KRAS-G12D protein inhibitor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a KRAS-G12D protein inhibitor as well as a preparation method and application thereof. The protein inhibitor comprises a compound I and pinocembrin, the mass ratio of the compound I to the pinocembrin is 1: (0.03-0.05). Wherein the compound I can be used for directly targeting and blocking the interaction between KRAS G12D and CRAF, and shows strong inhibitory activity on KRAS G12D mutation tumors on biochemical and cellular levels. Further research finds that the compound I and pinocembrin are combined for use to achieve a synergistic tumor inhibition effect, so that the use dosage of the compound I is remarkably reduced on the premise of maintaining the same curative effect, and a new scientific basis is provided for subsequent development of a low-toxicity and high-efficiency tumor treatment scheme.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a KRAS-G12D protein inhibitor and a preparation method and application thereof. BACKGROUND

[0002] Rat sarcoma viral oncogene homolog (RAS) is the first confirmed proto-oncogene in humans, belonging to the small GTPase in the RAS superfamily. RAS gene mainly includes three subtypes: Harvey rat sarcoma viral oncogene homolog (HRAS), Kirsten rat sarcoma viral oncogene homolog (KRAS) and neuroblastoma RAS viral oncogene homolog (NRAS). Among them, KRAS gene mutation accounts for 85% of all RAS gene mutations, and is known as the "king of targets" in cancer targeted therapy.

[0003] KRAS protein plays a key role in cell signal transduction, and its function is similar to that of a "molecular switch". When KRAS binds to guanosine triphosphate (GTP), it is activated, and then transmits signals to promote cell proliferation, differentiation and survival to the downstream; when GTP is hydrolyzed to guanosine diphosphate (GDP), KRAS is inactivated, and signal transduction is terminated. This cycle of activation and inactivation is precisely regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs): GEFs promote the exchange of GDP and GTP, activating KRAS; GAPs accelerate GTP hydrolysis, making it inactivated. However, once KRAS mutates, especially the 12th glycine is replaced by other amino acids (such as G12C or G12D), it will cause its continuous abnormal activation. This kind of mutation will make KRAS "stuck in the open state", and it is difficult to hydrolyze GTP to GDP, so it continuously transmits cell growth signals, and finally induces tumors. At present, a variety of covalent inhibitors have been successfully developed, which can irreversibly bind to KRAS G12C mutant protein and inhibit its activity.

[0004] The successful development of KRAS G12C covalent inhibitors is attributed to its unique chemical basis in structure: the cysteine residue side chain introduced by this mutation exposes a highly reactive thiol group under physiological pH conditions, which can undergo a regioselective Michael addition reaction with an alpha, beta-unsaturated amide warhead to form a thermodynamically stable, kinetically irreversible thioether covalent bond. In contrast, the G12D mutation replaces glycine with aspartic acid, whose side chain carboxylic acid group is fully deprotonated in the cell, both negatively charged causing electrostatic repulsion and lacking sufficient nucleophilicity, making it difficult for conventional acrylamide warheads to effectively bind. Even if the carboxylic acid is modified into an electrophilic center, it may cause non-specific modification of the widely existing carboxylic acid groups in the proteome, triggering the risk of systemic toxicity. Therefore, the covalent inhibition strategy applicable to G12C is not structurally chemically feasible for the G12D mutation.

[0005] Currently, inhibitors targeting KRAS G12D are mainly divided into two types: polypeptide and non-polypeptide. Polypeptide inhibitors generally have poor cell membrane penetration and poor drugability, so current research focuses more on the development of non-polypeptide small molecule KRAS G12D inhibitors.

[0006] Hepicidin, the chemical name of which is beta-D-allopyranose p-formyl phenyl glycoside, is an effective component isolated from the fruits of the mountain longan plant in Yunnan, China. It is a safe and effective sedative and hypnotic drug, and long-term use has not been found to be toxic or have other adverse reactions, and the drug efficacy is stable. Given its good biological activity and safety, structural modification and transformation using hepicidin as a lead compound have important research value for expanding its medical application range or developing new compounds with stronger activity and higher bioavailability.

[0007] Punicalin (PNC) is a hydrolysable tannin that can be isolated from pomegranate peel, pomegranate leaves and pomegranate juice, and has various pharmacological activities such as antioxidant, anti-liver injury, antibacterial, antiviral and anti-inflammatory activities. However, there is no research report that punicalin has the effect of enhancing the anti-tumor effect of KRAS G12D inhibitors. SUMMARY

[0008] In order to overcome the shortcomings of the prior art, one of the purposes of the present application is to provide a KRAS-G12D protein inhibitor capable of inhibiting the growth of KRAS G12D mutant CT26 and Panc1 tumor cells.

[0009] One of the purposes of the present application is achieved by using the following technical solutions: A KRAS-G12D protein inhibitor comprises Compound I and punicalin; the mass ratio of Compound I to punicalin is 1: (0.03-0.05); the structural formula of Compound I is as follows: .

[0010] Further, the preparation process of the compound I comprises the following steps: (1) adding methyl thieno[2,3-d]pyrimidine-2-carboxylate into ethanol, then adding hydrazine hydrate, and performing the reaction under heating; after the reaction is completed, the reaction solution is cooled, and after filtration, washing, and drying, the intermediate 1 is obtained; The structural formula of the intermediate 1 is as follows: (2) adding trichloroisocyanuric acid into ethanol and stirring until uniform, then adding β-D-allopyranose p-formylphenyl glycoside, and after stirring for 10-15 min, adding the intermediate 1 to perform the reaction; after the reaction is completed, purification is performed to obtain the compound I.

[0011] Further, the molar ratio of the methyl thieno[2,3-d]pyrimidine-2-carboxylate and the hydrazine hydrate in step (1) is 1: (1.2-1.6).

[0012] Further, the temperature of the heating in step (1) is 70-80°C; and the reaction time is 6-8 h.

[0013] Further, the molar ratio of the intermediate 1, the trichloroisocyanuric acid, and the β-D-allopyranose p-formylphenyl glycoside in step (2) is 1: (1-1.25): (1-1.5).

[0014] Further, the reaction time in step (2) is 3-5 h.

[0015] The second object of the present application is to provide a preparation method of the KRAS-G12D protein inhibitor, and the steps are simple.

[0016] The preparation method of the KRAS-G12D protein inhibitor comprises the following steps: According to the mass ratio, the compound I and pinocembrin are weighed and uniformly mixed.

[0017] The third object of the present application is to provide the application of the KRAS-G12D protein inhibitor, and the prospect is broad.

[0018] The third object of the present application is achieved by the following technical scheme: The application of the KRAS-G12D protein inhibitor is in the preparation of an antitumor drug.

[0019] Further, the tumor is a tumor related to KRAS-G12D mutation.

[0020] Further, the tumor is colon cancer, pancreatic cancer, kidney cancer, lung cancer, gastric cancer.

[0021] Compared with the prior art, the present application has the beneficial effects that: The compound I obtained by the present application can directly target and block the interaction of KRAS G12D and CRAF, and exhibit strong inhibitory activity on KRAS G12D mutant tumors in biochemical and cellular levels. Further research shows that the combination of compound I and pinocembrin has a synergistic tumor inhibition effect, which significantly reduces the dosage of compound I under the premise of maintaining the same therapeutic effect, and provides a new scientific basis for the subsequent development of low-toxicity and high-efficiency tumor treatment programs. DETAILED DESCRIPTION

[0022] The present application will be further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features between them can be combined in any manner to form new embodiments. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, such as those not specifically mentioned, are conventional products obtained through commercial channels.

[0023] (I) Preparation Example Preparation Example 1 The present preparation example provides a compound I, and the preparation process is as follows: (1) Thieno[2,3-d]pyrimidine-2-carboxylic acid methyl ester (CAS: 2111518-43-3, 5 mmol) was added to 15 mL of ethanol, and hydrazine hydrate (7 mmol) was added. After stirring uniformly, the temperature was raised to 75°C and reacted for 7h. After the reaction was completed, the reaction liquid was cooled to room temperature, the filter cake was washed with water, and dried to obtain intermediate 1. The nuclear magnetic resonance and mass spectrum results of intermediate 1 are as follows: 1 HNMR (C7H6N4OS, DMSO-d6, 400MHz) δ: 10.05 (s, 1H), 9.57 (s, 1H), 7.32 (d, 1H), 7.14 (d, 1H), 4.52 (s, 2H). MS m / z 194.03 (M), Found: 194.05. (2) Trichloroisocyanuric acid (TCCA, 2.2 mmol) was added to 20 mL of ethanol and stirred uniformly. Then β-D-allopyranose p-formyl phenyl glycoside (CAS: 80154-34-3, 2.5 mmol) was added, stirred for 12 min, and then intermediate 1 (2 mmol) was added. The reaction was carried out at room temperature for 4h. After the reaction was completed, the reaction liquid was concentrated and dispersed with water, and the aqueous phase was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, separated and purified by chromatography (v石油醚 : v 乙酸乙酯 = 5: 1), to obtain compound I, the nuclear magnetic and mass spectrum results of compound I are as follows: 1 HNMR(C 20 H 18 N4O7S, DMSO-d6, 400MHz) δ: 9.28 (s, 1H), 8.03 (d, 2H), 7.32 (d, 1H), 7.14 (d, 1H), 7.05 (d, 2H), 5.80 (d, 1H), 4.85 (s, 1H), 4.76 (s, 1H), 4.71 (s, 1H), 4.21 (t, 1H), 3.95 (s, 1H), 3.72 (t, 1H), 3.61-3.55 (m, 4H). MS m / z 459.09 (M+1), Found: 459.09. Preparation Example 2 The present preparation example provides a compound I, the preparation process is as follows: (1) thieno [2, 3-d] pyrimidine-2-carboxylic acid methyl ester (5mmol) is added to 15mL of ethanol, then hydrazine hydrate (6mmol) is added, after stirring uniformly, the temperature is raised to 70℃ and reacted for 8h; after the reaction is completed, the reaction liquid is cooled to room temperature, the reaction liquid is filtered and the filter cake is washed with water, dried to obtain intermediate 1, and the nuclear magnetic and mass spectrum results of intermediate 1 are the same as those of preparation example 1.

[0024] (2) trichloroisocyanuric acid (2mmol) is added to 20mL of ethanol and stirred uniformly, then β-D-allopyranose p-formyl phenyl glycoside (2mmol) is added, stirred for 10min, then intermediate 1 (2mmol) is added, and reacted for 3h at room temperature; after the reaction is completed, the reaction liquid is concentrated and dispersed with water, the aqueous phase is extracted with ethyl acetate, the organic phase is dried with anhydrous sodium sulfate, chromatographically separated and purified (v 石油醚 : v 乙酸乙酯 = 5: 1), to obtain compound I, the nuclear magnetic and mass spectrum results of compound I are as follows:

[0025] Preparation Example 3 The present preparation example provides a compound I, the preparation process is as follows: (1) thieno [2, 3-d] pyrimidine-2-carboxylic acid methyl ester (5mmol) is added to 15mL of ethanol, then hydrazine hydrate (8mmol) is added, after stirring uniformly, the temperature is raised to 80℃ and reacted for 6h; after the reaction is completed, the reaction liquid is cooled to room temperature, the reaction liquid is filtered and the filter cake is washed with water, dried to obtain intermediate 1, and the nuclear magnetic and mass spectrum results of intermediate 1 are the same as those of preparation example 1.

[0026] (2) trichloroisocyanuric acid (2.5 mmol) was added into 20 mL 75% ethanol solution and stirred uniformly, then β-D-allopyranose p-tolyl glycoside (3 mmol) was added, after stirring for 15 min, intermediate 1 (2 mmol) was added, and the reaction was carried out at room temperature for 5 h; after the reaction was completed, the reaction solution was concentrated and dispersed with water, and the water phase was extracted with ethyl acetate, and the organic phase was dried with anhydrous sodium sulfate and separated and purified by chromatography (v 石油醚 : v 乙酸乙酯 = 5: 1) to obtain compound I, and the nuclear magnetic resonance and mass spectrum results of compound I were the same as those of preparation example 1.

[0027] (II) Examples Example 1 The present example provides a KRAS-G12D protein inhibitor, which comprises compound I of preparation example 1 and pinocembrin, wherein the mass ratio of compound I to pinocembrin is 1:0.04.

[0028] The present example also provides a preparation method of the KRAS-G12D protein inhibitor, which specifically comprises the following steps: According to the above mass ratio, compound I and pinocembrin were weighed and uniformly mixed.

[0029] Example 2 The present example provides a KRAS-G12D protein inhibitor, which comprises compound I of preparation example 2 and pinocembrin, wherein the mass ratio of compound I to pinocembrin is 1:0.03.

[0030] The present example also provides a preparation method of the KRAS-G12D protein inhibitor, which specifically comprises the following steps: According to the above mass ratio, compound I and pinocembrin were weighed and uniformly mixed.

[0031] Example 3 The present example provides a KRAS-G12D protein inhibitor, which comprises compound I of preparation example 3 and pinocembrin, wherein the mass ratio of compound I to pinocembrin is 1:0.05.

[0032] The present example also provides a preparation method of the KRAS-G12D protein inhibitor, which specifically comprises the following steps: According to the above mass ratio, compound I and pinocembrin were weighed and uniformly mixed.

[0033] Comparative Example 1 The difference between the present comparative example and example 1 is that pinocembrin is omitted.

[0034] (III) Experimental Examples Experimental Example 1 The KRAS-G12D / CRAF binding kit was used to detect the biochemical activity of the compound I prepared in Preparation Example 1. The required buffer and reaction solution were prepared according to the instructions of the kit. The experimental process was as follows: The compound I was dissolved in DMSO to prepare a 10 mM stock solution, and then diluted with buffer by 3 times gradient. 0.1 μL of the gradient-diluted compound I was added to a 384-well plate, and 5 μL of Tag2-KRASG12D>P was added to each well, and then centrifuged at 1000 rpm for 1 min. 5 μL of Tag1-CRAF was added to each well, and then centrifuged at 1000 rpm for 1 min, and then incubated at 25℃ for 15 min. 10 μL of a mixture of anti-Tag1-TB3+ and anti-Tag2-XL665 was added to each well, and then centrifuged at 1000 rpm for 1 min, and then incubated at 4℃ for 3 h. The fluorescence intensity ratio at 665 / 615 nm was read by a microplate reader. The log value of the concentration of the compound I was taken as the abscissa, and the 665 / 615 nm ratio was taken as the ordinate. The data were analyzed and the IC 50 value was calculated. The results are shown in Table 1.

[0035] Table 1 As can be seen from Table 1, the compound I obtained by the present application has good inhibitory ability for the interaction between KRAS-G12D and CRAF protein.

[0036] Experimental Example 2 This experimental example investigates the inhibitory effect of the preparations obtained in Examples 1-3 and Comparative Example 1 on the proliferation of mouse Panc-1 cells and AGS. The experimental process was as follows: The preparations obtained in Examples 1-3 and Comparative Example 1 were added to DMSO according to the concentration of compound I, and a 20 mM stock solution was prepared, and then diluted with culture medium to prepare 5 μM, 10 μM, 25 μM, 50 μM and 100 μM test samples. 0.1% DMSO was used as a control group.

[0037] Panc-1 cells (containing KRAS G12D mutation) in the logarithmic growth phase were inoculated into a 96-well cell culture plate at a density of 1×10 4 cells / well, and cultured overnight in a 37℃, 5% CO2 incubator, and then the test samples were added for continuous culture for 48 h. After the culture was completed, 50 μL of CellTiter-Glo detection solution was added to each well, shaken for 5 min, and then allowed to stand for 10 min. The fluorescence intensity value of each well of the sample was read by a microplate reader, and the IC 50 value of the KRAS-G12D protein inhibitor for inhibiting cell proliferation was calculated. The results are shown in Table 2.

[0038] AGS cells in logarithmic growth phase (containing KRAS G12D mutation) were used at a dose of 1×10⁻⁶. 4 Seeds were planted at a density of cells / well into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator. Then, the test samples were added and cultured for another 48 hours. After incubation, 50 μL of CellTiter-Glo assay solution was added to each well, shaken for 5 min, and allowed to stand for 10 min. The fluorescence intensity values ​​of each well were read using a microplate reader, and the IC50 of the KRAS-G12D protein inhibitor on cell proliferation was calculated. 50 The values ​​are shown in Table 2.

[0039] Table 2 As shown in Table 2, the KRAS-G12D protein inhibitor obtained in this invention can inhibit the growth of Panc-1 and AGS tumor cells with KRAS G12D mutations.

[0040] Experimental Example 3 This experiment investigates the effects of the formulations obtained in Examples 1-3 and Comparative Example 1 on tumor growth in a mouse xenograft model of pancreatic cancer (KPC).

[0041] Take the logarithmic growth phase of Panc-1 (KRAS) G12D Cell suspension (1×10) 7 0.2 mL of the drug (number of tumor cells / mL) was subcutaneously injected into BALB / c nude mice. After excluding mice that failed to develop tumors, the tumors in the remaining mice were inoculated until the average tumor volume reached 200 mm². 3 Administer medication at the designated time. Select 40 animals and divide them into 5 groups of 8 animals each. Administer medication as follows.

[0042] The administration method is as follows: Model group: administered physiological saline by gavage for 28 consecutive days; Example 1 group: Intraperitoneal injection of the preparation of Example 1, 20 mg / kg / day, for 28 days; Example 2 group: Intraperitoneal injection of the preparation of Example 2, 20 mg / kg / day, for 28 days; Example 3 group: Intraperitoneal injection of the preparation of Example 3, 20 mg / kg / day, for 28 days; Comparative Example 1: The preparation of Comparative Example 1 was administered intraperitoneally at a dose of 20 mg / kg / day for 28 days.

[0043] Forty-eight hours after the last administration, nude mice were euthanized by cervical dislocation, the transplanted tumors were removed, and the tumor weight of each mouse was measured to calculate the tumor inhibition rate. Tumor inhibition rate (%) = (1 - tumor weight of experimental group / tumor weight of model group) × 100%. The results are shown in Table 3.

[0044] Table 3 As can be seen from the above, compared with Comparative Example 1, the KRAS-G12D protein inhibitor obtained by the present application can significantly inhibit the growth of pancreatic cancer tumor. The above results show that the use of compound I and pinocembrin in combination can inhibit the growth of pancreatic cancer tumor.

[0045] In summary, the compound I obtained by the present application can directly target and block the interaction of KRAS G12D and CRAF, and exhibit strong inhibitory activity on KRAS G12D mutant tumor in both biochemical and cellular levels. Further research shows that the combination of compound I and pinocembrin has a synergistic tumor inhibition effect, which significantly reduces the dosage of compound I under the premise of maintaining the same therapeutic effect, and provides a new scientific basis for the subsequent development of low-toxicity and high-efficiency tumor treatment scheme.

[0046] The above embodiments are only preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and replacements made by those skilled in the art on the basis of the present application shall fall within the scope of protection of the present application.

Claims

1. A KRAS-G12D protein inhibitor, characterized in that, The compound I is pinocembrin; a mass ratio of the compound I to pinocembrin is 1: (0.03-0.05); The compound I has the following structural formula: 。 2. The KRAS-G12D protein inhibitor of claim 1, wherein, The preparation process of the compound I comprises the following steps: (1) adding methyl thieno[2,3-d]pyrimidine-2-carboxylate into ethanol, then adding hydrazine hydrate, and performing reaction under heating; after the reaction is completed, cooling the reaction solution, and performing filtration, washing, and drying to obtain an intermediate 1; The intermediate 1 has the following structural formula: (2) adding trichloroisocyanuric acid into ethanol and stirring until uniform, then adding β-D-allopyranose p-methoxyphenyl glycoside, stirring for 10-15 min, and then adding the intermediate 1 to perform reaction; after the reaction is completed, performing purification to obtain the compound I.

3. The KRAS-G12D protein inhibitor of claim 2, wherein, In step (1), a molar ratio of the methyl thieno[2,3-d]pyrimidine-2-carboxylate to the hydrazine hydrate is 1: (1.2-1.6).

4. The KRAS-G12D protein inhibitor of claim 2, wherein, In step (1), a heating temperature is 70-80 ℃; and a reaction time is 6-8 h.

5. The KRAS-G12D protein inhibitor of claim 2, wherein, In step (2), a molar ratio of the intermediate 1 to the trichloroisocyanuric acid to the β-D-allopyranose p-methoxyphenyl glycoside is 1: (1-1.25): (1-1.5).

6. The KRAS-G12D protein inhibitor of claim 2, wherein, In step (2), a reaction time is 3-5 h.

7. A method of preparing a KRAS-G12D protein inhibitor according to any one of claims 1 to 6, characterized in that, The method comprises the following steps: According to the mass ratio, the compound I and pinocembrin are weighed and uniformly mixed, and then the mixture is obtained.

8. Use of a KRAS-G12D protein inhibitor according to any one of claims 1 to 6, characterized in that, The application in preparation of an antitumor drug.

9. The KRAS-G12D protein inhibitor for use according to claim 8, characterized in that, The tumor is a tumor related to KRAS-G12D mutation.

10. The KRAS-G12D protein inhibitor for use according to claim 9, characterized in that, The tumor is colon cancer, pancreatic cancer, renal cancer, lung cancer, or gastric cancer.

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