Application of pyrrolidine [3, 4-d] pyrimidine derivative in preparation of PARPs / ATR multi-target inhibitor

Abstract

The invention discloses an application of a pyrrolidine [3, 4-d] pyrimidine derivative in preparation of PARPs / ATR multi-target inhibition. The invention provides preparation of a pyrrolidine [3, 4-d] pyrimidine derivative with a structure as shown in a formula (I) as a PARPs / ATR multi-target inhibitor, a pharmaceutical composition and a hydrate containing the derivative as shown in the formula (I), and isotope derivatives, chiral isomers, allosteres, different salts, prodrugs, preparations and the like of the compounds. The invention also provides a preparation method of the pyrrolidine [3, 4-d] pyrimidine derivative as a PARPs / ATR multi-target inhibitor and application of the compounds in treatment of solid tumors and hematoma. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of pyrrolidine [3,4-d] pyrimidine derivatives in the preparation of PARPs / ATR multi-target inhibition. Background Art

[0002] The PARP (PolyADP-Ribose Polymerase) protein family consists of 17 members, whose function is to catalyze the poly(ADP-ribosyl)ation of proteins and transfer ADP-ribose to substrate proteins. According to the number of ADP-ribose transferred, members of the PARP family are further divided into monoPARP and polyPARP. Among them, PARP1 / 2, which we are most familiar with, belongs to polyPARP, and another member of the family that is relatively hotly studied, PARP7, belongs to monoPARP. PARP1 and PARP2 have 60% sequence homology in the catalytic domain, so the drugs currently on the market are also PARP1 / 2 dual-target inhibitors. PARP7 inhibitors have a dual mechanism of action of directly inhibiting tumor growth and enhancing anti-tumor immunity. Currently, only one inhibitor, RBN-2397, is in clinical research.

[0003] ATR inhibitors were once considered one of the most promising synthetic lethal targets after PARP inhibitors, and are new therapeutic drugs for patients with ATM mutations. ATR stands for "ataxia telangiectasia mutated gene Rad3-associated kinase", which belongs to the serine-threonine kinase of the phosphatidylinositol 3-kinase (PIKK) family. After activation, ATR kinase can regulate cell biological processes through various signals, including cell cycle arrest, inhibition of replication origin, promotion of deoxynucleotide synthesis, initiation of replication forks, and repair of DNA double-strand breaks. The results in ovarian cancer cells with BRCA1 / 2 mutations and acquired resistance to PARP1 inhibitors showed that the combination of olaparib and ATRi AZD6738 can effectively inhibit tumor cell proliferation, relieve G2 / M phase inhibition, and lead to tumor cell apoptosis.

[0004] At present, there are many studies on the combined use of ATR-related inhibitors and PARP inhibitors for tumor treatment, but no single molecule has been found to simultaneously target PARPs and ATR. The in vitro test results of the compounds involved in the present invention show that this type of structure simultaneously targets PARP1, PARP2 and PARP7. It is a kind of pan-PARP inhibitor. At the same time, these compounds also have good inhibitory ability against ATR kinase. These compounds have good anti-tumor proliferation ability and have not been reported yet. Summary of the invention

[0005] The first object of the present invention is to address the deficiencies of the prior art and provide an application of pyrrolidine [3,4-d] pyrimidine derivatives in the preparation of PARPs / ATR multi-target inhibitors.

[0006] Use of a pyrrolidine [3,4-d] pyrimidine derivative, or its optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate in the preparation of a PARPs / ATR multi-target inhibitor, wherein the structure of the derivative is shown in formula (I):

[0007]

[0008] in:

[0009] R l and R 2 are independently selected from hydrogen, alkyl, hydroxy, aryl, heteroaryl, cycloalkyl or heterocycle;

[0010] R 3 Selected from the following excerpts:

[0011]

[0012] Where m = 0 or 1, n = 0 or 1; R 4 and R 5 are independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, cyano, halogen or NR 6 , R 6 is selected from hydrogen, chlorine, alkyl, cycloalkyl, alkoxy, amino or halogen; R 4 , R 5 and R 6 wherein the alkyl, cycloalkyl and alkoxy groups are each independently substituted by one or more substituents selected from halogen, hydroxy, carboxyl, amino, nitro, cyano, C1-C6 acylamino, C1-C6 acyloxy, C1-C6 alkoxy, aryloxy, alkylthio, C1-C6 alkyl, C1-C6 acyl, C6-C10 aryl, C3-C8 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, heterocyclyl, heteroaryl, methylenedioxy, urea, thiol, azido, carbonyl, alkylsulfonyl, sulfamoyl, dialkylsulfamoyl and alkylsulfinyl;

[0013] As a preference, R l , R 2 is H or methyl;

[0014]

[0015] Preferably, a pyrrolidine [3,4-d] pyrimidine derivative having PARPs and ATR activity is any one of compounds 1 to 15 shown in the following structures:

[0016]

[0017] The second object of the present invention is to provide a PARPs / ATR multi-target inhibitor, including a pyrrolidine [3,4-d] pyrimidine derivative having a structural formula as shown in formula (I), or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof.

[0018] The third object of the present invention is to provide a method for preparing a pyrrolidine [3,4-d] pyrimidine derivative, which is specifically as follows:

[0019]

[0020] The raw materials a1 and a2 undergo substitution reaction to obtain an intermediate a3; a3 undergoes Suzuki coupling reaction or Buchwald-Hartwig reaction to obtain an intermediate a6; a6 removes the boc protecting group to obtain a7, and a7 and the intermediate a10 undergo acid-ammonia condensation to obtain a compound represented by formula (I), namely the pyrrolidine [3,4-d] pyrimidine derivative.

[0021] The preparation method of the compound of the general formula (I) of the present invention is specifically described above, but these specific methods do not constitute any limitation to the present invention. The compound of the general formula (I) described above can be synthesized using standard synthesis techniques or known techniques combined with the methods described herein. In addition, the solvent, temperature and other reaction conditions mentioned herein can be changed. The starting materials used for the synthesis of the compound can be obtained by synthesis or from commercial sources. At the same time, the compounds described herein and other related compounds with different substituents can be synthesized using known techniques and raw materials.

[0022] On the other hand, the compounds described herein are prepared according to methods known in the art. However, the conditions of the methods, such as reactants, solvents, bases, the amount of the compounds used, reaction temperature, reaction time, etc., are not limited to the following explanations. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily performed by those skilled in the art to which the present invention belongs.

[0023] The fourth object of the present invention is to provide a pyrrolidine [3,4-d] pyrimidine derivative as shown in formula (I), or its optical isomers, racemates, single enantiomers, possible diastereomers, or its pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, solvates for use in the preparation of drugs for treating or preventing tumors.

[0024] The fifth object of the present invention is to provide an anti-tumor drug containing a safe and effective amount of the pyrrolidine [3,4-d] pyrimidine derivative having PARPs and ATR activities, or its optical isomers, racemates, single enantiomers, possible diastereomers, or its pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, and solvates.

[0025] Preferably, the anti-tumor drug may further include a pharmacologically acceptable salt and a pharmacologically acceptable excipient or carrier.

[0026] Preferably, in the application and the anti-tumor drug, the tumor includes solid tumors and hematological tumors.

[0027] Since the compounds of the present invention have the activity of inhibiting the proliferation of various tumor cell lines, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate various diseases, including various cancers.

[0028] The "safe and effective amount" of the present invention means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 5-1000 mg of the compound of the present invention per dose. Preferably, the "one dose" is one capsule or tablet.

[0029] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0030] The administration method of the compound or pharmaceutical composition of the present invention is not particularly limited. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and topical administration.

[0031] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (c) humectants, such as glycerol; (d) disintegrators, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates and sodium carbonate; (e) solubilizers, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0032] Solid dosage forms, such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.

[0033] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0034] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0035] Suspensions, in addition to the active compounds, may contain suspending agents, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0036] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0037] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0038] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0039] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 5000 mg, preferably 5 to 2000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0040] Compared with the prior art, the present invention has the following main advantages:

[0041] The present invention provides a pyrrolidine [3,4-d] pyrimidine derivative with PARPs / ATR multi-target inhibition, which is synthesized by a simple method and has excellent biological activity, filling the gap of the current single molecule dual-targeting PARPSs and ATR. The pyrrolidine [3,4-d] pyrimidine derivative with PARPs and ATR activity in the present invention is expected to become an anti-tumor drug candidate for treating various cancers, such as solid tumors and blood tumors. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples where no specific conditions are indicated are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0043] Example 1: Preparation of Compound 1

[0044]

[0045] Step 1: 1a (2000.0 mg, 6.89 mmol, 1.00 equiv), (R)-3-Methylmorpholine (767.0 mg, 7.58 mmol, 1.10 equiv) and DIPEA (1780.0 mg, 13.79 mmol, 2.00 equiv) were added to DMF (10 mL), and the mixed solution was stirred at 50 ° C for 12 hours. After the reaction was completed by TLC, the reaction solution was cooled to room temperature, and water (100 mL) was added to quench the reaction. The resulting solution was extracted with EtOAc (3×50 mL). The combined organic layer was purified by Na 2 SO 4 Dry, filter to remove the desiccant, and concentrate under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: petroleum ether = 1:1). The collected fractions were combined and concentrated to obtain an oily substance, and a light yellow solid 1b (2300.0 mg, yield 85.2%) was obtained. LCMS m / z = 355.8 [M+H] + .

[0046] Step 2: 1b (2200.0 mg, 6.20 mmol, 1.00 equiv), 1c (2260.0 mg, 9.29 mmol, 1.50 equiv) and K 3 PO 4 (3940.0 mg, 18.59 mmol, 3.00 equiv) was added to DMF (250 mL) and H 2 In a mixed solvent of O, N 2 The oxygen was removed for 10 min, and then Pd(PPh 3 ) 4 (716.1 mg, 0.62 mmol, 0.10 equivalent), the mixed solution was stirred at 100 ° C for 3 hours under nitrogen protection. After the reaction was complete as detected by TLC, the reaction solution was cooled to room temperature, water (100 mL) was added to quench the reaction, and the resulting solution was extracted with EtOAc (3×50 mL). The combined organic layer was purified by Na 2 SO 4 Dry, filter to remove the desiccant, and concentrate under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: petroleum ether = 1:1). The collected fractions were combined and concentrated to obtain a light yellow solid, and a light yellow solid 1d (2300.0 mg, yield 87.4%) was obtained. LCMS m / z = 436.5 [M+H] + .

[0047] Step 3: Add HCl·dioxane (5 mL, 4 M) dropwise to a solution of 1d (2300.0 mg, 5.28 mmol, 1.00 equiv) in MeOH (10 mL) at room temperature. Stir the resulting solution at room temperature for 4 hours. After the reaction was completed, a large amount of yellow solid was found to precipitate by TLC monitoring. The yellow solid was then filtered to obtain a yellow solid 1e (1950.0 mg, yield 99.2%). LCMS m / z=336.4[M+H] + .

[0048] Step 4: To a solution of 1e (100 mg, 0.27 mmol, 1.00 equiv) and 1f (80.2 mg, 2.7 mmol, 1.00 equiv) in DMF (5 mL) was added HOBt (47.2 mg, 0.35 mmol, 1.30 equiv) and EDCI (134.0 mg, 2.60 mmol, 1.00 equiv) at room temperature. DIPEA (139.0 mg, 1.08 mmol, 4.00 equiv) was then added, and the reaction was stirred at room temperature for 4 hours. Water (100 mL) was added to quench the reaction, and the resulting solution was extracted with EtOAc (3×50 mL). The combined organic layers were purified by Na 2 O 4 3 O 4 6 0 4 0 0 4 0 0 2 SO 4 The residue was dried, filtered to remove the desiccant, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column to obtain a white solid 1 (73.0 mg, yield 44.1%). 1 H NMR (500 MHz, DMSO-d 6 )δ12.61(s,1H),11.38–11.08(m,1H),8.27(ddd,J=7.9,3.6,1.4Hz,1H),8.09–7.97(m,2H),7.97–7.7 9(m,2H),7.59–7.46(m,3H),7.44(dt,J=7.8,2.8Hz,1H),7.37–7.24(m,2H),7.17(q,J=7.7Hz,1H),5. 11–4.94(m,1H),4.89–4.64(m,1H),4.59–4.42(m,2H),4.36(d,J=9.5Hz,2H),4.17(d,J=12.7Hz,1H), 4.00(dd,J=11.3,3.5Hz,1H),3.88–3.62(m,2H),3.61–3.38(m,2H),1.24(dd,J=81.0,6.7Hz,3H).LCMS m / z=616.7[M+H] + .

[0049] Example 2: Preparation of Compound 2

[0050]

[0051] Step 1: 2a (220.0 mg, 0.62 mmol, 1.00 equiv), 2b (226.0 mg, 0.93 mmol, 1.50 equiv) and K 3 PO 4 (394.0 mg, 1.86 mmol, 3.00 equiv) was added to DMF (20 mL) and H 2 In a mixed solvent of O, N 2 The oxygen was removed for 10 min, and then Pd(PPh 3 ) 4 The mixed solution was stirred at 100°C for 3 hours under nitrogen protection. After the reaction was complete as detected by TLC, the reaction solution was cooled and filtered, and then the reaction was quenched by adding water (100 mL). The resulting solution was extracted with EtOAc (3×50 mL). The combined organic layer was purified by Na 2 SO 4 Dry, filter to remove the desiccant, and concentrate under reduced pressure. The residue was chromatographed on a silica gel column (ethyl acetate: petroleum ether = 1:1). The collected fractions were combined and concentrated to obtain a light yellow solid, and a light yellow solid 2c (243.0 mg, yield 89.8%) was obtained. LCMS m / z = 437.5 [M+H] + .

[0052] Step 2: HCl·dioxane (5 mL, 4 M) was added dropwise to a solution of 2c (243.0 mg, 5.28 mmol, 1.00 equiv) in MeOH (10 mL) at room temperature. The resulting solution was stirred at room temperature for 4 hours. After the reaction was completed, a large amount of yellow solid was found to precipitate, which was then filtered to obtain a yellow solid 2d (208.0 mg, yield 99.9%). LCMS m / z=337.4[M+H] + .

[0053] Step 3: To a solution of 2d (100 mg, 0.27 mmol, 1.00 equiv) and 2e (80.8 mg, 2.7 mmol, 1.00 equiv) in DMF (5 mL) was added HOBt (47.2 mg, 0.35 mmol, 1.30 equiv) and EDCI (134.0 mg, 2.60 mmol, 1.00 equiv) at room temperature. DIPEA (139.0 mg, 1.08 mmol, 4.00 equiv) was then added, and the reaction was stirred at room temperature for 4 hours. The reaction was then quenched by adding water (50 mL), and the resulting solution was extracted with EtOAc (3×50 mL). The combined organic layers were purified by Na 2 SO 4Dry, filter to remove the desiccant, and concentrate under reduced pressure. The residue was chromatographed on a silica gel column to obtain a white solid 2 (83.0 mg, yield 50%). LCMS m / z = 617.7 [M+H] + .

[0054] Example 3: Preparation of Compound 3

[0055]

[0056] Referring to the synthesis method of compound 2, in the first step, compound 3a (220.0 mg, 0.62 mmol, 1.00 equivalent) was reacted with compound 3b (226.0 mg, 0.93 mmol, 1.50 equivalent) to obtain yellow solid compound 3c (190.0 mg, yield 70.6%) LCMS m / z = 436.5 [M+H] + ; Step 2 3c (190.0 mg, 0.44 mmol, 1.00 equivalent) was detert-butyloxycarbonylated with dioxane hydrochloride solution to obtain a crude yellow solid compound 3d (145.0 mg, yield 89.3%) LCMS m / z = 336.4 [M+H] + Step 3 Compound 3d (145.0 mg, 0.39 mmol, 1.00 equivalent) and compound 3e (200.6 mg, 0.39 mmol, 1.00 equivalent) were condensed with acid and ammonia to obtain white solid 3 (175.0 mg, yield 72.9%) LCMS m / z = 616.7 [M+H] + ;

[0057] Example 4: Preparation of Compound 4

[0058]

[0059] Referring to the synthesis method of compound 2, in the first step, compound 4a (500.0 mg, 1.41 mmol, 1.00 equivalent) was reacted with compound 4b (417.0 mg, 2.82 mmol, 2.00 equivalent) to obtain yellow solid compound 4c (123.0 mg, yield 20.1%) LCMS m / z = 336.4 [M+H] + LCMS m / z=437.5[M+H] + Step 2: 4c (123.0 mg, 0.28 mmol, 1.00 equivalent) was detert-butyloxycarbonylated with dioxane hydrochloride solution to obtain a crude yellow solid compound 4d (103.0 mg, yield 99.3%). LCMS m / z = 337.4 [M+H] +Step 3 Compound 4d (103.0 mg, 0.28 mmol, 1.00 equivalent) and compound 4e (82.6 mg, 0.28 mmol, 1.00 equivalent) were condensed with acid and ammonia to obtain white solid 4 (75.0 mg, yield 44.1%) LCMS m / z = 617.7 [M+H] + ;

[0060] Example 5: Preparation of Compound 5

[0061]

[0062] Referring to the synthesis method of compound 2, the first step was to obtain yellow solid compound 5c (100 mg, yield 25.2%) by substitution reaction. LCMS m / z = 451.5 [M+H] + ; In the second step, the tert-butyloxycarbonyl group was removed from the dioxane hydrochloride solution to obtain a crude yellow solid compound 5d (73 mg, yield 86.9%). LCMS m / z = 351.4 [M+H] + ; In the third step, compound 5d and compound 5e were condensed by acid-ammonia to obtain white solid 5 (23.0 g, yield 27.6%) LCMS m / z = 631.7 [M+H] + ; 1 H NMR (500 MHz, DMSO-d 6 )δ12.61(d,J=3.2Hz,1H),8.30–8.23(m,1H),8.07–7.98(m,2H),7.93–7.87(m,1H),7.83(dt,J=15. 2,7.6Hz,1H),7.62–7.56(m,1H),7.50(dd,J=7.3,5.0Hz,2H),7.34–7.20(m,3H),5.14–4.97(m,2H), 4.86–4.71(m,1H),4.49(s,2H),4.36(d,J=6.6Hz,2H),4.03–3.96(m,1H),3.77(d,J=11.5Hz,1H),3. 71–3.62(m,1H),3.58–3.50(m,1H),2.79(d,J=6.5Hz,3H),1.33(d,J=6.8Hz,2H),0.89–0.76(m,3H).

[0063] Example 6: Test of the inhibitory activity of compounds on PARP1, PARP2 and PARP7 enzymes

[0064] 1. Experimental Materials and Instruments

[0065] Experimental materials: PARP1 (BPS, Cat. No. 80501), PARP2 (BPS, Cat. No. 80502), PARP7 (BPS,

[0066] Cat.No.80527), Histone (Active Motif, Cat.No.81167), SuperSignal ELISAFemto Substrate (THERMO PIERCE, Cat.No.37074), Biotin-NAD+ (R&D, Cat.No.6573), Strep-HRP (Thermo Pierce, Cat.No.21127)

[0067] Instrument: SpectraMax Paradigm multi-function microplate reader;

[0068] 2. Experimental Methods

[0069] Prepare histone-coated 384-well plate, add 25 μL histone solution to each well, incubate overnight at 4°C; prepare PBST buffer, blocking buffer, and detection buffer; wash histone-coated 384-well plate 3 times with PBST buffer. Block with 50 μL blocking buffer at room temperature for 1 hour. Wash plate 3 times with PBST buffer. Prepare 2000x compound in source plate. Transfer 50nl of compound from source plate to 96-well middle plate with 19.95ul detection buffer. 1000 rpm, 1min. Transfer 5μL DMSO / compound to each well. Enzyme reaction: incubate enzyme mix at 25°C for 10min. Add 10μL enzyme mix (desired minimum control), incubate compound at room temperature for 10min, add 10μL detection buffer. Add 10μL 2.5x biotin-nad+ to each well, incubate at 25°C for 90min. Wash plate 3 times with PBST buffer.

[0070] Probe: Add 25 μL of Stre-HRP. Incubate at room temperature for 1 hour and wash the plate 3 times with PBS buffer. Add 25 μL of Quantitative Enhancer Mix. Incubate for 10 minutes. Add 2.5 μL of Quantitative Red Stop Solution and shake the plate for 10-30 seconds to stop peroxidase activity.

[0071] Data processing:

[0072] The data were fitted in Excel using equation (1) to obtain inhibition values.

[0073] Equation (1): Inh%=(Max-Signal) / (Max-Min)*100.

[0074] Fit the data in XL-Fit using equation (2) to obtain IC 50 value.

[0075] Equation (2): Y = Bottom + (Top - Bottom) / (1 + (IC 50 / X)*HillSlope)

[0076] Where Y is the inhibition percentage and X is the compound concentration

[0077] 3. Experimental results

[0078] The inhibitory effects of some target compounds on PARP1 enzyme were determined according to the above experimental method, and the results are shown in Table 1.

[0079] Table 1 Half-maximal inhibitory concentration of target compounds on PARP1 enzyme

[0080]

[0081]

[0082] Note: + indicates compound IC 50 >50nM

[0083] ++ indicates compound 50nM>IC 50 >5nM

[0084] +++ indicates compound IC 50 <5nM

[0085] Conclusion: The representative compounds of the present invention can effectively inhibit the enzyme activities of PARP1, PARP2 and PARP7.

[0086] Example 15: Test of the ATR enzyme inhibitory activity of compounds

[0087] 1. Experimental Materials and Instruments

[0088] Experimental materials: ATR (BPS, Cat. No. 50051); Tris buffer; ATR (BPS, Cat. No. 50006); 384-well plate (Perkin Elmer, Cat. No. 6007279); DMSO (Coolaber);

[0089] Instrument: SpectraMax Paradigm multi-function microplate reader;

[0090] 2. Experimental Methods

[0091] Prepare 1x Assay Buffer: Prepare 1x Assay Buffer (modified Tris buffer); Compound serial dilution: Transfer compounds to assay plate by Echo in 100% DMSO, DMSO content does not exceed 1%; Prepare enzyme solution: Prepare enzyme solution in 1x Assay Buffer; Prepare substrate solution: Add trypsin and acetylated peptide substrate to 1x Assay Buffer to make substrate solution; Transfer 15 μL enzyme solution to assay plate or for low control transfer 15 μL 1x Assay Buffer; Incubate at room temperature for 15 minutes; Add 10 μL substrate solution to each well to start the reaction; Read plate on Paradigm with excitation wavelength of 355 nm and emission wavelength of 460 nm; Curve Fitting: Fit data in Excel using equation (1) to obtain inhibition values ​​Equation (1): Inh% = (Max-Signal) / (Max-Min)*100 Fit data in XL-Fit using equation (2) to obtain IC 50 Value equation (2): Y = bottom + (top - bottom) / (1 + (IC 50 / X)*HillSlope). Where Y is the inhibition percentage and X is the compound concentration.

[0092] 3. Experimental results

[0093] The inhibitory effects of some target compounds on ATR enzyme were determined according to the above experimental method. The results are shown in Table 2.

[0094] Table 2 Half-maximal inhibitory concentration of target compounds on ATR enzyme

[0095] Compound No. <![CDATA[ATRIC 50 (nM)]]> 1 +++ 2 + 3 ++ 4 +++ 5 ++ AZ20 +++

[0096] Note: + indicates compound IC 50 >50.0nM

[0097] ++ indicates compound 50.0nM>IC 50 >5.0nM

[0098] +++ indicates compound IC 50 <5nM

[0099] Conclusion: The representative compounds of the present invention can effectively inhibit ATR enzyme activity.

[0100] Example 16: Test of the inhibitory activity of some compounds on tumor cells

[0101] 1. Cell lines and cell culture.

[0102] Human triple-negative breast cancer cells (MDA-MB-231) and colorectal cancer cells (LoVo) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. The cells were cultured in modified RPMI medium supplemented with 10% (vol / vol) fetal bovine serum, 50 μg / mL penicillin, and 50 μg / mL streptomycin. All cell lines were kept at 37°C with 5% CO 2 Incubate in a humidified atmosphere.

[0103] 2. Cell viability assay.

[0104] The cells were seeded into two 384-well white-walled tissue culture treated plates at the previously optimized seeding density and placed in a 5% CO 2 Incubator overnight. After 5 days of drug treatment, the cell growth viability was detected using the CellTiter-Glo luminescent activity detector (Promega). Cell viability was measured using Cell TiterGlo (CTG, Promega, G7573) immediately after administration (day 0) and after 6 days of incubation. Relative viability was calculated by normalizing the raw luminescence counts with cells treated with DMSO control (Ctrl.). Graphpad / Prism 8 software was used to calculate the half-maximal inhibitory concentration (IC50) value and fit the sigmoid dose-response curve.

[0105] Table 3 Half inhibitory concentration of target compounds on tumor cells

[0106]

[0107] Note: + indicates compound IC 50 >10.0μM

[0108] ++ indicates compound 10.0μM>IC 50 >1.0μM

[0109] +++ indicates compound IC 50 <1μM

[0110] Conclusion: The representative compounds of the present invention can effectively inhibit MDA-MB-231 cells and LoVo cells.

Claims

1. Use of a pyrrolidine [3,4-d] pyrimidine derivative, or its optical isomer, racemate, single enantiomer, possible diastereomer, or its pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate in the preparation of PARPs / ATR multi-target inhibitors, characterized in that: The structural formula of the pyrrolidine [3,4-d] pyrimidine derivative is shown in formula (I): in: R l and R 2 are independently selected from hydrogen, alkyl, hydroxy, aryl, heteroaryl, cycloalkyl or heterocycle; R 3 Selected from the following excerpts: Where m = 0 or 1, n = 0 or 1; R 4 and R 5 are independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, cyano, halogen or NR 6 , R 6 is selected from hydrogen, chlorine, alkyl, cycloalkyl, alkoxy, amino or halogen; R 4 , R 5 and R 6 The alkyl, cycloalkyl and alkoxy groups are each independently substituted by one or more substituents selected from halogen, hydroxy, carboxyl, amino, nitro, cyano, C1-C6 acylamino, C1-C6 acyloxy, C1-C6 alkoxy, aryloxy, alkylthio, C1-C6 alkyl, C1-C6 acyl, C6-C10 aryl, C3-C8 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, heterocyclyl, heteroaryl, methylenedioxy, urea, thiol, azido, carbonyl, alkylsulfonyl, sulfamoyl, dialkylsulfamoyl and alkylsulfinyl.

2. The use according to claim 1, characterized in that: In formula (I): R l , R 2 is H or methyl; R 3 for 3. The use according to claim 2, characterized in that: The chemical structural formula of the pyrrolidine [3,4-d] pyrimidine derivative is any one of the following formulas 1 to 15:

4. A PARPs / ATR multi-target inhibitor, characterized in that: The invention comprises a pyrrolidine [3,4-d] pyrimidine derivative having a structural formula as shown in formula (I), or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof.

5. A method for preparing a pyrrolidine [3,4-d] pyrimidine derivative, characterized in that: The method is specifically as follows: The raw materials a1 and a2 undergo substitution reaction to obtain an intermediate a3; a3 undergoes Suzuki coupling reaction or Buchwald-Hartwig reaction to obtain an intermediate a6; a6 removes the boc protecting group to obtain a7, and a7 and the intermediate a10 undergo acid-ammonia condensation to obtain a compound represented by formula (I), namely the pyrrolidine [3,4-d] pyrimidine derivative.

6. Use of a pyrrolidine [3,4-d] pyrimidine derivative, or its optical isomer, racemate, single enantiomer, possible diastereomer, or its pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate in the preparation of an anti-tumor drug, characterized in that: The structural formula of the pyrrolidine [3,4-d] pyrimidine derivative is shown in formula (I): in: R l and R 2 are independently selected from hydrogen, alkyl, hydroxy, aryl, heteroaryl, cycloalkyl or heterocycle; R 3 Selected from the following excerpts: wherein m=0 or 1, n=0 or 1; R4 and R5 are independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, cyano, halogen or NR6, and R6 is selected from hydrogen, chlorine, alkyl, cycloalkyl, alkoxy, amino or halogen; the alkyl, cycloalkyl and alkoxy in R4, R5 and R6 are independently substituted by one or more substituents selected from halogen, hydroxyl, carboxyl, amino, nitro, cyano, C1-C6 acylamino, C1-C6 acyloxy, C1-C6 alkoxy, aryloxy, alkylthio, C1-C6 alkyl, C1-C6 acyl, C6-C10 aryl, C3-C8 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, heterocyclyl, heteroaryl, methylenedioxy, ureido, thiol, azido, carbonyl, alkylsulfonyl, sulfamoyl, dialkylsulfamoyl and alkylsulfinyl.

7. The use according to claim 6, characterized in that: The tumors include solid tumors and hematological tumors.

8. An anti-tumor drug, characterized in that: Contains a safe and effective amount of a pyrrolidine [3,4-d] pyrimidine derivative having a structural formula as shown in formula (I), or its optical isomers, racemates, single enantiomers, possible diastereomers, or its pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, and solvates.

9. The anti-tumor drug according to claim 8, characterized in that: It also includes pharmacologically acceptable salts and pharmacologically acceptable excipients or carriers.

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