CN107722013A - Denitrification purine compound and its pharmaceutical composition, preparation method and purposes - Google Patents

Abstract

The present invention relates to a class of deazapurine compounds with the following general formula 1 structure, a preparation method thereof, a pharmaceutical composition comprising the compound, and uses thereof. The compound has selective B-Raf V600E mutant tumor cell inhibitory activity, so the deazapurine compound or its pharmaceutical composition of the present invention can be used to prepare a drug for treating tumor or cancer.

Description

Deazapurine compound and its pharmaceutical composition, preparation method and application

technical field

The present invention relates to the field of medicinal chemistry, in particular to a class of deazapurine compounds, a preparation method thereof and a pharmaceutical composition comprising the compound, and the preparation of the deazapurine compound or its pharmaceutical composition for the treatment of tumors or cancers use in medicines.

Background technique

Cancer is a serious threat to human health and is one of the leading causes of death in the world. According to the statistics of the World Health Organization, the number of people who died of cancer in the world in 2008 reached 7.6 million (accounting for about 13% of all deaths). As the environment continues to deteriorate, the number of people dying of cancer worldwide will continue to increase. It is predicted that the number of people dying of cancer will reach 11 million by 2030.

The RAF kinase has been linked to cancer since the first discovery of C-Raf in 1983, then called RAF1. As of 2002, B-Raf mutations have been identified in a variety of tumors, including malignant melanoma (50-60%), thyroid cancer (45-59%), colorectal cancer (4.7-14.8%), lung cancer (3 %), almost all papillary craniopharyngiomas, typical hairy cell leukemias, cases of metanephric adenomas and some other tumors. Among cancers with B-Raf mutations, V600E mutations are the most common, accounting for about 80-90%.

Metastatic melanoma is a very serious disease among malignant tumors, which is extremely insensitive to chemoradiotherapy. The effective rate of single-drug Dacarbazine, the gold standard of chemotherapy drugs, is only 7.5%-12.2%, and the progression-free survival (PFS) is less than 2 months. Cutaneous melanoma is the leading cause of dermatological death, and patients with advanced disease have a poor prognosis. In 2012, 232,000 people worldwide were diagnosed with melanoma, and 55,000 people died from the disease.

At present, there are two main types of inhibitors for B-Raf. One is a broad-spectrum RAF inhibitor. , Platelet-derived growth factor receptor (PDGFR), multiple kinases including receptor tyrosine kinase (KIT) have inhibitory effects. The other is a selective inhibitor against B-RafV600E mutant, typical representatives are Vemurafenib and Dabrafenib. Targeted drugs that have been clinically approved for the treatment of melanoma with B-Raf V600E mutation have good curative effect on melanoma, and can significantly prolong progression-free survival and overall survival compared with chemotherapy and radiotherapy.

The success of B-Raf inhibitors indicates that molecular markers can be used to subdivide patients, clarify the target drug users, and then strengthen the clinical effect of targeted therapy. Although B-Raf mutations exist in many kinds of tumors, such as colorectal cancer, thyroid cancer, non-small cell lung cancer, malignant glioma, etc., B-Raf inhibitors are only in a small part of malignant melanoma with B-Raf mutations. effective in cancer patients. The targeted therapy of B-Raf has just started, and there are still many challenges in the future. The compound of this patent application has a novel structure, and has better B-Raf V600E mutant enzyme inhibitory activity, and also shows good inhibitory activity on B-RafV600E mutant melanoma and colon cancer cells, and has been further developed into an anti- Oncology Drug Potential.

Contents of the invention

One object of the present invention is to provide a deazapurine compound represented by general formula 1 or a pharmaceutically acceptable salt thereof. The deazapurine compound or its pharmaceutically acceptable salt can inhibit the growth of various tumor cells, so it can be developed into a drug for treating tumor or cancer.

Another object of the present invention is to provide the use of the deazapurine compound represented by the general formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumor or cancer.

Another object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of one or more deazapurine compounds represented by general formula 1 or pharmaceutically acceptable salts thereof.

Another object of the present invention is to provide the use of the above pharmaceutical composition in the preparation of medicines for treating tumors or cancers.

The present invention provides a deazapurine compound represented by general formula 1 or a pharmaceutically acceptable salt thereof:

in,

R can be amino or halogen ^; preferably amino or chlorine;

R can be ^hydrogen , halogen, substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₂ -C ₆ alkenyl, substituted or unsubstituted C ₁ -C ₆ alkylaryl, or Substituted or unsubstituted C ₆ -C ₁₂ aryl; preferably hydrogen, substituted or unsubstituted C ₁ -C ₆ alkyl, allyl or nitrobenzyl; more preferably, hydrogen, methyl, Nitrobenzyl or allyl; most preferably, can be hydrogen, methyl, 4-nitrobenzyl or 1-allyl; wherein the substituents can be selected from halogen, C ₁ -C ₆ alkyl , cyano, nitro, amino (NH ₂ ), hydroxyl, carboxyl and mercapto, preferably selected from halogen, C ₁ -C ₄ alkyl and nitro, said halogen comprising fluorine, chlorine, bromine or iodine;

R ³ can be hydrogen, hydroxyl, amino, mercapto, halogen, C ₁ -C ₆ alkyl, C ₆ -C ₁₂ aryl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylamino, C ₁ - C ₆ alkylthio, C ₆ -C ₁₂ aryl ether, C ₆ -C ₁₂ arylamino or C ₆ -C ₁₂ arylthio; preferably hydrogen, halogen, C ₁ -C ₆ alkyl, C ₁ - C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₆ -C ₁₂ aryl ether or C ₆ -C ₁₂ arylthio; further preferably hydrogen or halogen, most preferably fluorine;

R ⁴ can be hydrogen, substituted or unsubstituted C ₁ -C ₆ alkyl acyl, substituted or unsubstituted C ₆ -C ₁₂ aryl acyl, substituted or unsubstituted C ₁ -C ₆ alkylsulfonyl, substituted or unsubstituted C ₆ -C ₁₂ arylsulfinyl, substituted or unsubstituted C ₁ -C ₆ alkylsulfinyl, substituted or unsubstituted C ₆ -C ₁₂ arylsulfinyl, substituted or unsubstituted C ₁ -C ₆ alkyl phosphoryl or substituted or unsubstituted C ₆ -C ₁₂ aryl phosphoryl; preferably hydrogen, substituted or unsubstituted C ₁ -C ₆ alkyl acyl, substituted or unsubstituted C ₆ -C ₁₂ aryl acyl, substituted or unsubstituted C ₁ -C ₆ alkylsulfonyl or substituted or unsubstituted C ₆ -C ₁₂ arylsulfonyl; more preferably, can be hydrogen, substituted or unsubstituted C ₁ -C ₆ alkylsulfonyl, in particular, C ₆ -C ₁₂ arylsulfonyl substituted with one or more halogens; more preferably, hydrogen, propanesulfonyl or difluorophenylsulfonyl ; Most preferably hydrogen, propane-1-sulfonyl or 2,6-difluorophenylsulfonyl; wherein the substituents can be selected from halogen, C ₁ -C ₆ alkyl, cyano, nitro , amino, hydroxyl, carboxyl and mercapto, preferably selected from halogen, C ₁ -C ₄ alkyl and nitro, said halogen comprising fluorine, chlorine, bromine or iodine;

n can be 1, 2, 3 or 4; preferably, n is 2 or 3; most preferably n is 2.

In the present invention, the term "C ₆ -C ₁₂ aryl" refers to an aromatic ring group with 6 to 12 carbon atoms that does not contain heteroatoms on the aromatic ring, such as: phenyl, naphthyl, biphenyl Wait. When there is a substituent on the aromatic ring, the substituent is selected from halogen, C ₁ -C ₆ alkyl, cyano, nitro, amino, hydroxyl, carboxyl and mercapto, preferably selected from halogen, C ₁ -C ₄ alkane base and nitro. Examples of substituted aryl groups include, for example, p-methylphenyl, p-methoxyphenyl, o-methoxyphenyl, p-nitrophenyl, and the like.

In the present invention, the term "C ₁ -C ₆ alkyl" refers to a straight chain or branched chain alkyl group having 1 to 6 carbon atoms in the main chain, including without limitation methyl, ethyl, propyl, Isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.; preferably ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl base.

Preferably, the compound represented by the general formula 1 of the present invention is a deazapurine compound represented by one of the following general formulas:

Wherein, R ² , R ³ , R ⁴ and n are the same as defined in Formula 1.

In the present invention, the deazapurine compound is more preferably selected from the following compounds:

In yet another aspect of the present invention, a pharmaceutical composition is provided, which comprises a therapeutically effective amount of one or more deazapurine compounds represented by general formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The drug may further include a pharmaceutically acceptable carrier.

Another aspect of the present invention provides a use of the deazapurine compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the compound in the preparation of a medicament for preventing or treating tumor or cancer.

The present invention also provides a method for preventing or treating tumor or cancer, said method comprising administering a therapeutically effective amount of deazapurine compounds according to the present invention and its pharmaceutically acceptable One or more of the salts, or the drug according to the present invention comprising one or more of the deazapurine compounds and their pharmaceutically acceptable salts according to the present invention in a therapeutically effective amount as active ingredients combination.

In the present invention, non-limiting examples of the tumor or cancer include lung cancer, gastric cancer, esophageal cancer, colon cancer, liver cancer, cervical cancer, breast cancer, leukemia, malignant lymphoma, nasopharyngeal cancer, and the like.

Beneficial effect

The present invention designs and synthesizes a class of deazapurine compounds, which are found to have selective B-Raf V600E mutant tumor cell inhibitory activity through activity tests on various tumor cells, indicating that this type of compound has potential for treating B-Raf Use of a V600E mutant tumor or cancer disease.

detailed description

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited.

Example of compound preparation

The sample data were determined by the following instruments: the melting point was determined by SGW X-4B melting point apparatus, and the temperature was not corrected; the proton nuclear magnetic resonance spectrum ( ¹ H NMR) was measured by a Bruker Avance III 300 or 400 nuclear magnetic resonance instrument, and the carbon nuclear magnetic resonance spectrum ( ¹³ C NMR ) was measured on a Bruker Avance III 400 or 500 type nuclear magnetic resonance instrument, with TMS as the internal standard, and the chemical shift unit was ppm; the mass spectrum was measured with Finnigan LCQ/DECA and Micromass Ultra Q-TOF (ESI) mass spectrometer; The liquid phase model is Agilent 1260infinity; Jingke WFH-203B three-purpose ultraviolet analyzer is used for color development, and the wavelengths are 254nm and 365nm.

The reverse phase column is YMC GEL ODS-AQ-HG (12nm, S-50μm) YMC Co., Ltd.; column chromatography silica gel (100-200 mesh, 300-400 mesh) is produced by Qingdao Ocean Chemical Factory; TLC silica gel plate HSGF-254 thin-layer chromatography silica gel plate produced for Yantai Chemical Factory, the thickness of the thin-layer chromatography plate used for thin-layer chromatography is 0.2±0.03mm, and the thickness of the thin-layer chromatography pre-prepared plate used for pre-preparation is 0.4-0.5mm ;Petroleum ether (boiling range 60-90°C), dichloromethane, ethyl acetate, and methanol are all analytically pure, provided by Shanghai Titan Technology Co., Ltd. or Sinopharm Chemical Reagent Co., Ltd., unless otherwise specified. , without special treatment.

TLC is thin layer chromatography;

DCM is dichloromethane;

DME is ethylene glycol dimethyl ether;

DMF is dimethylformamide;

DMAP is 4-dimethylaminopyridine;

Boc ₂ O is di-tert-butyl dicarbonate.

Preparation of compound intermediates:

Preparation of 2-amino-3-methyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2304)

Preparation of 2-amino-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2303)

Add bromoacetaldehyde dimethyl acetal (9.4mL, 1.0 equivalent) to water (40mL), add hydrochloric acid (2mL) under stirring, heat to 90°C for 1 hour, cool in ice water after the reaction, add sodium acetate trihydrate (12 g, 1.1 equivalents) were stirred to obtain a reaction solution. Then add 2,4-diamino-6-hydroxypyrimidine compound 2302 (10g, 0.080mol) into water (80mL), then add sodium acetate trihydrate (7.6g), then add the aforementioned reaction solution, and heat to 80°C , reacted overnight. TLC detection showed that the reaction was complete. The reaction solution was stirred under ice water for two hours, filtered, and the filter cake was washed with water. The filter cake was dissolved in 95% ethanol as much as possible, dissolved three times, the ethanol phase was combined, and the solution was spin-dried to obtain a purple solid 2303 (4.7 g, yield: 40%).

¹ H NMR (400MHz,DMSO-d ₆ )δ10.98(s,1H),10.24(s,1H),6.61(dd,J=3.4,2.2Hz,1H),6.18(dd,J=3.4,2.2 Hz,1H), 6.06(s,2H).

LRMS (EI): 150[M] ⁺ .

IR(KBr)v 3183, 1629, 1606, 1589 cm ^-1 .

Preparation of 2-amino-3-methyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2304)

Compound 2303 (2.0g, 0.012mol) was dissolved in 0.1M NaOH (147mL, 1.0eq), cooled in ice water, then added dimethyl sulfate (6.3mL, 5.0eq) and stirred at natural temperature. After stirring for 2 hours, 1M NaOH (53mL, 4.0 equivalents) was stirred for 2 hours. After rotary evaporation and spin-drying, methanol was added to dissolve, and the sample was mixed with silica gel. Column chromatography (DCM:MeOH=10:1) gave light yellow solid compound 2304 (0.84g, yield: 38%).

¹ H NMR (400MHz, DMSO-d ₆ )δ10.90(s,1H),6.62(dd,J=3.3,2.3Hz,1H),6.57(s,2H),6.19(dd,J=3.3,2.2 Hz,1H), 3.30(s,3H).

¹ H NMR (400MHz, DMSO-d ₆ +D ₂ O) δ 6.63 (d, J=3.4Hz, 1H), 6.21 (d, J=3.4Hz, 1H), 3.30 (s, 3H).

LRMS(EI): 164[M] ⁺ .

IR (KBr) v 3490, 3351, 1673, 1610, 1573, 1540, 1517, 748 cm ⁻¹ .

Preparation of acetamido deazapurine compound (2306a or 2306b)

Preparation of N-(7-acetyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-yl)acetamide (2305a)

Compound 2303 (10 g, 0.067 mol) was dissolved in acetic anhydride (100 mL), then added acetic acid (10 mL), and heated to 130° C. for 2 hours. The reaction solution was evaporated under reduced pressure to remove acetic anhydride and acetic acid to obtain a brown solid 2305a, which was directly used in the next reaction.

Preparation of N-(7-acetyl-3-methyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-yl)acetamide (2305b)

Compound 2304 (0.84g, 7.3mmol) was dissolved in acetic anhydride (15mL), then added acetic acid (1.5mL), and heated to 130°C for 2 hours. The reaction solution was evaporated under reduced pressure to remove acetic anhydride and acetic acid to obtain a brown solid 2305b, which was directly used in the next reaction.

Preparation of N-(4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-yl)acetamide (2306a)

Compound 2305a was dispersed in methanol (100 mL), ammonia water (5 mL) was added, and stirred for 0.5 hour. TLC detection showed that the reaction was complete. Methanol and ammonia water were distilled off under reduced pressure. The residue was dissolved in methanol as much as possible, filtered, and the filter cake was washed several times with methanol, and the combined filtrate was spin-dried to obtain khaki solid 2306a (6.5 g, two-step yield: 51%).

¹ H NMR (400MHz,DMSO-d ₆ )δ11.68(brs,3H),6.94(d,J=3.4Hz,1H),6.40(d,J=3.4Hz,1H),2.16(s,3H) .

LRMS(EI): 192[M] ⁺ .

Preparation of N-(3-methyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-yl)acetamide (2306b)

Compound 2305b was dissolved in methanol (20 mL), ammonia water (0.5 mL) was added, and stirred for 2 hours. TLC detection showed that the reaction was complete. After methanol and ammonia water were distilled off under reduced pressure, compound 2306b (1.0 g, two-step yield: 95%) was obtained by silica gel column chromatography.

¹ H NMR (400MHz,DMSO-d ₆ )δ11.78(s,1H),10.50(s,1H),7.03(s,1H),6.44(s,1H),3.38(s,3H),2.10( s, 3H).

LRMS(EI):206[M] ⁺ .

Preparation of 2,6-difluoro-3-substituted-benzoyl chloride (2313)

Preparation of 2,6-difluoro-3-nitrobenzoic acid (2308)

2,6-difluorobenzoic acid compound 2307 (2.0g, 13mmol) was dissolved in concentrated nitric acid (20mL, 65%-68%), the above reaction solution was placed in ice water to cool, and then slowly dripped with a dropping funnel Concentrated sulfuric acid (20 mL, 98%) was added, the temperature was raised naturally, and the mixture was stirred for 6 hours. TLC detection showed that the reaction was complete. The reaction solution was slowly poured into ice water, extracted 3 times with ethyl acetate, combined the organic phases, washed 3 times with water, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain the crude compound 2308 (2.6 g, yield: 100%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.39 (td, J=5.6, 9.0 Hz, 1H), 7.49 (td, J=1.6, 9.0 Hz, 1H).

Preparation of methyl 2,6-difluoro-3-nitrobenzoate (2309)

Dissolve 2,6-difluoro-3-nitrobenzoic acid compound 2308 (20.0 g, 0.10 mol) in methanol (200 mL), add concentrated sulfuric acid (10 mL), and heat to reflux overnight. After the reaction solution was rotary evaporated to remove methanol, water and ethyl acetate were added to the residue, and the liquids were separated. The aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated sodium bicarbonate, washed with water, washed with saturated brine, anhydrous sodium sulfate Dry and evaporate to dryness under reduced pressure to obtain light yellow solid compound 2309 (19.0 g, yield: 89%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.46 (td, J=9.0, 5.5 Hz, 1H), 7.53 (td, J=9.1, 1.7 Hz, 1H), 3.95 (s, 3H).

Preparation of methyl 3-amino-2,6-difluorobenzoate (2310)

Iron powder (15.5g, 4.0 equivalents) was added to 100mL of water, 100mL of acetic acid was added under stirring, and 2,6-difluoro-3-nitrobenzoic acid methyl ester compound 2309 (15.0g, 0.069mol) was dissolved in ethanol (100mL), add a small amount of acetic acid to aid dissolution, slowly add to the above mixed solution, heat to 110 ° C, TLC detection shows that the reaction is complete. The reaction solution was evaporated under reduced pressure, ethyl acetate was added to the residue, filtered, and the filtrate was washed with saturated sodium bicarbonate, water, and saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain compound 2310 (13.2 g, yield: 100%), directly used in the next step without purification.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 6.99–6.84 (m, 2H), 5.28 (s, 2H), 3.86 (s, 3H).

Preparation of methyl 2,6-difluoro-3-(N-(propylsulfonyl))benzoate (2311a)

Dissolve 2,6-difluoro-3-aminobenzoic acid methyl ester compound 2310 (3.7g, 0.020mol) in dichloromethane (40mL), then add triethylamine (8.4mL, 3.0 equivalents), ice water cooling Propanylsulfonyl chloride (4.7 mL, 2.1 equivalents) was slowly added under low temperature, stirred at room temperature for 3.5 hours, TLC detection showed that the reaction was complete. The reaction solution was quenched with water, extracted three times with dichloromethane, combined organic phases, washed with 1M hydrochloric acid, washed with saturated aqueous sodium bicarbonate solution, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain oily compound 2311a, without Further purified and directly used in the next reaction.

¹ H NMR (400MHz, CDCl ₃ ) δ7.51(td, J=8.5, 5.5Hz, 1H), 7.07(td, J=8.9, 1.5Hz, 1H), 3.99(s, 3H), 3.66(ddd, J＝13.8, 9.0, 6.6Hz, 2H), 3.51(ddd, J＝13.8, 8.9, 6.7Hz, 2H), 2.03–1.91(m, 4H), 1.12(t, J＝7.4Hz, 2×CH ₃ ).

Preparation of methyl 3-(N-((2,6-difluorophenyl)sulfonyl))-2,6-difluorobenzoate (2311b)

The operation steps refer to the synthesis of compound 2311a.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.03-7.89 (m, 2H), 7.86-7.75 (m, 1H), 7.53-7.37 (m, 5H).

Preparation of 2,6-difluoro-3-(propylsulfonamido)benzoic acid (2312a)

The compound 2311a obtained in the previous step was dissolved in tetrahydrofuran (60 mL), then an aqueous solution (15 mL) of sodium hydroxide (3.2 g, 4.0 equivalents) was added, heated to reflux, and reacted for 2 hours. TLC detection showed that the reaction was complete. After the reaction solution was evaporated to remove the organic solvent under reduced pressure, the aqueous phase was adjusted to pH = 1, extracted three times with ethyl acetate, the organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain compound 2312a (5.3 g , two-step total yield: 94%).

¹ H NMR (300MHz,DMSO-d ₆ )δ14.06(brs,1H),9.73(s,1H),7.53(td,J=9.0,5.8Hz,1H),7.20(td,J=9.1,1.6 Hz, 1H), 3.15–3.01(m, 2H), 1.82–1.64(m, 2H), 0.97(t, J=7.4Hz, 3H).

LRMS (ESI): m/z 278[MH] ^- .

Preparation of 3-(2,6-difluorophenylsulfonylamino)-2,6-difluorobenzoic acid (2312b)

The operation steps refer to the synthesis of compound 2312a.

¹ H NMR (400MHz, DMSO-d ₆ )δ14.06(brs,1H),10.91(brs,1H),7.75(tt,J=8.4,6.1Hz,1H),7.45(td,J=8.9,5.8 Hz, 1H), 7.30 (t, J = 9.0Hz, 2H), 7.21 (td, J = 9.1, 1.5Hz, 1H).

¹³ C NMR (125MHz, DMSO-d ₆ ) δ162.0, 159.1 (dd, J _CF = 257.5, 3.8 Hz), 157.6 (dd, J _CF = 252.2, 5.6 Hz), 154.1 (dd, J _CF = 255.8, 7.4 Hz ), 136.6(t, J _CF ＝11.0Hz), 131.1(d, J _CF ＝10.4Hz), 120.8(dd, J _CF ＝13.4, 3.5Hz), 117.7(t, J _CF ＝16.2Hz), 113.9( dd, J _CF =22.9, 3.6 Hz), 113.3 (d, J _CF =19.4 Hz), 112.9 (dd, J _CF =23.3, 4.4 Hz).

LRMS(ESI):348[MH] ^- .

HRMS(ESI): The theoretical value of C ₁₃ H ₆ O ₄ NF ₄ S is 347.9959[MH] ^- , and the observed value is 347.9956[MH] ^- .

Preparation of 2,6-difluoro-3-(propylsulfonamido)benzoyl chloride (2313a)

Compound 2312a (5.0g, 0.018mol) was dissolved in toluene, then thionyl chloride (3.9mL, 3.0eq) was added, heated to reflux for 3 hours, and the solvent was spin-dried to obtain compound 2313a (5.3g, yield: 100%) , directly used in the next reaction without further purification.

Preparation of 3-(2,6-difluorophenylsulfonamido)-2,6-difluorobenzoyl chloride (2313b)

Compound 2312b (0.70g, 2.0mmol) was dissolved in toluene, then thionyl chloride (0.4mL, 3.0eq) was added, heated to reflux for 3 hours, and the solvent was spin-dried to obtain compound 2313b (0.74g, yield: 100%) , directly used in the next reaction without further purification.

Preparation of 2,6-difluoro-3-nitrobenzoyl chloride (2313c)

2,6-Difluoro-3-nitrobenzoic acid compound 2308 (4.1g, 20mmol) was dissolved in anhydrous dichloromethane (40mL), then DMF (catalytic amount) was added, and then oxalyl chloride (3.4mL , 2.0 equivalents), stirred overnight, and for about 18 hours, dichloromethane and excess oxalyl chloride were distilled off under reduced pressure to obtain yellow oily compound 2313c (4.4 g, yield: 100%), which was directly used in the next step without further purification step response.

Preparation of 2-chloro-3-methyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2318a)

Preparation of 2-chloro-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2315)

Compound 2314 (3.0 g, 0.016 mol) was dissolved in 1M NaOH (120 mL) aqueous solution, heated to 80° C., and reacted overnight. TLC detection showed that the reaction was complete, and then the reaction solution was cooled with ice water, and the pH value was adjusted to about 6 with acetic acid. A solid was precipitated, the filter cake was washed with water and dried to obtain a brown solid 2315 (2.2 g, yield: 82%).

¹ H NMR (400MHz,DMSO-d ₆ )δ12.78(brs,1H),12.03(brs,1H),7.07(t,J=2.8Hz,1H),6.45(t,J=2.6Hz,1H) .

LRMS (ESI): 170 [M+H] ⁺ .

Preparation of tert-butyl 2-chloro-4-oxo-3H-pyrrolo[2,3-d]pyrimidine-7(4H)-carbonate (2316)

Compound 2315 (0.68g, 4.0mmol) was dissolved in redistilled tetrahydrofuran (10mL), triethylamine (0.56mL, 4.0mmol) was added, DMAP (34mg) was added and cooled in an ice bath for 30 minutes, and Boc ₂ O solution in tetrahydrofuran was stirred at room temperature overnight. TLC detection showed that after the reaction was complete, the sample was directly mixed, and silica gel column chromatography gave white solid compound 2316 (0.90 g, yield: 83%).

¹ H NMR (400MHz,DMSO-d ₆ )δ13.37(s,1H),7.45(d,J=3.8Hz,1H),6.63(d,J=3.8Hz,1H),1.59(s,9H) .

LRMS (ESI): 270[M+H] ⁺ , 268[MH]-.

Preparation of tert-butyl 2-chloro-3-methyl-4-oxo-3H-pyrrolo[2,3-d]pyrimidine-7(4H)-carbonate (2317a)

KF preparation method of diatomite dispersion:

Potassium fluoride dihydrate (3.2 g) and diatomaceous earth (2.0 g) were added to water (80 mL), stirred for 3 hours, spin-dried, and dried in a vacuum oven at 55° C. overnight.

Compound 2316 (0.54g, 2.0mmol) was dissolved in acetonitrile (10mL), KF (1.2g, 10mmol) and iodomethane (0.12mL, 2.0mmol) dispersed in diatomaceous earth were added, and stirred overnight at room temperature. Diatomaceous earth was removed by filtration, and silica gel column chromatography (eluent: DCM:MeOH=100:1) after mixing the sample gave compound 2317a (0.33 g, yield 58%).

¹ H NMR (400MHz, CDCl ₃ ) δ7.31(d, J=3.8Hz, 1H), 6.65(d, J=3.8Hz, 1H), 3.75(s, 3H), 1.65(s, 9H).

Preparation of 2-chloro-3-methyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2318a)

Compound 2317a (0.33g, 1.2mmol) was dissolved in dichloromethane (20mL), then concentrated hydrochloric acid (1.5mL) was added, and stirred overnight at room temperature. TLC detection showed that after the reaction was complete, dichloromethane and concentrated hydrochloric acid were distilled off under reduced pressure to obtain a pink solid 2318a (0.20 g, yield: 95%).

¹ H NMR (400MHz, CDCl ₃ ) δ9.42 (brs, 1H), 6.95 (t, J=2.8Hz, 1H), 6.72 (t, J=2.8Hz, 1H), 3.78 (s, 3H).

¹ H NMR (400MHz, DMSO-d ₆ ) δ12.01(s, 1H), 7.08(dd, J=2.4, 3.3Hz, 1H), 6.48(dd, J=2.3, 3.3Hz, 1H), 3.60( s, 3H).

¹ H NMR (400 MHz, DMSO-d ₆ +D ₂ O) δ 7.07 (dd, J=2.4, 3.3 Hz, 1H), 6.51-6.44 (m, 1H), 3.59 (s, 3H).

¹³ C NMR (125 MHz, DMSO-d ₆ ) δ 158.6, 145.7, 143.6, 121.8, 105.6, 102.9, 33.0.

LRMS (ESI): 184[M+H] ⁺ .

HRMS (ESI): C ₇ H ₆ N ₃ ONaCl [M+Na] ⁺ calc. 206.0097, found 206.0092.

IR (KBr) v 3216, 3110, 2925, 1660, 1558, 1490, 1419, 1286, 1219, 1068, 904, 740, 596 cm ^-1 .

Preparation of 2-chloro-3-(4-nitrobenzoyl or allyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2318b or 2318c)

Preparation of 2-chloro-3-(4-nitrobenzoyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2318b)

Compound 2316 (100mg, 0.37mmol) was dissolved in a mixed solvent of anhydrous DME (4mL) and DMF (1mL), stirred under ice water for 10 minutes, then added NaH (17mg, 0.43mmol), stirred for 20 minutes and then added LiBr (64mg, 0.74mmol) was warmed up to room temperature, and p-nitrobenzyl bromide (92mg, 0.43mmol) was added after 15 minutes to be warmed up to 65°C and stirred overnight. After TLC detection showed that the reaction was complete, the reaction solution was added with water, extracted three times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain a waxy solid. The scraper obtained compound 2318b (53 mg, yield: 47%).

¹ H NMR (400MHz, DMSO-d ₆ )δ12.21(s, 1H), 8.22(d, J=8.7Hz, 2H), 7.49(d, J=8.6Hz, 2H), 7.17(t, J= 2.7Hz, 1H), 6.60–6.52(m, 1H), 5.56(s, 2H).

¹ H NMR (400MHz, DMSO-d ₆ +D ₂ O) δ8.21(d, J=8.4Hz, 2H), 7.48(d, J=8.4Hz, 2H), 7.16(d, J=3.3Hz, 1H), 6.55 (d, J=3.3Hz, 1H), 5.56 (s, 2H).

¹³ C NMR (125 MHz, DMSO-d ₆ ) δ 158.5, 147.2, 145.8, 144.7, 143.0, 128.2, 124.3, 122.4, 105.6, 103.3, 48.4.

IR (KBr) v 3224, 1664, 1556, 1515, 1423, 1344, 1197, 1108, 950, 732 cm ^-1 .

LRMS(ESI):303[MH] ^- .

HRMS (ESI) ^{: C13H8O3N4Cl[MH] -} _{calc . 303.0285} , found _303.0280 .

Preparation of 3-allyl-2-chloro-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2318c)

Compound 2316 (0.27g, 1.0mmol) was dissolved in a mixed solvent of anhydrous DME (4mL) and DMF (1mL), stirred under ice water for 10 minutes, then NaH (48mg, 1.2mmol) was added, stirred for 20 minutes and then LiBr (0.17 g, 2.0 mmol) was added and the temperature was raised to room temperature. After 15 minutes, propene bromide (17 μL, 1.2 mmol) was added and the temperature was raised to 65° C. and stirred overnight. After TLC detection showed that the reaction was complete, the reaction solution was added with water, extracted three times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain a waxy solid. The scraper obtained compound 2318c (115 mg, yield: 55%).

¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),7.11(s,1H),6.50(s,1H),5.95(m,1H),5.19(d,J=10.5Hz, 1H), 5.01 (d, J=17.2Hz, 1H), 4.81 (d, J=4.9Hz, 2H).

¹³ C NMR (125 MHz, DMSO-d ₆ ) δ 158.1, 145.7, 143.1, 132.8, 122.0, 117.0, 105.6, 103.1, 47.7.

LRMS (ESI): 210 [M+H] ⁺ .

HRMS (ESI): C ₉ H ₈ N ₃ ONaCl [M+Na] ⁺ calc. 232.0254, found 232.0249.

Preparation of 2-chloro-5 or 6-(2,6-difluoro-3-nitrobenzoyl)-3H-pyrazolo[2,3-d]pyrimidin-4(7H)-one (2319)

Anhydrous aluminum trichloride (42.8g, 0.32mol) was dissolved in nitrobenzene (200mL), stirred and dissolved, and compound 2315 (6.8g, 40mmol) was added under ice water cooling, and after compound 2315 was dissolved, added 2,6-Difluoro-3-nitrobenzoyl chloride (13.3g, 60mmol) was stirred, heated to 90°C, and reacted overnight. The reaction solution was slowly poured into ice water to quench. Extract with ethyl acetate 4 times, combine the organic phases, wash with hydrochloric acid at pH=1, wash with saturated sodium bicarbonate, wash with water, wash with saturated brine, dry over anhydrous sodium sulfate, evaporate to dryness under reduced pressure, add a large amount of petroleum ether to the residue, The upper layer was decanted, methanol was added to the remaining solid, and light brown solid compound 2319a (8.8 g, yield: 62%) was obtained by filtration. After the mother liquor was concentrated, ethyl acetate was added to stir, and filtered to obtain light brown solid compound 2319b (0.6 g, yield: 4%).

2-Chloro-5-(2,6-difluoro-3-nitrobenzoyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2319a)

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.19(s, 1H), 13.10(s, 1H), 8.40(td, J=5.4, 8.8Hz, 1H), 8.07(s, 1H), 7.46( q,J=8.6Hz,1H).

¹ H NMR (400 MHz, DMSO-d ₆ +D ₂ O) δ 8.41–8.28 (m, 1H), 7.98 (s, 1H), 7.44–7.30 (m, 1H).

¹³ C NMR (125MHz, DMSO-d ₆ ) δ179.2, 162.2 (dd, J _CF = 257.4, 7.4 Hz), 158.3, 153.6 (dd, J _CF = 264.0, 9.3 Hz), 150.5, 144.4, 134.4 (d, J _CF = 4.1 Hz), 132.4, 129.2 (d, J _CF = 11.2 Hz), 121.3 (t, J _CF = 23.1 Hz), 119.8, 113.4 (dd, J _CF = 24.0, 2.7 Hz), 103.5.

LRMS(ESI):353[MH] ^- .

HRMS (ESI): C ₁₃ H ₄ O ₄ N ₄ F ₂ Cl [MH] ^- calc. 352.9889, found 352.9880.

IR(KBr) vmax 3224, 3124, 1664, 1556, 1515, 1423, 1344, 1319, 1197, 1108, 950, 829, 732, 590 cm ^-1 .

2-Chloro-6-(2,6-difluoro-3-nitrobenzoyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2319b)

¹ H NMR (400MHz, DMSO-d ₆ )δ13.39(s,1H),13.21(s,1H),8.47(td,J=8.9,6.3Hz,1H),7.54(t,J=8.7Hz, 1H), 7.43(s, 1H).

¹³ C NMR (125MHz, DMSO-d ₆ ) δ175.7, 162.0 (dd, J _CF = 257.8, 6.4 Hz), 159.2, 153.4 (dd, J _CF = 265.0, 8.6 Hz), 151.4, 147.2, 134.9, 131.6, 130.2 (d, J _CF =11.7 Hz), 118.2 (t, J _CF =23.6 Hz), 117.3, 113.7 (dd, J _CF =23.5, 3.2 Hz), 108.5.

LRMS(ESI):353[MH] ^- .

_HRMS (ESI) ^{: C13H4N4O4F2Cl[MH] -} _{calc . 352.9889} , found _352.9882 .

Compound preparation:

N-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-2,4-difluorobenzoyl)- Preparation of 2,6-difluorophenylsulfonamide (2301a)

Anhydrous aluminum trichloride (278mg, 2.3mmol) was dissolved in nitromethane (3mL), after stirring to dissolve, compound 2306a (50mg, 0.29mmol) was added, and after compound 2306a was dissolved, compound 2313b (143mg, 0.44 mmol) stirred, heated to 80-90°C, and reacted overnight. The reaction solution was poured into water, extracted three times with ethyl acetate, the organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and silica gel column chromatography to obtain compound 2301a (21mg, yield: 17%) .

¹ H NMR (400MHz,DMSO-d ₆ )δ12.24(s,1H),10.86(s,2H),7.64(s,1H),7.42–7.30(m,1H),7.27–7.16(m,2H ), 7.14–7.03(m,1H), 6.97(s,1H), 6.80(s,2H).

¹ H NMR (400MHz, DMSO-d ₆ +D ₂ O) δ7.71–7.54(m,1H),7.44–7.30(m,1H),7.27–7.12(m,2H),7.12–7.03(m, 1H), 6.98(s, 1H).

¹³ C NMR (125MHz, DMSO-d ₆ )δ182.,4,175.0,169.8,160.3,160.3,158.2,158.2,158.1,158.1,154.9,154.6,130.9,130.1,120.5,119.5,1168.1,113.0,113 112.3, 112.1, 102.0, 97.3.

LRMS (ESI): 504[M+Na] ⁺ , 480[MH] ^- .

HRMS (ESI): C ₁₉ H ₁₁ O ₄ N ₅ F ₄ NaS [M+Na] ⁺ calc. 504.0360, found 504.0359.

N-(3-(2-Amino-3-methyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-2,4-difluoro Preparation of phenyl)-2,6-difluorophenylsulfonamide (2301b)

Anhydrous aluminum trichloride (518mg, 3.9mmol) was dissolved in nitromethane (5mL), stirred and dissolved, and compound 2306b (100mg, 0.49mmol) was added. After compound 2306b was dissolved, compound 2313b (214mg, 0.59 mmol), heated to 100-105°C, and reacted overnight. The reaction solution was poured into water, extracted three times with ethyl acetate, the organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The crude product of compound 2301b was obtained by scraping, and the crude product was washed with petroleum ether: ethyl acetate = 1:1, and filtered to obtain pure compound 2301b (72 mg, yield: 30%).

¹ H NMR (400MHz,DMSO-d ₆ )δ11.97(s,1H),10.78(s,1H),7.72–7.61(m,1H),7.44–7.31(m,1H),7.28–7.18(m ,2H), 7.17–7.03(m,2H), 7.01–6.86(m,2H), 3.24(s,3H).

¹ H NMR (400MHz,DMSO-d ₆ +D ₂ O)δ7.73–7.62(m,1H),7.44–7.35(m,1H),7.29–7.17(m,2H),7.17–6.88(m, 2H), 3.23(s, 3H).

¹³ C NMR (125MHz, DMSO-d ₆ ) δ179.5, 159.2 (dd, J _CF = 257.0, 3.2 Hz), 157.2, 154.7, 153.6 (dd, J _CF = 250.7, 6.9 Hz), 153.2 (dd, J _CF = 249.2,9.8Hz), 152.6,135.9,130.0,121.4,120.5,120.2(t,J _CF ＝23.9Hz),118.3,113.7(d,J _CF ＝23.1Hz),112.2(d,J _CF ＝24.0Hz) , 111.8 (d, J _CF =22.6 Hz), 96.3, 28.4.

LRMS (ESI): 518 [M+Na] ⁺ .

HRMS (ESI): C ₂₀ H ₁₃ O ₄ N ₅ F ₄ NaS [M+Na] ⁺ calc. 518.0517, found 518.0521.

Preparation of 2-amino-5-(2,6-difluoro-3-nitroformyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2301c)

Anhydrous aluminum trichloride (0.53mg, 4.0mmol) was dissolved in nitromethane (3mL), and after stirring to dissolve, compound 2306a (96mg, 0.50mmol) was added. After compound 2306a was dissolved, compound 2313c (166mg, 0.75 mmol), heated to 80°C and reacted overnight. The reaction solution was poured into water, filtered to obtain a solid, dissolved in methanol, mixed, and subjected to silica gel column chromatography to obtain compound 2301c (50 mg, yield: 30%).

¹ H NMR (400MHz,DMSO-d ₆ )δ12.22(s,1H),10.48(s,1H),8.40–8.29(m,1H),7.65(s,1H),7.45–7.34(m,1H ), 6.45(s,2H).

¹³ C NMR (125MHz, DMSO-d ₆ ) δ 178.6, 162.2 (dd, J _CF = 256.6, 7.8 Hz), 158.0, 154.6, 154.3, 153.6 (dd, J _CF = 263.2, 9.6 Hz), 134.3 (t, J _CF =3.1 Hz), 130.1, 128.7 (d, J _CF =11.4 Hz), 122.0 (t, J _CF =24.4 Hz), 120.1, 113.2 (d, J _CF =26.7 Hz), 97.11.

LRMS(ESI):334[MH] ^- .

_HRMS (ESI) ^{: C13H6O4N5F2[MH] -} _{calc . 334.0388} , found _334.0383 .

Preparation of 2-amino-5-(3-amino-2,6-difluorobenzoyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2301d)

Add reduced iron powder (44mg, 0.79mmol, 4.0 equivalent) to water (2mL), add acetic acid (2mL) under stirring, dissolve compound 2301c (66mg, 0.20mmol) in absolute ethanol (2mL), add a small amount of acetic acid to help dissolve , slowly added to the above reaction solution, heated to 110°C, and reacted for 0.5 hours. After the reaction was detected by TLC, water, ethanol and acetic acid in the reaction solution were distilled off under reduced pressure, the pH was adjusted to weak alkaline with NaHCO ₃ , water was distilled off under reduced pressure, dissolved with methanol, filtered, and repeated twice. The filtrate was sequentially eluted with water, 10%, 20%, and 30% methanol solution on a reverse-phase column to obtain compound 2301d (37 mg, yield: 41%).

¹ H NMR (400MHz,DMSO-d ₆ )δ11.89(s,1H),10.44(s,1H),7.16(s,1H),6.92–6.71(m,2H),6.34(s,2H), 5.09(s,2H).

¹³ C NMR (125MHz, DMSO-d ₆ ) δ180.7, 157.9, 154.7, 154.2, 150.0 (dd, J _CF = 234.1, 6.5 Hz), 146.7 (dd, J _CF = 240.5, 8.0 Hz), 133.5 (d, J _CF = 13.3 Hz), 130.3, 121.1, 119.3 (t, J _CF = 22.3 Hz), 116.5 (t, J _CF = 6.9 Hz), 111.3 (dd, J _CF = 22.1, 2.7 Hz), 97.6.

LRMS(ESI):304[MH] ^- .

_HRMS (ESI) ^{: C13H8O2N5F2[MH] -} _{calc . 304.0646} , found _304.0652 .

N-(3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-2,4-difluorophenyl)propanyl - Preparation of 1-sulfonamide (2301e)

The operation steps refer to the synthesis of compound 2301a.

¹ H NMR (300MHz,DMSO-d ₆ )δ12.07(s,1H),10.45(s,1H),7.45(d,J=12.6Hz,2H),7.12(t,J=8.8Hz,1H) , 6.41 (s, 2H), 3.14–2.92 (m, 2H), 1.82–1.56 (m, 2H), 0.92 (t, J=7.3Hz, 3H).

¹³ C NMR (100MHz, DMSO-d ₆ ) δ179.89, 157.47, 153.97, 153.68, 128.82, 128.46, 128.37, 121.37, 120.25, 119.92, 111.77, 111.53, 96.89, 52.97, 16.810, 12.

LRMS (ESI): 434.2 [M+Na] ⁺ ; 410.1 [MH] ^- .

HRMS (ESI): C ₁₆ H ₁₅ O ₄ N ₅ F ₂ SNa[M+Na] ⁺ Calc.: 434.0711; Found: 434.0692.

N-(3-(2-Amino-3-methyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-2,4-difluoro Preparation of phenyl) propane-1-sulfonamide (2301f)

Dissolve aluminum trichloride (1.2g, 8.6mmol, 10.0 equivalents) into nitrobenzene (5mL), add compound 2306b (0.18g, 0.86mmol) after dissolving, and continue stirring to dissolve, then add compound 2313a ( 0.31g, 1.0mmol, 1.2 equivalents), heated to 100°C and reacted overnight. After TLC detection showed that the reaction was complete, the reaction solution was poured into ice water, and 1M hydrochloric acid was added to adjust the pH to about 1, extracted three times with ethyl acetate, the organic phases were combined, washed with water, washed with saturated saline, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure , silica gel column chromatography to obtain the crude product of compound 2301f, the crude product was stirred with a mixed solvent (petroleum ether: ethyl acetate=1:2, v/v), and filtered to obtain a light yellow solid, namely compound 2301f (0.12g, yield : 33%).

¹ H NMR (400MHz, DMSO-d ₆ ) δ12.01 (brs, 1H), 9.66 (brs, 1H), 7.53–7.41 (m, 2H), 7.13 (t, J=8.9Hz, 1H), 6.91 ( brs,2H), 3.21(s,3H), 3.12–2.99(m,2H), 1.80–1.61(m,2H), 0.89(m,3H).

¹ H NMR (400MHz, DMSO-d ₆ +D ₂ O) δ7.52–7.40 (m, 2H), 7.13 (t, J=8.8Hz, 1H), 3.21 (s, 3H), 3.08–2.97 (m , 2H), 1.76–1.59 (m, 2H), 0.86 (t, J=7.4Hz, 3H).

¹³ C NMR (125MHz, DMSO-d ₆ ) δ180.5, 157.4, 157.0 (dd, J _CF = 246.2, 6.6 Hz), 154.6, 153.4 (dd, J _CF = 248.7, 8.2 Hz), 152.4, 129.2, 129.0 (d , J _CF ＝9.3Hz), 121.6(d, J _CF ＝13.7Hz), 120.7(t, J _CF ＝22.9Hz), 120.6, 112.1(d, J _CF ＝22.1Hz), 96.2, 53.4, 28.3, 17.2 ,12.9.

LRMS (ESI): 426[M+H] ⁺ , 448[M+Na] ⁺ , 424[MH] ^- .

HRMS (ESI): C ₁₇ H ₁₈ O ₄ N ₅ F ₂ S [M+H] ⁺ calc. 426.1042, found 426.1038.

N-(3-(2-chloro-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-2,4-difluorophenyl)-2 , Preparation of 6-difluorophenylsulfonamide (2320a)

Dissolve anhydrous aluminum trichloride (0.32g, 2.3mmol) in nitromethane (3mL), stir to dissolve and add compound 2315 (50mg, 0.29mmol), then add compound 2313b (130mg, 0.35mmol) and stir , heated to 60°C, and reacted overnight. After TLC detection showed that the reaction was complete, the reaction solution was slowly poured into ice water, extracted three times with ethyl acetate, combined the organic phases, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Compound 2320a (60 mg, yield: 41%) was obtained by silica gel column chromatography.

¹ H NMR (400MHz,DMSO-d ₆ )δ13.00(brs,2H),10.84(brs,1H),7.76–7.65(m,1H),7.57(s,1H),7.49–7.39(m,1H ), 7.25 (t, J=9.4Hz, 2H), 7.19 (t, J=9.1Hz, 1H).

LRMS(ESI):499[MH] ^- .

HRMS (ESI) ^{: C19H8N4O4F4SCl[MH] -} _{calc . 498.9891 ,} found _498.9897 .

N-(3-(2-Chloro-3-methyl-4-oxo-4,7-dihydro-3H-pyrazolo[2,3-d]pyrimidine-5-carbonyl)-2,4-di Preparation of fluorophenyl)-2,6-difluorophenylsulfonamide (2320b)

Anhydrous aluminum trichloride (538mg, 4.0mmol) was dissolved in nitromethane (10mL), after stirring and dissolving, compound 2318a (74mg, 0.40mmol) was added, and compound 2,6-difluoro-3-(2 ,6-Difluorobenzenesulfonylamino)benzoyl chloride (219mg, 0.60mmol) was stirred, heated to 90-95°C, and reacted overnight. The reaction solution was quenched with ice water, extracted three times with ethyl acetate, combined organic phases, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Add a large amount of petroleum ether and stir, pour off the upper layer, add methanol to dissolve the residue through a silica gel column, and use a volume ratio of 5:1 and 3:1 petroleum ether-ethyl acetate mixed solution, and a volume ratio of 20:1 dichloromethane- Methanol mixed solution was used as gradient elution to obtain the crude product of compound 2320b. Then, the crude product 2320b was gradiently eluted with water, 40%, 50%, 60%, and 80% methanol solution on a reverse-phase column to obtain compound 2320b (82 mg, yield 40%).

¹ H NMR (400MHz,DMSO-d ₆ )δ12.97(brs,1H),10.81(brs,1H),7.75–7.65(m,1H),7.62(s,1H),7.48–7.38(m,1H ), 7.24(t, J=9.0Hz, 2H), 7.16(t, J=9.1Hz, 1H), 3.52(s, 3H).

LRMS(ESI):513[MH] ^- .

HRMS (ESI) ^{: C20H10N4O4F4SCl[MH] -} _{calc . 513.0047 ,} found _513.0045 .

N-(3-(2-chloro-3-(4-nitrobenzoyl)-4-oxo-4,7-dihydro-3H-pyrazolo[2,3-d]pyrimidine-5-carbonyl )-2,4-difluorophenyl)-2,6-difluorophenylsulfonamide (2320c) preparation

Anhydrous aluminum trichloride (400mg, 3.0mmol) was dissolved in nitrobenzene (4mL), and after stirring to dissolve, compound 2318b (61mg, 0.20mmol) was added. After compound 2318b was dissolved, compound 2,6-di Fluoro-3-(2,6-difluorobenzenesulfonylamino)benzoyl chloride (110mg, 0.30mmol) was stirred, heated to 100-105°C, and reacted for 48 hours. The reaction solution was quenched with ice water, extracted three times with ethyl acetate, combined the organic phases, washed with hydrochloric acid at pH = 2, washed with saturated aqueous sodium bicarbonate solution, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and added A large amount of petroleum ether, the upper layer was decanted to remove nitrobenzene, and the residual solid was filtered with methanol to obtain the crude product. The NMR and mass spectra of the crude product show that there is a residual fragment of the right carboxylic acid in the structure of the compound. The crude product is dissolved in DMSO, added to an aqueous solution of sodium bicarbonate, centrifuged, and then washed with distilled water, centrifuged to obtain compound 2320c (30mg , yield: 24%).

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.12(s, 1H), 10.84(s, 1H), 8.21(d, J=8.3Hz, 2H), 7.72–7.63(m, 1H), 7.61( s,1H),7.50(d,J＝7.8Hz,2H),7.45–7.34(m,1H),7.28–7.17(m,2H),7.17–7.05(m,1H),5.49(s,2H) .

¹ H NMR (400MHz, DMSO-d ₆ +D ₂ O) δ8.20 (d, J = 8.6 Hz, 2H), 7.73–7.59 (m, 1H), 7.56 (s, 1H), 7.48 (d, J =8.5Hz, 2H), 7.44–7.34 (m, 1H), 7.19 (t, J=9.0Hz, 2H), 7.15–7.06 (m, 1H), 5.48 (s, 2H).

LRMS (ESI): 636[M+H] ⁺ ,658[M+Na] ⁺ ,634[MH] ^- .

HRMS (ESI) ^{: C26H13O6N5ClF4S[MH] -} _{calc . 634.0217 ,} found _634.0220 .

N-(3-(3-allyl-2-chloro-4-oxygen-4,7-dihydro-3H-pyrazol[2,3-d]pyrimidine-5-carbonyl)-2,4-di Preparation of fluorobenzoyl)-2,6-difluorophenylsulfonamide (2320d)

Anhydrous aluminum trichloride (321mg, 2.4mmol) was dissolved in nitrobenzene (4mL), and after stirring to dissolve, compound 2318c (42mg, 0.20mmol) was added. After compound 2318c was dissolved, compound 2,6-di Fluoro-3-(2,6-difluorobenzenesulfonylamino)benzoyl chloride (110mg, 0.30mmol) was stirred, heated to 80°C, and reacted for 36 hours. The reaction solution was quenched with water, extracted three times with ethyl acetate, combined organic phases, pH = 2, washed with hydrochloric acid, washed with saturated aqueous sodium bicarbonate solution, washed with water, washed with saturated saline, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, added a large amount of petroleum Ether, the upper layer was decanted to remove nitrobenzene, the residual solid was stirred and filtered with methanol, the filter cake was washed with water, and dried over anhydrous sodium sulfate. Compound 2320d (25 mg, yield: 23%) was obtained as a pale brown solid.

¹ H NMR (400MHz,DMSO-d ₆ )δ13.04(s,1H),10.84(s,1H),7.76–7.66(m,1H),7.60(s,1H),7.50–7.39(m,1H ), 7.26(t, J=9.3Hz, 2H), 7.19(t, J=8.7Hz, 1H), 5.98–5.83(m, 1H), 5.19(d, J=10.1Hz, 1H), 5.04(d , J=17.1Hz, 1H), 4.73 (d, J=4.7Hz, 2H).

¹³ C NMR (125MHz, DMSO-d ₆ ) δ180.0, 159.2 (dd, J _CF ＝257.4, 2.7Hz), 157.5 (dd, J _CF ＝248.3, 6.5Hz), 156.2, 153.8 (dd, J _CF ＝251.8, 8.4Hz), 148.4, 145.9, 136.5(t, J _CF ＝10.8Hz), 132.3, 132.1, 130.3(d, J _CF ＝6.3Hz), 120.5, 120.4(d, J _CF ＝14.0Hz), 119.6(t , J _CF =23.1 Hz), 117.6 (t, J _CF =16.0 Hz), 117.4, 113.8 (dd, J _CF =22.8, 1.9 Hz), 112.7 (d, J _CF =23.0 Hz), 102.9, 48.1.

LRMS (ESI): 541[M+H] ⁺ , 563[M+Na] ⁺ .

HRMS (ESI): C ₂₂ H ₁₄ O ₄ N ₄ ClF ₄ S [M+H] ⁺ calc. 541.0355, found 541.0368.

Preparation of 5-(3-amino-2,6-difluorobenzoyl)-2-chloro-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2320e)

Add reduced iron powder (0.22mg, 4.0mmol) to water (10mL), add acetic acid (10mL) under stirring, dissolve compound 2319a (0.36g, 1.0mmol) in absolute ethanol, add a small amount of acetic acid to dissolve, slowly add Into the above reaction solution, heated to 110°C, and reacted for 0.5 hours. After the reaction was detected by TLC, the reaction liquid water, ethanol and acetic acid were evaporated under reduced pressure, the pH was adjusted to weak alkaline with saturated NaHCO ₃ aqueous solution, extracted three times with ethyl acetate, the insoluble matter was filtered, the organic phase was washed with water, washed with saturated saline, and anhydrous Dry over sodium sulfate and evaporate to dryness under reduced pressure to obtain white solid compound 2320e (257 mg, yield: 78%).

¹ H NMR (500MHz,DMSO-d ₆ )δ12.96(s,2H),7.70(s,1H),6.93–6.85(m,1H),6.85–6.79(m,1H),5.12(s,2H ).

LRMS(ESI):323[MH] ^- .

HRMS (ESI): C ₁₃ H ₈ O ₂ N ₄ F ₂ Cl [M+H] ⁺ calc. 325.0304, found 325.0300.

Preparation of 6-(3-amino-2,6-difluorobenzoyl)-2-chloro-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2320f)

Add reduced iron powder (25mg, 0.44mmol) to water (2mL), add acetic acid (2mL) under stirring, dissolve compound 2319b (40mg, 0.11mmol) in absolute ethanol, add acetic acid to aid dissolution, and slowly add to the above reaction solution, heated to 110°C, and reacted for 0.5 hours. After the reaction was detected by TLC, the acetic acid was evaporated under reduced pressure, the residue was added with saturated sodium bicarbonate, extracted 5 times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and silica gel column layer Analysis gave white solid 2320f (8 mg, yield: 22%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 13.24 (brs, 1H), 13.17 (brs, 1H), 7.01-6.87 (m, 3H), 5.27 (brs, 2H).

¹³ C NMR (125MHz, DMSO-d ₆ ) δ178.7, 159.4, 150.8, 150.0 (dd, J _{CF = 236.2, 6.0 Hz), 146.7, 146.4 (dd, J CF =} 242.4, 7.2 Hz), 133.9 (d, J _CF = 12.9 Hz), 132.4, 117.9 (t, J _CF = 8.2 Hz), 116.0 (dd, J _CF = 22.9, 19.5 Hz), 114.7, 111.9 (dd, J _CF = 21.7, 3.0 Hz), 108.0.

LRMS(ESI):323[MH] ^- .

HRMS (ESI): C ₁₃ H ₆ O ₂ N ₄ F ₂ Cl [MH] ^- calc. 323.0147, found 323.0156.

N-(3-(2-chloro-3-methyl-4-oxygen-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-2,4-difluoro Benzoyl)propane-1-sulfonamide (2320j) and 5-(3-amino-2,6-difluorobenzoyl)-2-chloro-3-methyl-3H-pyrrolo[2,3- d] Preparation of pyrimidin-4(7H)-one (2320g)

Anhydrous aluminum trichloride (538mg, 4.0mmol) was dissolved in nitromethane (10mL), after stirring to dissolve, compound 2318a (74mg, 0.4mmol) was added, and compound 2,6-difluoro-3-propanesulfonate was added Amidobenzoyl chloride (144mg, 0.6mmol) was stirred, heated to 100-105°C, and reacted overnight. The reaction solution was quenched with ice water, extracted three times with ethyl acetate, combined organic phases, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Add a large amount of petroleum ether to wash twice, and pour off the supernatant. The solid was dissolved in methanol, and the sample was mixed for silica gel column chromatography, and the mixed solution of petroleum ether-ethyl acetate with a volume ratio of 5:1 and 3:1 was used as the eluent for gradient elution, and the less polar components were collected. Concentration afforded compound 2320j as a pale brown solid. Then, the dichloromethane-methanol mixed solution with a volume ratio of 20:1 was eluted as an eluent to obtain a more polar component, which was concentrated to obtain 2320 g of compound.

2320j

¹ H NMR (600MHz, DMSO-d ₆ )δ7.89(s,1H),7.50(q,J=8.7Hz,1H),7.15(t,J=8.7Hz,1H),3.49(s,3H) , 3.08–3.01 (m, 2H), 1.74–1.65 (m, 2H), 0.89 (t, J=7.4Hz, 3H).

LRMS(ESI):443[MH] ^- .

HRMS (ESI): C ₁₇ H ₁₄ N ₄ O ₄ F ₂ SCl [MH] ^- calc. 443.0392, found 443.0395.

2320g

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.67 (s, 1H), 6.84 (d, J=7.4 Hz, 2H), 5.11 (s, 2H), 3.56 (s, 3H).

¹ H NMR (400 MHz, DMSO-d ₆ +D ₂ O) δ 7.61 (s, 1H), 6.93–6.75 (m, 2H), 3.55 (s, 3H). LRMS (ESI): 339[M+H] ⁺ , 361[M+Na] ⁺ , 337[MH] ^- .

HRMS (ESI): C ₁₄ H ₉ O ₂ N ₄ F ₂ NaCl [M+Na] ⁺ calc. 361.0280, found 361.0285.

N-(3-(2-chloro-3-(4-nitrobenzyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)- 2,4-difluorophenyl)propyl-1-sulfonamide (2320k) and 5-(3-amino-2,6-difluorobenzoyl)-2-chloro-3-(4-nitrobenzyl base)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (2320h)

Anhydrous aluminum trichloride (526mg, 3.9mmol) was dissolved in nitromethane (5ml), after stirring to dissolve, compound 2321b (80mg, 0.26mmol) was added, and compound 2,6-difluoro-3-(2 ,6-Difluoropropanesulfonylamino)benzoyl chloride (117mg, 0.39mmol) was stirred, heated to 100-105°C, and reacted for 48 hours. TLC showed that the reaction of the raw material was basically complete. The reaction solution was quenched with water, extracted three times with ethyl acetate, combined organic phases, washed with hydrochloric acid at pH = 2, washed with saturated aqueous sodium bicarbonate solution, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and column chromatography , using a dichloromethane-methanol mixed solution with a volume ratio of 50:1 as an eluent to obtain two compounds with similar polarity, add methanol to dissolve as much as possible, and filter to obtain a solid, that is, compound 2320k (30mg, yield: 20% ), the filtrate was passed through a reverse-phase column (MeOH%=60%) to obtain a light brown solid, namely compound 2320h (4 mg, yield: 3%).

2320k

¹ H NMR (400MHz,DMSO-d ₆ )δ13.16(s,1H),9.73(s,1H),8.21(d,J=8.4Hz,2H),7.96(s,1H),7.50(t, J=10.1Hz, 3H), 7.18(t, J=8.9Hz, 1H), 5.48(s, 2H), 3.03(t, J=7.5Hz, 2H), 1.67(q, J=7.7Hz, 2H) ,0.88(t,J=7.3Hz,3H). ¹³ C NMR(125MHz,DMSO-d ₆ )δ180.6,157.0,156.9(dd,J _CF =247.2,6.8Hz),153.2(dd,J _CF =249.6, 8.1Hz), 148.4, 147.3, 145.8, 144.3, 132.5, 129.4(d, J _CF ＝9.7Hz), 128.2, 124.3, 122.0(d, J _CF ＝14.6Hz), 120.8, 119.7(t, J _CF ＝22.2 Hz), 112.4 (d, J _CF =22.3 Hz), 103.0, 53.6, 48.8, 17.2, 13.00.

LRMS (ESI): 588[M+Na] ⁺ ,564[MH] ^- .

HRMS (ESI): C ₂₃ H ₁₈ O ₆ N ₅ ClF ₂ NaS [M+Na] ⁺ calc. 588.0532, found 588.0523.

2320h

¹ H NMR (400MHz, DMSO-d ₆ )δ8.22(d, J=8.8Hz, 2H), 7.71(s, 1H), 7.52(d, J=8.7Hz, 2H), 6.91–6.79(m, 2H), 5.52(s, 2H), 5.13(s, 2H). ¹³ C NMR (125MHz, DMSO-d ₆ ) δ181.5, 156.8, 150.1 (dd, J _CF ＝235.7, 6.8Hz), 148.5, 147.2, 146.8 (dd, J _CF ＝241.9,7.9Hz),145.8,144.4,133.4(dd,J _CF ＝11.5,6.5Hz),132.7,128.3,124.3,121.1,118.7(dd,J _CF ＝22.5,19.6Hz), 117.2 (t, J _CF =7.3 Hz), 111.5 (dd, J _CF =21.9, 2.9 Hz), 103.1, 48.9.

LRMS (ESI): 460[M+H] ⁺ ,458[MH] ^- .

HRMS (ESI): C ₂₀ H ₁₃ O ₄ N ₅ ClF ₂ [M+H] ⁺ calc. 460.0624, found 460.0630.

N-(3-(2-chloro-4-oxo-4,7-hydrogen-3H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-2,4-difluorobenzoyl)propyl - Preparation of 1-sulfonamide (2320i)

Dissolve anhydrous aluminum trichloride (0.53g, 4.0mmol) in nitromethane (5ml), stir to dissolve, add compound 2315 (85mg, 0.5mmol), then add compound 2313b (179mg, 0.6mmol) Stir, heat to 80°C, react overnight, TLC monitoring shows that the reaction is complete. The reaction solution was slowly poured into ice water, extracted three times with ethyl acetate, the organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Column chromatography gave a white solid, compound 2320i (100 mg, yield: 46%).

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.04(s, 2H), 9.72(s, 1H), 7.92(s, 1H), 7.58–7.45(m, 1H), 7.18(t, J=9.2 Hz, 1H), 3.12–2.99 (m, 2H), 1.80–1.62 (m, 2H), 0.92 (t, J=7.4Hz, 3H).

LRMS(ESI):429[MH] ^- .

_HRMS (ESI) ^{: C16H12N4O4F2SCl [MH] -} _{calc . 429.0236} , found _429.0247 .

N-(3-(3-allyl-2-chloro-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-2,4-di Preparation of fluorobenzoyl) propane-1-sulfonamide (2320l)

Dissolve anhydrous aluminum trichloride (321mg, 3.0mmol) in nitrobenzene (4ml), stir to dissolve, add compound 2318c (42mg, 0.2mmol), then add compound 2,6-difluoro-3-propanesulfonate Amidobenzoyl chloride (89mg, 0.3mmol) was stirred, heated to 70°C, and reacted for 24 hours. The reaction solution was quenched with water, extracted three times with ethyl acetate, the organic phases were combined, washed with hydrochloric acid at pH=2, washed with saturated aqueous sodium bicarbonate solution, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Column chromatography, eluting with a dichloromethane-methanol mixed solution with a volume ratio of 100:1 as the eluent gave a solid, that is, compound 2320l (41 mg, yield 44%).

¹ H NMR (400MHz, DMSO-d ₆ )δ13.08(s,1H),9.73(s,1H),7.93(s,1H),7.52(m,1H),7.17(t,J＝8.9Hz, 1H), 5.89(m, 1H), 5.18(d, J=10.7Hz, 1H), 5.03(d, J=16.5Hz, 1H), 4.73(d, J=4.7Hz, 2H), 3.13–2.99( m,2H),1.71(m,2H),0.92(t,J=7.4Hz,3H). ¹³ C NMR(125MHz,DMSO-d ₆ )δ180.7,156.9(dd,J _CF =247.0,6.7Hz), 156.5, 153.2(dd, J _CF ＝249.9, 8.3Hz), 148.3, 145.7, 132.3, 132.1, 129.5(d, J _CF ＝9.5Hz), 121.90(d, J _CF ＝16.7Hz), 120.7, 119.8(t , J _CF =22.1 Hz), 117.4, 112.4 (d, J _CF =22.5 Hz), 102.9, 53.6, 48.1, 17.2, 13.0.

LRMS (ESI): 471[M+H] ⁺ , 493[M+Na] ⁺ , 469[MH] ^- .

HRMS (ESI): C ₁₉ H ₁₇ O ₄ N ₄ ClF ₂ NaS [M+Na] ⁺ calc. 493.0519, found 493.0524.

Activity Test Experimental Example

The compounds of the present invention have effects on the activity of tumor cells and enzymes.

Experimental principle:

MTT, 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-DiphenylTetrazolium Bromide) colorimetric method uses MTT thiazole blue as the color reagent, which is a kind of detection of cell survival and This method has been widely used in the screening of anticancer drugs.

MTT is a light yellow powder with good water solubility, which can easily penetrate the cell membrane and enter the cell. It is a dye that accepts hydrogen ions. It can act on the mitochondrial respiratory chain of living cells. Under the action of cytochrome C and succinate dehydrogenase, the tetrazolium ring is cracked to generate blue formazan crystals, which are deposited in the cells. Due to the disappearance of succinate dehydrogenase in dead cells, MTT cannot be reduced, so the amount of formazan crystals generated by reduction is only proportional to the number of living cells. Subsequently, the generated formazan crystals were dissolved with DMSO, and the optical density OD value at 492 nm was measured by a microplate reader, which could reflect the number of viable cells. Therefore, within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number.

The MTT method is simple, fast and accurate, and has a good correlation with the cell counting method, and can also be used to determine the cell growth curve. MTT method is widely used in cytotoxicity test, tumor radiosensitivity test and new drug screening and other applications.

experimental method:

The cells were made into a single suspension with H-DMEM medium containing 10% (v/v) fetal bovine serum, seeded in a 96-well plate at a density of 2500-3500 cells/well, and added to the To test the compound, set up triplicate wells, the concentration of the primary screening is 10 μM, and add an equal amount of DMSO to the control wells. After the compound was acted for 72 hours, MTT solution was added to each well with a final concentration of 0.5 mg/mL, and incubated at 37°C for 4 hours. Discard the medium, add 100 μL/well DMSO, shake for 10 minutes, use a microplate microplate reader to measure the light absorption value of each well at 492 nm, and then calculate the inhibitory rate of the compound on tumor cells. Compounds whose inhibition rate was greater than 50% at 10 μM primary screening were selected for further IC ₅₀ test, and Sorafenib and Vemurafenib were positive drugs. The experimental results are shown in Table 1 and Table 2.

Table 1 Deazapurine compounds inhibitory activity on tumor cells and B-Raf ^V600E mutant

^a Tested at a concentration of 100 μM.

Table 1 tests the inhibitory activity of some compounds of this patent application on B-RafV600E mutated melanoma cell A375, B-RafV600E mutated colorectal cancer cell HT29 and B-RafWT wild-type colorectal cancer cell HCT116, preferably the inhibition rate Better compounds were tested for IC ₅₀ , and compounds with better inhibitory activity against A375 cells were also tested for inhibition rate of B-Raf V600E mutant. As a result, it was found that the compounds had good anti-B-Raf V600E mutant tumor activity.

The tumor cell activity selectivity of table 2 compound 2301f

In addition, because the B-RafV600E mutation has a high proportion (4.7-14.8%) in colorectal cancer, we also tested the inhibitory activity of compound 2301f on multiple colorectal cancer cell lines (Table 2). The results showed that the IC ₅₀ value of 2301f on the B-RafV600E mutant colorectal cancer cell HT-29 reached 0.55 μM, and the IC ₅₀ value was better than that of the marketed drug vemurafenib (IC ₅₀ =0.69 μM). In addition, from the inhibition results of other colorectal cancer cells, we can see that 2301f has an IC ₅₀ >10 μM on B-Raf wild-type colorectal cancer cells, and vemurafenib has an inhibitory effect on LOVO, HCT-15 and HCT-8 The inhibition rates were 8.88 μM, 7.75 μM and 5.84 μM, respectively. This also shows that this type of active compound has good selectivity and has the potential to be further developed into related anti-tumor drugs.

Claims (10)

1. A deazapurine compound represented by the following general formula 1:

wherein,

R¹is amino or halogen;

R²is hydrogen, halogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₂-C₆Alkenyl, substituted or unsubstituted C₁-C₆Alkylaryl, or substituted or unsubstituted C₆-C₁₂An aryl group;

R³is hydrogen, hydroxy, amino, mercapto, halogen, C₁-C₆Alkyl radical, C₆-C₁₂Aryl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkylamino radical, C₁-C₆Alkylthio radical, C₆-C₁₂Aryl ether group, C₆-C₁₂Arylamino or C₆-C₁₂An arylthio group;

R⁴is hydrogen, substituted or unsubstituted C₁-C₆Alkanoyl, substituted or unsubstituted C₆-C₁₂Aroylyl, substituted or unsubstituted C₁-C₆Alkylsulfonyl, substituted or unsubstituted C₆-C₁₂Arylsulfonyl, substituted or unsubstituted C₁-C₆Alkylsulfinyl, substituted or unsubstituted C₆-C₁₂Arylsulfinyl, substituted or unsubstituted C₁-C₆Alkylphosphoryl or substituted or unsubstituted C₆-C₁₂An aryl phosphoryl group;

wherein the substituent is selected from halogen and C₁-C₆Alkyl, cyano, nitro, amino, hydroxyl, carboxyl and mercapto;

n is 1, 2,3 or 4.

2. The deazapurine compound of claim 1, or a pharmaceutically acceptable salt thereof,

R²is hydrogen, substituted or unsubstituted C₁-C₆Alkyl, allyl or nitrobenzyl;

R³is hydrogen, halogen, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio radical, C₆-C₁₂Aryl ether group or C₆-C₁₂An arylthio group;

R⁴is hydrogen, substituted or unsubstituted C₁-C₆Alkanoyl, substituted or unsubstituted C₆-C₁₂Aroylyl, substituted or unsubstituted C₁-C₆Alkylsulfonyl or substituted or unsubstituted C₆-C₁₂An arylsulfonyl group;

wherein the substituent is selected from halogen and C₁-C₄Alkyl and nitro, the halogen being fluorine, chlorine, bromine or iodine;

n is 2 or 3.

3. The deazapurine compound of claim 1, or a pharmaceutically acceptable salt thereof,

R²is hydrogen, methyl, nitrobenzyl or allyl;

R³is hydrogen or halogen;

R⁴is hydrogen, substituted or unsubstituted C₁-C₆Alkylsulfonyl or C substituted by one or more halogens₆-C₁₂An arylsulfonyl group.

4. The deazapurine compound of claim 1, or a pharmaceutically acceptable salt thereof,

R¹is amino or chlorine;

R²is hydrogen, methyl, 4-nitrobenzyl or 1-allyl;

R³is fluorine;

R⁴hydrogen, propylsulfonyl, difluorophenylsulfonyl; preferably hydrogen, propyl-1-sulfonyl, 2, 6-difluorophenylsulfonyl;

n is 2.

5. The deazapurine compound or pharmaceutically acceptable salt thereof as claimed in claim 1, wherein the compound represented by the general formula 1 is a deazapurine compound represented by one of the following general formulae:

wherein R is²、R³、R⁴And n is the same as defined in formula 1.

6. The deazapurine compound or pharmaceutically acceptable salt thereof as claimed in claim 1, wherein said deazapurine compound is selected from the group consisting of:

7. a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of one or more deazapurine compounds selected from the group consisting of the deazapurine compounds of any one of claims 1-6 or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition of claim 7, further comprising a pharmaceutically acceptable carrier.

9. Use of the deazapurine compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prevention or treatment of a tumour or cancer.

10. Use of the pharmaceutical composition of claim 7 or 8 in the manufacture of a medicament for the prevention or treatment of a tumor or cancer.