymphoblastic leukemia (ALL, including acute T-lymphoblastic leukemia (T-ALL) and acute B-lymphoblastic leukemia (B-ALL)) and acute myeloid leukemia (AML). CL can be divided into chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), and rarer types of leukemia such as hairy cell leukemia and prolymphocytic leukemia.

[0052] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

[0053] Example 1: Animal In Vitro Cell Metabolite Identification Experiment with AST-3424

[0054] AST-3424 at a concentration of 10 μM was incubated in human, monkey, rat, and mouse hepatocytes for 0 min or 120 min.

[0055] Hepatocyte samples were collected at 0 min and 120 min, and 100 μL / well were combined according to time points. 200 μL of acetonitrile was added, and the samples were vortexed to disrupt the cells. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was dried under an air stream at 45°C. The dried hepatocyte samples were reconstituted with 100 μL of acetonitrile / water (10 / 90, v / v). 2.0 μL of the reconstituted sample was analyzed by UPLC-UV / Q-TOF MS, and another 10.0 μL was analyzed by UPLC-MS / MS to detect AST-2660.

[0056] UPLC-UV / Q-TOF MS conditions

[0057] Leveraging Waters ACQUITY The chromatographic separation was performed using a System ultra-high performance liquid chromatography (UHPLC) system (Waters, Milford, MA, USA). The chromatographic column was an ACQUITY HSS T3 column with a particle size of 1.8 μm (2.1 × 100 mm). The column temperature was 45 °C, the flow rate was 0.4 mL / min, and the UV detection wavelength was 200-600 nm. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile:methanol containing 0.1% formic acid in a 9:1 (v / v) ratio. The mobile phase gradient started at 5% B, maintained for 2 min, then linearly increased to 70% B within 7 min, followed by a linear increase to 95% B within 2 min, and finally decreased directly to 5% B. The system equilibration time was 2 min. The total injection time was 13 min.

[0058] Mass spectrometry was performed using a Synapt G2-Si Q-TOF MS quadrupole-time-of-flight tandem mass spectrometer (Waters Laboratories, Milford, MA, USA). The ionization mode was positive ion electrospray ionization. The scan range was m / z 50–1000 Da. Leucine-enkephalin (m / z 556.2771) was used as an external standard for mass-to-charge ratio correction. The source temperature was 120 °C, the desolvent gas flow rate was 750 L / h, the desolvent gas temperature was 350 °C, the capillary voltage was 3.0 kV, and the cone voltage was 30 V. The desolvent gas was nitrogen, and the collision gas was argon.

[0059] To obtain MSE data, mass spectrometry data acquisition uses two independent scan functions that can be switched rapidly within 10 ms. The difference between them lies in the collision energy (CE). During low-energy scans, the transport collision energy is 2 eV and the trap collision energy is 2 eV; during high-energy scans, the trap collision energy is 15-30 eV and the transport collision energy is 10-15 eV.

[0060] UPLC-MS / MS conditions

[0061] Leveraging Waters ACQUITY Chromatographic separation was performed using a System ultra-high performance liquid chromatography (UHPLC) system (Waters Corporation, Milford, MA, USA). The chromatographic column was an ACQUITY BEH Amide column with a particle size of 1.7 μm (2.1 × 100 mm). The column temperature was 20 °C, and the flow rate was 0.4 mL / min. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile:methanol containing 0.1% formic acid in a 9:1 (v / v) ratio. The mobile phase gradient started at 95% B, maintained for 0.3 min, then linearly decreased to 10% B within 0.8 min, followed by a linear decrease to 5% B within 0.9 min, and finally directly increased to 95% B. The system equilibration time was 1.5 min. The total injection time was 3.5 min. Mass spectrometry detection was performed using a Waters Xevo TQ-S MS triple quadrupole tandem mass spectrometer (MS / MS). The ion source is an electrospray ionization source (ESI), and multiple reaction monitoring (MRM) is used for detection in negative ion mode. The detection ion pair of AST-2660 is m / z 147.0→62.9.

[0062] Figure 1-5 shows the UPLC-UV detection results of AST-3424 standard and four cell incubation samples, with a detection wavelength of 254 nm.

[0063] The above UPLC-UV detection results were analyzed by UPLC-Q-TOF MS, and the simulated ion chromatograms obtained after comparison and subtraction with blank samples are shown in Figures 6-9.

[0064] The corresponding ion peaks in the simulated ion chromatogram were analyzed by MS / MS and quantified to obtain the results of metabolite identification and relative content, as shown in Table 1 below.

[0065] Table 1: Identification results and relative contents of AST-3424 metabolites in hepatocytes of different species

[0066] M15* is AST-2660.

[0067] - indicates that it was not detected.

[0068] M11 is metabolite I-1, and M8 is metabolite I-2. M11 and M15 (also known as AST-2660) are common metabolites in hepatocytes of all species. M11 has the highest content of metabolites in hepatocytes of all four species (approximately 21% of AST-3424 in human hepatocytes, 195% in monkey hepatocytes, 27% in rat hepatocytes, and 78% in mouse hepatocytes), indicating that this metabolite is not highly species-specific. After further metabolism of M11 to demethylate M8, its content is lower (approximately 1.25% of AST-3424 in human hepatocytes, 16.10% in monkey hepatocytes, undetectable in rat hepatocytes, and approximately 5.91% in mouse hepatocytes).

[0069] Example 2: Identification Experiment of Blood Metabolites in Humans Using AST-3424

[0070] Nine samples from six patients before and after the first dose (day 1), at a dose level of 6 mg / m². 2 (Group 1) and 8 mg / m 2 (Group 2): Blood samples were collected before administration and 15 minutes after the start of infusion, and within 1 minute, 5 minutes, 30 minutes, 60 minutes, 2 hours, 4 hours and 6 hours after the end of infusion.

[0071] Pooling strategy: Samples taken at 0 hours (before administration) will serve as a negative control and will not be pooled. Other post-administration samples will be pooled according to the Hamilton AUC strategy. Each of the 6 patients was pooled separately.

[0072] After mixing, 6 negative control samples and 6 test samples were obtained.

[0073] For 6 negative controls and 6 pooled test samples, 100 μL of blood sample was added to 400 μL of acetonitrile. The mixture was vortexed for at least 60 s, then centrifuged at 16,000 g, 4 °C for 20 min to precipitate proteins. 400 μL of the supernatant was transferred and placed in a nitrogen evaporator under a stable nitrogen stream at room temperature until dry. The dried residue was reconstituted with 100 μL of diluent (ACN:H₂O (v / v) = 1:1). The mixture was then centrifuged at 16,000 g for 10 min, the supernatant was transferred, and the samples were analyzed using UHPLC-MS / MS. The results are shown in Table 2 below.

[0074] Table 2: Results of AST-3424 blood metabolite identification experiments in subjects with different identification numbers

[0075] AST-3424 (6 mg / m²) was administered intravenously to patients with relapsed or refractory acute lymphoblastic leukemia using liquid chromatography-mass spectrometry. 2 Dosage group and 8 mg / m 2 Metabolite identification was performed on plasma samples from the dosage groups (Chinese Clinical Trial Registry No. CTR20201908). The parent drug AST-3424 was the most abundant substance in all subjects, accounting for 78.78%–90.8% of the relative peak area. A total of four metabolites were identified: one metabolite (M17) was detected in the plasma of subjects 1101 and 1201; two metabolites (M16 and M17) were detected in the plasma of subjects 1103, 1105, and 1203; and four metabolites (M16, M17, M18, and M11) were detected in the plasma of subject 1202.

[0076] Therefore, the common metabolite in plasma and hepatocytes is M11, namely metabolite I-1; in addition, considering the possibility of further demethylation, metabolite I-2 can also be detected after a longer period of administration.

[0077] Examples 1 and 2 clearly show that metabolites I-1 and I-2 are the main metabolic products of AST-3424 in the blood and the liver, the main metabolic organ, after it enters the human body.

[0078] To conduct more in-depth research, it is necessary to synthesize and prepare compounds I-1 and I-2 with higher purity.

[0079] Example 3: Chemical Synthesis and HPLC Detection of AST-3424 Metabolites I-1 / I-2

[0080] Add raw material II (0.5 g, 1.6 mmol, 1.0 eq, prepared according to patent publications WO2020172506 and WO2016145092), 15 ml acetonitrile, 3-hydroxy-N-methylbenzamide (0.39 g, 2.6 mmol, 1.6 eq), and cesium carbonate (0.66 g, 4.7 mmol, 3.0 eq) sequentially to a dry single-necked flask. After addition, the reaction mixture was reacted overnight at room temperature. LC-MS was used to confirm that the reaction of raw material II was complete. The mixture was filtered, and the residue was washed with a small amount of acetonitrile. The filtrate and washings were combined and concentrated to dryness under reduced pressure. The residue was purified by normal-phase preparative liquid chromatography to obtain 0.62 g of compound I-1 (oily substance), with a yield of 88%. 1H-NMR (400MHz, Chloroform-d3): δ7.95(d,J=8.4Hz,1H),7.63(dt,J=7.7,1.3Hz,1H),7.42(d,J=8.0Hz,1H),7.36(t,J=2.1Hz,1H),7.32(q,J=4.8Hz,1 H),7.23-7.14(m,2H),7.12(d,J=1.7Hz,1H),5.51(p,J=6.7Hz,1H),2.89(d ,J=4.8Hz,3H),2.19-2.05(m,4H),2.04-1.91(m,5H),1.55(d,J=6.5Hz,3H). MS:447.21[M+H + ].

[0081] Add starter II (0.5 g, 1.6 mmol, 1.0 eq), 15 mL acetonitrile, 3-hydroxybenzamide (0.53 g, 2.6 mmol, 1.6 eq), and cesium carbonate (0.65 g, 4.7 mmol, 3 eq) sequentially to a dry single-necked flask. After addition, the reaction mixture was allowed to react overnight at room temperature. LC-MS was used to confirm that starter II had reacted completely. The mixture was filtered, and the filtrate and washings were combined with a small amount of pre-washed residue. The mixture was concentrated to dryness under reduced pressure. The residue was then purified by normal-phase preparative liquid chromatography to obtain 0.58 g of compound I-2 (oily substance), with a yield of 84%. 1 H-NMR (400MHz, Chloroform-d3): δ8.00(d,J=8.4Hz,1H),7.67(dt,J=7.8,1.3Hz,1H),7.48(t,J=7.9Hz,1H),7.44(t,J=2.1Hz,1H),7.25 (ddd,J=10.1,8.4,2.3Hz,2H),7.18(d,J=1.7Hz,1H),5.57(p,J=6.7Hz,1H),2.24-2.08(m,4H),2.08-1.95(m,4H),1.59(d,J=6.6Hz,3H). MS:433.32[M+H + ].

[0082] The mass spectrometry data in the above spectral characterization are consistent with the high-resolution mass spectrometry test data in Examples 1 and 2.

[0083] I-1 purity test

[0084] The purity of compound I-1 was determined by HPLC using the following conditions and instruments.

[0085] Table 3: HPLC detection conditions for AST-3424 metabolites I-1 / I-2

[0086] An appropriate amount of sample I-1 was dissolved in acetonitrile to prepare a solution with a concentration of approximately 1 mg / ml. The solution was then injected for testing. The results are shown in Figure 10. The HPLC purity of compound I-1 prepared above is 99.93%, which indicates that compound I-1 prepared above has extremely high chemical purity and is suitable as a standard in the quality control process of AST-3424 active pharmaceutical ingredient / formulation.

[0087] In fact, the same test I-2 yielded similar results, as shown in Figure 11: the HPLC purity of the compound I-2 prepared above is 99.96%, which indicates that the compound I-2 prepared above has extremely high chemical purity and is suitable as a standard in the quality control process of AST-3424 active pharmaceutical ingredient / formulation.

[0088] Further research revealed that the HPLC detection conditions described in Table 3 above showed high resolution for the detection of AST-3424 and its metabolites I-1 / I-2. This indicates that the HPLC detection method is suitable for use as a method for detecting AST-3424 and its metabolites I-1 / I-2 in the quality control process of AST-3424 raw materials / preparations.

[0089] Example 4: In vitro cancer cell proliferation activity assay of AST-3424 metabolites I-1 / I-2

[0090] 1) Add 100 μL of H460 cell suspension to each well of a 96-well plate, with a cell density of 2000 cells / well.

[0091] 2) The cells were cultured overnight at 37°C in a 5% CO2 incubator.

[0092] 3) Compound treatment

[0093] Monotherapy: 24 hours after cell seeding, add 99.5 μL of growth medium to each well. Add 0.5 μL of different concentrations of the test compound AST-3424 or its metabolites I-1 / I-2, gently shake to ensure thorough mixing, and then place in a 37°C, 5% CO2 incubator.

[0094] Combined treatment (pretreatment with AKR1C3 enzyme inhibitor AST-3021 for 2 h, followed by co-treatment with AST-3424 or its metabolites I-1 / I-2 for 72 h): 24 h after cell seeding, add 99 μL of growth medium to each well. Add 0.5 μL of the corresponding concentration of AST-3021, gently shake to ensure homogeneity, and incubate for 2 h. Then add 0.5 μL of different concentrations of the compound, gently shake to ensure homogeneity, and incubate at 37°C in a 5% CO2 incubator.

[0095] 4) Place the cell plate in an incubator for 72 hours.

[0096] 5) Place the cell test plate at room temperature for 30 minutes to equilibrate, and discard 100 μL of culture medium from each well.

[0097] 6) Add 25 μL of CTG reagent to each well, shake with a rapid shaker for 2 minutes, and let stand at room temperature in the dark for 30 minutes.

[0098] 7) Use a multi-functional microplate reader to read the chemiluminescence signal value. The reading time is 1000ms.

[0099] 8) Calculate ICs using GraphPad Prism 5 software 50 The IC of the compound is obtained using the following nonlinear fitting formula. 50 The (half-maximal inhibitory concentration) values ​​are shown in Table 4 below.

[0100] Table 4: Results of in vitro assay of AST-3424 and its metabolites I-1 / I-2 inhibiting the proliferation of AKR1C3-dependent cancer cells in vitro.

[0101] The cytotoxicity of the AST-3424 demonomethyl metabolite I-1 was not significantly different from that of AST-3424, but AKR1C3 was more selective: the activation fold of I-1 was 443.06-fold, while that of AST-3424 was 230.19-fold.

[0102] AST-3424 dedimethyl metabolite I-2 is 8 times less cytotoxic than AST-3424, IC50. 50 =2.57nM, and the activation selectivity of AKR1C3 is significantly lower than that of AST-3424, and the IC after the addition of AST-3021 is... 50 The value is only 43.42 times that of AST-A098 alone.

[0103] The metabolites I-1 / I-2 of AST-3424 are similar to AST-3424, both being DNA alkylating agents activated by the AKR1C3 enzyme and exhibiting selective cell proliferation inhibition dependent on the AKR1C3 enzyme.

Claims

1. The demethylated metabolite of AST-3424 of formula I-2, 2. The use of the I-2 compound according to claim 1 in the preparation of a medicament for treating tumors and cancer.

3. A pharmaceutical composition comprising the I-2 compound of claim 1, further comprising pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is used to treat tumors or cancer.

5. The pharmaceutical composition according to claim 4 or the application according to claim 2, wherein the tumor or cancer is selected from liver cancer or leukemia.

6. Uses of compounds I-1 and I-2 as standards or reference substances:

7. Uses of compounds I-1 and I-2 in the quality control of formulations / active pharmaceutical ingredients containing AST-3424:

8. The use according to claim 7, wherein, The formulation containing AST-3424 is a concentrated solution of AST-3424 for injection, and the active pharmaceutical ingredient containing AST-3424 is an ethanol solution with a mass percentage of 30-40%.

9. A method for preparing compounds I-1 and I-2, characterized in that, Prepared by contact reaction of AST-3424 with human cells:

10. The preparation method according to claim 9, characterized in that, The human cells mentioned include human liver cells.

11. A method for preparing compounds I-1 and I-2, characterized in that, Prepared by contact reaction of AST-3424 with human liver microsomes:

12. A method for preparing compounds I-1 and I-2, characterized in that... Compound II is reacted with the corresponding 3-hydroxy-N-methylbenzamide / 3-hydroxybenzamide in acetonitrile solvent under the action of Cs2CO3 to prepare compounds of formula I-1 and I-2 above:

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