7-Methylthiazolo[5,4-d]pyrimidine compounds, preparation methods and uses thereof

Abstract

The present invention discloses a 7-methylthiazole [5,4-d] pyrimidine compound, a preparation method and its use; including a compound shown in formula (I) or a physiologically acceptable salt thereof: #imgabs0# wherein R1 is hydrogen, substituted or unsubstituted alkyl, cycloalkyl, substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted cycloalkylcarbonyl; R2 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic group. The PI3K inhibitor compound of the present invention has a novel structure, excellent PI3K kinase inhibitory activity, and significantly improves the selectivity of PI3Kα / mTOR. By regulating the mTOR enzyme activity, the safety risk caused by the strong inhibition of mTOR kinase and the risk caused by the induction of some RTK enzymes can be reduced, and the drug efficacy and toxicity are better.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to 7-methylthiazolo[5,4-d]pyrimidine compounds, preparation methods and uses thereof. Background Art

[0002] The phosphatidylinositol 3-kinase (PI3K) family is a class of kinases that specifically catalyze the phosphorylation of the 3-hydroxyl group of phosphatidylinositol and produce inositol lipid substances with second messenger functions. The signaling pathway composed of PI3K and its downstream molecular signaling protein kinase B (Akt) / rapamycin target protein (mTOR) is one of the most important intracellular signaling pathways in mammals and is essential for many important physiological functions, including cell cycle, cell survival, protein synthesis and growth, metabolism, movement and angiogenesis. As a family of lipid kinases, PI3K can be divided into three main categories (I, II and III) according to their structure and substrate specificity. Class I PI3K is further divided into two major subclasses: Class IA and Class IB. Class IA PI3K includes PI3Kα, PI3Kβ, and PI3Kδ, which are composed of their corresponding catalytic subunits p110 (p110α, p110β, p110δ) and regulatory subunits p85; Class IB (PI3Kγ) is composed of the catalytic subunit p110γ and the regulatory subunits p101 or p87.

[0003] On July 23, 2014, the U.S. FDA approved Idelalisib, developed by Gilead Sciences, for marketing. On September 18, 2014, the European EMA approved its marketing under the trade name Idranib is a PI3Kδ kinase inhibitor that can induce apoptosis and inhibit cell proliferation. It is used to treat relapsed chronic lymphocytic leukemia (CLL), relapsed follicular B-cell non-Hodgkin's lymphoma (FL), and relapsed small lymphocytic lymphoma (SLL).

[0004] On September 14, 2017, the U.S. FDA accelerated the approval of Bayer's Copanlisib for marketing under the trade name Copanlisib is a PI3K inhibitor that inhibits both PI3K-α and PI3K-δ kinase isoforms and is used to treat adult patients with relapsed follicular lymphoma who have received at least two systemic therapies.

[0005] Buparlisib (BKM120) is a pan-class I PI3K inhibitor developed by Novartis that acts on p110α / β / δ / γ. It is used to treat postmenopausal estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative locally advanced or metastatic breast cancer in clinical research. The company is also conducting Phase II clinical studies for the treatment of follicular lymphoma, gastrointestinal stromal tumors, mantle cell lymphoma, prostate cancer, diffuse large B-cell lymphoma, non-small cell lung cancer, hepatocellular carcinoma, myelofibrosis, advanced endometrial cancer, melanoma, bladder cancer, pancreatic cancer and malignant glioma. In addition, duvelisib and alpelisib are FDA-approved PI3K inhibitors, and phosphatidylinositol-3-kinase inhibitors (PI3K) have broad application prospects. It is worth noting that currently marketed PI3K inhibitors have been observed to have significant toxic side effects in clinical applications, such as autoimmune dysfunction, opportunistic infections, skin toxicity, hypertension and hyperglycemia, and both idelalisib and duvelisib have been given black box warnings by the FDA. In addition, inhibition of PI3K signaling mediates negative feedback loops and / or induces activation of other proliferation signaling pathways, thereby weakening the anti-tumor efficacy of PI3K inhibitors. In order to overcome these major obstacles, it is necessary to develop new PI3K inhibitor compounds with different structures, better efficacy and lower toxicity. Summary of the invention

[0006] The purpose of the present invention is to develop a 7-methylthiazolo[5,4-d]pyrimidine compound, a preparation method and its use. The 7-methylthiazolo[5,4-d]pyrimidine PI3K inhibitor compound has a completely new structure different from existing compounds, shows antiproliferative activity, and is therefore used to prevent or treat diseases associated with excessive proliferation, especially for the preparation of drugs for inhibiting PI3 kinase.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a 7-methylthiazolo[5,4-d]pyrimidine compound, including a compound represented by formula (I) or a physiologically acceptable salt thereof:

[0009]

[0010] Wherein, R1 is hydrogen, substituted or unsubstituted alkyl, cycloalkyl, substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted cycloalkylcarbonyl;

[0011] R2 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aromatic heterocyclic group.

[0012] As one embodiment of the present invention, R1 is selected from any one of hydrogen, acetyl, propionyl, cyclopropanecarbonyl, deuterated acetyl, methyl, and cyclopropyl; R2 is selected from any one of 2,4-difluoro-substituted benzene ring group, 4-fluoro-substituted benzene ring group, methyl, thiophen-2-yl, and cyclopropyl.

[0013] As an embodiment of the present invention, any one of the following compounds is selected:

[0014]

[0015]

[0016] The present invention also provides a method for preparing a 7-methylthiazolo[5,4-d]pyrimidine compound, the method comprising the following steps:

[0017] S1. Compound With compound Coupling reaction to obtain amino target compounds

[0018] Or S2, amino compounds With R b X undergoes acylation reaction to obtain the acylated target compound R b is an alkyl acyl group, and X is a halogen atom;

[0019] or S3, compound With compound The coupling reaction gave the compound Further with R a -NH2 undergoes amination reaction to obtain alkyl or cycloalkyl substituted amino target compounds Among them, R a is an alkyl group or a cycloalkyl group.

[0020] As an embodiment of the present invention, a method for preparing a 7-methylthiazolo[5,4-d]pyrimidine compound comprises the following steps:

[0021] Compound With compound The coupling reaction gave the compound Further amination reaction gave the compound

[0022] or compound With R b X undergoes acylation to give compound Among them, R b is an alkyl acyl group, and X is a halogen atom.

[0023] As one embodiment of the present invention, the coupling reaction is carried out in the presence of potassium carbonate, dioxane and PdCl2.

[0024] As an embodiment of the present invention, the acylation reaction is carried out with DCM as solvent in the presence of pyridine.

[0025] As an embodiment of the present invention, the amination reaction is carried out using NMP as solvent and reacting with aqueous ammonia.

[0026] As one embodiment of the present invention, the compound It is prepared by the following steps:

[0027] A1. 2-Bromo-4-amino-5-methoxypyridine and compounds The compound was obtained by reaction in the presence of DMAP and pyridine.

[0028] A2. Compounds In the presence of potassium acetate, PdCl2 and dioxane, it reacts with diboric acid pinacol ester to obtain the compound

[0029] As one embodiment of the present invention, the compound It is prepared by the following steps:

[0030] B1. Compounds The compound is obtained by reacting in the presence of iron powder and acetic acid.

[0031] B2. Compounds The compound is obtained by reacting in the presence of potassium thiocyanate and acetic acid.

[0032] B3, compound The compound was prepared by reaction with isoamyl nitrite

[0033] B4. Compounds The compound was obtained by reaction in the presence of dibenzylamine, BINAP, Pd2(dba)3 and Cs2CO3.

[0034] B5. Compounds Reaction with citric acid to obtain compound

[0035] B6. Compounds Bromination to give compound

[0036] As one embodiment of the present invention, the compound It is prepared by the following steps:

[0037] C1. Compound The compound is obtained by reacting in the presence of iron powder and acetic acid.

[0038] C2. Compound The compound is obtained by reacting in the presence of potassium thiocyanate and acetic acid.

[0039] C3. Compound Under the conditions of tert-butyl nitrite and acetonitrile, bromination reaction occurs to obtain the compound

[0040] The present invention also provides the use of 7-methylthiazolo[5,4-d]pyrimidine PI3K inhibitor compounds in the preparation of drugs for treating PI3 kinase-related diseases. In particular, the use of 7-methylthiazolo[5,4-d]pyrimidine PI3K inhibitor compounds in the preparation of drugs for treating tumor diseases. The compounds of the present invention show excellent inhibitory effects in various tumor cell lines, especially compound Id has an IC of 1.17kDa in the lymphoma cell line DOHH2. 50 The value is 0.063μm, which is much better than the control drug BKM120 of 0.53μm.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1) The 7-methylthiazolo[5,4-d]pyrimidine PI3K inhibitor compound of the present invention has a novel structure, excellent PI3K kinase inhibitory activity, and significantly improved PI3Kα / mTOR selectivity. By regulating the mTOR enzyme activity, it can reduce the safety risks caused by the strong inhibition of mTOR kinase, such as hyperglycemia, and the risks caused by the induction of some RTK enzymes, and has better efficacy and lower toxicity.

[0043] 2) The tumor inhibition experiment in animals showed that the compounds of the present invention can achieve excellent tumor inhibition effects at relatively low doses, and the weight of the animals is basically unchanged, which shows good safety. At the same time, the compounds show good metabolic properties.

[0044] 3) The present invention also provides a simple preparation method for 7-methylthiazolo[5,4-d]pyrimidine PI3K inhibitor compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0046] Figure 1The effects of compounds Ic and Id on the body weight changes of nude mice with human gastric cancer HGC27;

[0047] Figure 2 The experimental therapeutic effects of compounds Ic and Id on human gastric cancer HGC27 nude mouse transplanted tumors. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several adjustments and improvements can be made without departing from the concept of the present invention. These all belong to the scope of protection of the present invention. All publications mentioned are incorporated by reference in their entirety.

[0049] One embodiment of the present invention includes compounds of formula (I):

[0050]

[0051] or a physiologically acceptable salt thereof, wherein:

[0052] R1 is hydrogen, substituted or unsubstituted alkyl, cycloalkyl, substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted cycloalkylcarbonyl;

[0053] R2 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aromatic heterocyclic group.

[0054] The alkyl group is a C1-C8 alkyl group, the cycloalkyl group is a C3-C8 cycloalkyl group, the alkylcarbonyl group is a C2-C8 alkylcarbonyl group, and the cycloalkylcarbonyl group is a C4-C8 cycloalkylcarbonyl group; the aryl group is a phenyl group or a naphthyl group, the aromatic heterocyclic group is a thienyl group, a thiazolyl group, a pyrazolyl group, a pyridyl group or a furyl group, and the substituent group is a halogen group or an alkyl group.

[0055] In a preferred embodiment, the present invention includes compounds of formula (I), wherein R1 is selected from any one of hydrogen, acetyl, propionyl, cyclopropanecarbonyl, deuterated acetyl, methyl, and cyclopropyl.

[0056] In another preferred embodiment, the present invention includes compounds of formula (I), wherein R2 is selected from any one of 2,4-difluoro-substituted phenyl ring group, 4-fluoro-substituted phenyl ring group, methyl, thiophen-2-yl, and cyclopropyl.

[0057] In another preferred embodiment, the present invention includes any one of the compounds of the following formulae or a physiologically acceptable salt thereof.

[0058] Table 1

[0059]

[0060]

[0061]

[0062]

[0063] If there is a discrepancy between the chemical name and the illustrated chemical structure, the illustrated chemical structure is preferred to the given chemical name.

[0064] Without being limited by theory or mechanism, the compounds of the present invention exhibit unexpected phosphatidylinositol-3-kinase inhibitory activity and chemical and structural stability. This unexpected activity is believed to be based on the structure of the compounds, particularly the core of these compounds (7-methylthiazolo[5,4-d]pyrimidine compounds). In addition, considering the activity in vivo, appropriate selection of R1 and R2 provides the necessary activity for the appropriate isoform.

[0065] The present invention also provides a method for preparing a 7-methylthiazolo[5,4-d]pyrimidine PI3K inhibitor compound.

[0066] Preparation of compounds Ic and Id: Compound 1 is used as the starting material to obtain intermediate 3, which is then brominated to obtain intermediate 8, coupled to obtain intermediate 10, and aminized to obtain the target compound Ic; Ic is acetylated to obtain the target compound Id. Specific reaction route 1:

[0067]

[0068]

[0069] Reaction Scheme 1

[0070] Using compound 1 as the starting material, intermediate 3 is prepared, and key intermediate 7 is further prepared. See reaction scheme 2 for details:

[0071]

[0072] Reaction Scheme 2

[0073] Amino target compound: 5-bromo-2-methoxypyridin-3-amine is used to prepare boric acid ester intermediate 9, which is then coupled with intermediate 7 to obtain the amino target compound. For details, see reaction scheme 3.

[0074]

[0075] Reaction Scheme 3

[0076] Wherein R2 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aromatic heterocyclic group, preferably 2,4-difluoro substituted phenyl ring group, 4-fluoro substituted phenyl ring group, methyl, thiophen-2-yl, cyclopropyl;

[0077] The amino target compounds include compound Ic, compound Ia, compound Ie and compound If, etc.

[0078] Preparation of alkyl-substituted amino target compounds: In reaction route 4, 5-bromo-2-methoxypyridin-3-amine is used to prepare borate intermediate 9, which is then coupled with intermediate 8 to obtain target compound 10, which is then further prepared to obtain alkyl-substituted amino compounds.

[0079]

[0080] Reaction Scheme 4

[0081] Wherein Ra is an alkyl group or a cycloalkyl group, preferably a methyl group or a cyclopropyl group;

[0082] R2 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aromatic heterocyclic group, preferably a 2,4-difluoro-substituted phenyl ring group, a 4-fluoro-substituted phenyl ring group, a methyl group, a thien-2-yl group, or a cyclopropyl group;

[0083] The alkyl-substituted amino compounds include compounds In, Ip, Im and Io.

[0084] Preparation of the acylated target compound: Using the amino target compound as a substrate, an acylation reaction is carried out to obtain the acylated target compound. For details, see Reaction Scheme 5.

[0085]

[0086] Reaction Scheme 5

[0087] Where R b is an alkyl acyl group, preferably an acetyl group, a propionyl group, a cyclopropanecarbonyl group, or a deuterated acetyl group;

[0088] X is chlorine, bromine or fluorine,

[0089] R2 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aromatic heterocyclic group, preferably a 2,4-difluoro-substituted benzyl group, a 4-fluoro-substituted benzyl group, a methyl group, a thien-2-yl group, a cyclopropyl group, or the like.

[0090] The acylated target compound includes compound Ij, compound Il, compound Ir, compound Ib, compound Ii, compound Ik, compound Iq, compound Ig and compound Ih.

[0091] Example 1. Preparation of 5-chloro-7-methylthiazolo[5,4-d]pyrimidin-2-amine

[0092]

[0093] Compound 1 (4.16 g, 1 eq) was dissolved in 100 ml of acetic acid, cooled to 5-10°C in an ice-water bath, and iron powder (5 g, 4.5 eq) was added in batches. The reaction was continued at room temperature for 2 h. Post-treatment: The reaction solution was filtered and the filtrate was concentrated. The residual liquid was dissolved in water, extracted with ethyl acetate three times, washed once with saturated sodium chloride aqueous solution, and the organic phase was concentrated under reduced pressure to obtain compound 2 with a yield of 85.2%. HPLC purity 99.56%; MS [M+1]: 178.

[0094] Compound 2 (3.56 g, 1 eq) was dissolved in 30 ml of acetic acid, potassium thiocyanate (1.94 g, 1 eq) was added, and the mixture was heated under reflux for 3 h. After the reaction was completed, the reaction solution was cooled to room temperature, and water was added dropwise. Solids gradually precipitated. The earth-colored solids, compound 3, were obtained by filtration. The yield was 90.6%. HPLC purity was 94.27%; MS [M+1]: 201.

[0095] Example 2: Preparation of 2-bromo-5-chloro-7-methylthiazolo[5,4-d]pyrimidine

[0096]

[0097] Compound 3 (4.0 g, 1 eq) was dissolved in 10 ml of acetonitrile, cooled to 0°C in an ice-water bath, and tert-butyl nitrite (3.0 g, 1.5 eq) was slowly added, followed by copper bromide (5.9 g, 1.25 eq) in batches. The mixture was kept at 0°C for 30 min, and then naturally warmed to room temperature and continued to react for 2.5 h. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate three times. The organic layer was washed twice with a saturated sodium chloride aqueous solution. The organic phase was concentrated under reduced pressure to obtain compound 8, with a yield of 88.5%. HPLC purity was 87.54%; MS [M+1]: 264.

[0098] Example 3. Preparation of N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)sulfonamide (Compound 9)

[0099] Using sulfonic anhydride or sulfonyl chloride, the following boric ester compounds were prepared using a similar method:

[0100]

[0101] Wherein R2 is 2,4-difluoro, methyl, cyclopropyl, thiophene-2-yl, and compound 9 corresponds to 9a, 9b, 9c and 9d, respectively, as shown in Table 2 below:

[0102] Table 2

[0103]

[0104] 9a: Preparation of 2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide

[0105] Step 1: Add 2-bromo-4-amino-5-methoxypyridine (10.15 g, 0.05 mol), DMAP (1.22 g), pyridine (7.12 g, 0.09 mol) to a three-necked flask and dissolve in DCM (61 ml), cool to -10-0 °C, and replace with N2 three times. Add 2,4-difluorobenzenesulfonyl chloride (13.8 g, 0.065 mol) in DCM (61 mL) dropwise, and monitor the reaction by TLC until the reaction is complete; pour the reaction solution into 100 ml of water, stir and separate. The organic phase is washed once with water (100 ml), saturated sodium bicarbonate (100 ml), and water (100 ml) in turn. The organic phase is concentrated to dryness under reduced pressure, and the concentrated residue is recrystallized from ethyl acetate. Intermediate S9a is obtained with a yield of 82.6%. HPLC: 96.86%. 1 HNMR (400MHz, DMSO) δ7.92-7.86(m,2H),7.82(m,1H),7.22(s,1H),7.01-6.91(m,2H),3.89(s,3H).

[0106] Step 2: In a three-necked flask, add the product S9a (18.9 g) from the previous step, potassium acetate (14.7 g), bipyralidin (15.6 g), PdCl2 (dppf) (1.24 g), and dioxane (330 ml), and stir. Replace with nitrogen three times. Heat to 90°C and react overnight.

[0107] After the reaction was completed, ethyl acetate (300 ml) and 300 ml of water were added to the reaction solution, stirred and separated, and the aqueous layer was extracted once with 150 ml of ethyl acetate. The organic phases were combined, filtered through diatomaceous earth, and the filtrate was washed once with saturated brine. Activated carbon was added and stirred at high temperature for 1 hour; filtered, concentrated under reduced pressure, purified by silica gel column chromatography, and recrystallized from acetonitrile-water to obtain compound 9a with a yield of 77.5%. HPLC: 98.37%; 1 H NMR (400MHz, DMSO) δ10.19(s,1H),8.19-8.16(m,1H),7.69(m,1H),7.68-7. 64(m,1H),7.58-7.52(m,1H),7.20-7.15(m,1H),3.58(s,3H),1.27(s,12H).

[0108] 9b: Preparation of N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methanesulfonamide

[0109] Step 1: Add 2-bromo-4-amino-5-methoxypyridine (10.15 g, 0.05 mol), DMAP (1.22 g), pyridine (7.12 g, 0.09 mol) and dissolve in DCM (61 ml) in a three-necked flask, cool to -10-0°C, and replace with nitrogen three times. Add a DCM solution (61 mL) of methanesulfonic anhydride (11.37 g, 0.065 mol) dropwise, react at room temperature, and monitor the reaction by TLC until the reaction is complete.

[0110] After the reaction was completed, the reaction solution was poured into 100 ml of water and stirred for separation. The organic phase was washed with water (100 ml), saturated sodium bicarbonate (100 ml), and water (100 ml) in sequence. The organic phase was concentrated to dryness under reduced pressure and recrystallized from ethyl acetate to obtain compound S9b with a yield of 81.3%. HPLC: 98.69%. 1 HNMR (400MHz, DMSO) δ7.97(m,1H),7.89(m,1H),6.76(s,1H),3.99(s,3H),3.04(s,3H).

[0111] Step 2: Add S9b (28.1 g), potassium acetate (29.4 g), bipyralidin (31.2 g), and PdCl2 (dppf) (2.5 g) to a three-necked flask and dissolve in dioxane (660 ml). Replace with nitrogen three times. Heat to 90°C and react overnight.

[0112] After the reaction is completed, add ethyl acetate (600 ml) and 600 ml of water, stir and separate the layers, and extract the water layer with 300 ml of ethyl acetate. Filter the organic phase with diatomaceous earth, and wash the filtrate with saturated brine. Add activated carbon and stir at high temperature for 1 hour; filter and concentrate. Pass the residual liquid through a column and recrystallize in acetonitrile-water. Dry at 60°C. Compound 9b is obtained with a yield of 75.8%. HPLC: 97.43%. 1 HNMR (400MHz, DMSO) δ9.25(s,1H),8.18(m,1H),7.75(m,1H),3.92(s,3H),2.99(s,3H),1.28(s,12H).

[0113] 9c: Preparation of N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)cyclopropanesulfonamide

[0114] Cyclopropanesulfonyl chloride was substituted for 2,4-difluorobenzenesulfonyl chloride, and the rest of the process was the same as steps 1 and 2 of Example 9a to obtain compound 9c with a two-step yield of 61.6%. HPLC: 97.79%. 1 H NMR (400MHz, DMSO) δ9.27(s,1H),8.19(m,1H),7.77(m,1H),3.92(s,3H),2.65-2.58(m,1H),1.27(s,12H),0.92-0.81(m,4H).

[0115] 9d: Preparation of N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)thiophene-2-sulfonamide

[0116] Thiophene-2-sulfonyl chloride was substituted for 2,4-difluorobenzenesulfonyl chloride, and the rest of the process was the same as steps 1 and 2 of Example 9a to obtain compound 9d with a two-step yield of 58.8%. HPLC: 97.48%. 1 H NMR (400MHz, DMSO) δ10.02(s,1H),8.18(m,1H),7.90-7.88(m,1H),7.75(m,1H),7.41-7.40(m,1H),7.12-7.09(m,1H),3.65(s,3H),1.27(s,12H).

[0117] Example 4. Preparation of N-(5-(5-chloro-7-methylthiazolo[5,4-d]pyrimidin-2-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide

[0118] 4-1 Preparation of Compound 10a

[0119]

[0120] Compound 8 (0.265g, 1eq), compound 9a (0.511g, 1.2eq), potassium carbonate (0.553g, 4eq), water (2ml), dioxane (6ml), stirred to dissolve the solid, and replaced with nitrogen 3 times. PdCl2 (dppf) (0.074g, 0.1eq) was added, and the nitrogen was replaced 3 times. The temperature was raised to 80°C and kept warm for 8 hours. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with ethyl acetate 3 times, and the organic layer was washed once with saturated brine. The organic phase was concentrated to dryness under reduced pressure and purified by silica gel column chromatography to obtain solid compound 10a with a yield of 45.3%. HPLC purity 95.62%; 1H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 8.72 (s, 1H), 8.23 ​​(s, 1H), 7.80 (m, 1H), 7.60 (m, 1H), 7.24 (m, 1H), 3.33 (s, 3H), 2.86 (s, 3H). MS [M+1]: 484.

[0121] 4-2 Preparation of Compound 10b

[0122]

[0123] Compound 8 (0.265g, 1eq), compound 9b (0.393g, 1.2eq), potassium carbonate (0.553g, 4eq), water (2ml), dioxane (6ml), stirred, and replaced with nitrogen 3 times. PdCl2 (dppf) (0.074g, 0.1eq) was added, and the nitrogen was replaced 3 times. The temperature was raised to 80°C and kept for 8 hours. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with ethyl acetate 3 times. The organic layer was washed once with saturated brine, and the organic phase was concentrated to dryness under reduced pressure. Solid compound 10b was purified by silica gel column chromatography with a yield of 52.1%. HPLC purity 96.44%; MS [M+1]: 386.

[0124] Example 5. Preparation of N-(5-(5-amino-7-methylthiazolo[5,4-d]pyrimidin-2-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (Compound Ic)

[0125]

[0126] Compound 10a (7.02 g, 14.5 mmol) was dissolved in NMP (50 ml), 28% aqueous ammonia (10 ml) was added, the temperature was raised to 70°C, and the reaction was carried out for 3 h. After the reaction was completed, the temperature was lowered to room temperature, and a solid was precipitated. The solid was filtered, and the filter cake was washed with water. PE:EA (2:1) was used as the mobile phase, and the compound Ic (yield 85%) was obtained by silica gel column chromatography. 1H NMR (400MHz, Acetone-d6) δ9.06(s,1H),8.54(s,1H),8.34(s,1H),7.95(m,1H),7.35(m,1H),7.20(m,1H),6.32(s,2H),3.86(s,3H),2.76(s,3H). MS[M+1]:465.

[0127] Example 6. Preparation of N-(2-(5-((2,4-difluorophenyl)sulfonylamino)-6-methoxypyridin-3-yl)-7-methylthiazolo[5,4-d]pyrimidin-5-yl)acetamide (Compound Id)

[0128]

[0129] Compound Ic (0.929 g, 1 eq) and pyridine (0.475 g, 3 eq) were dissolved in DCM (5 ml), cooled to 0°C, and a solution of acetyl chloride (0.172 g, 1.2 eq) in DCM (2 ml) was added dropwise. The temperature was maintained at 0-5°C and the reaction was continued for 6-8 h. After the reaction was completed, the reaction solution was washed with 10% hydrochloric acid, saturated sodium bicarbonate, and water, respectively. The organic phase was concentrated to dryness under reduced pressure, and PE:EA (2:1) was used as the mobile phase. Compound Id was purified by silica gel column chromatography with a yield of 60.5%. 1 H NMR(400MHz, Acetone-d6)δ9.03(s,1H),8.51(s,1H),8.36(s,1H),7.94(m,1H) , 7.34 (m, 1H), 7.22 (m, 1H), 6.30 (s, 1H), 3.88 (s, 3H), 2.73 (s, 3H), 2.31 (s, 3H). MS[M+1]:507.

[0130] Example 7. Preparation of Compound 4

[0131]

[0132] A mixture of compound 3 (4g, 20mmol, 1.0eq) in acetonitrile (30mL) was added to isoamyl nitrite (7.0g, 60mmol, 3.0eq), and stirred at room temperature for 3h. LCMS showed that the reaction was complete. The mixture was diluted with ethyl acetate, washed with water and brine, dried over Na2SO4, and concentrated. Residue was purified by column chromatography to obtain compound 4 (2g, 50% yield) in the form of a yellow solid.

[0133] Example 8. Preparation of Compound 5

[0134]

[0135] A mixture of compound 4 (2g, 10.8mmol, 1.0eq), benzhydrylamine (2.4g, 12.9mmol, 1.2eq) in toluene (20.0ml) was added to BINAP (684mg, 1.1mmol, 0.1eq), Pd2(dba)3 (1g, 1.1mmol, 0.1eq) and Cs2CO3 (7g, 21.6mmol, 2.0eq). The mixture was stirred at 120°C for 16 hours. LCMS showed that the reaction was complete. The mixture was diluted with EA, washed with water and brine, dried over Na2SO4, and concentrated. Pre-TLC purification of the residue gave compound 5 (1g, 36% yield) in the form of a yellow solid.

[0136] Example 9. Preparation of 2-bromo-7-methylthiazolo[5,4-d]pyrimidin-5-amine (Compound 7)

[0137]

[0138] To a mixture of compound 5 (1 g, 3 mmol, 1.0 eq) and acetonitrile (3 ml) was added citric acid (1.2 g, 6 mmol, 2.0 eq) and the mixture was stirred at room temperature for 5 h. LCMS showed that the reaction was complete. The mixture was distilled under reduced pressure. The residue was diluted with DCM and washed with brine, dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give yellow solid compound 6 (400 mg, 80% yield).

[0139] A mixture of compound 6 (400 mg, 2.4 mmol, 1.0 eq) and NBS (854 mg, 4.8 mmol, 2.0 eq) in DMF (15 ml) was stirred at room temperature for 5 h. LCMS showed that the reaction was complete. The resulting product was diluted with ethyl acetate, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by pre-TLC to obtain compound 7 (165 mg, 35% yield) in the form of a yellow solid. 1 HNMR (400 MHz, DMSO): 2.55 (s, 3H), 7.06 (s, 2H). LCMS: Rt: 1.287 min; MS [M+1]: 245, 247.

[0140] Example 10: Preparation of amino target compounds

[0141]

[0142] Wherein R2 is 2,4-difluorophenyl, methyl, cyclopropyl and 2-thienyl, and compound 9 corresponds to 9a, 9b, 9c and 9d, which react with compound 7 to prepare compounds Ic, Ia, Ie and If respectively.

[0143] Preparation of Compound Ic

[0144] Add compound 7 (0.66 g, 1 eq), compound 9a (1.28 g, 1.2 eq), potassium carbonate (1.4 g, 4 eq), water (2.5 ml), and dioxane (5 ml) to a three-necked flask, and replace the atmosphere with nitrogen three times. Add PdCl2 (dppf) (0.09 g). Replace the atmosphere with N2 three times. React at 85-95°C for 4-6 hours.

[0145] The reaction solution was poured into water and extracted with ethyl acetate three times; washed once with saturated brine; the organic phase was dried with an appropriate amount of anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by column to obtain the product compound Ic, with a yield of 50.7%, HPLC: 97.16%, MS [M+1]: 465.

[0146] Preparation of Compound Ia

[0147] Compound 9b was used as the reactant and the preparation method was the same as that of Ic in this example to obtain compound Ia with a yield of 43.2%. HPLC: 95.65%, MS [M+1]: 367.

[0148] Preparation of Compound Ie

[0149] Compound 9c was used as the reactant and the preparation method was the same as that of Ic in this example to obtain compound Ie with a yield of 45.1%. HPLC: 95.10%, 1H NMR (400MHz, Acetone-d6) δ7.57 (m, 1H), 7.47 (s, 1H), 4.04 (m, 2H), 3.84 (s, 3H), 2.89 (s, 3H), 1.41 (m, 1H), 1.04 (m, 2H), 0.86 (m, 2H). MS [M+1]: 393.

[0150] Preparation of compound If

[0151] Compound 9d was used as the reactant and the preparation method was the same as that of Ic in this example to obtain compound If in a yield of 39.8%. HPLC: 96.59%, 1H NMR (400MHz, Acetone-d6) δ8.51 (s, 1H), 8.41 (s, 1H), 7.90 (m, 1H), 7.66 (m, 1H), 7.17 (m, 1H), 6.30 (s, 2H), 3.86 (s, 3H), 2.71 (s, 3H). MS [M+1]: 435.

[0152] Example 11: Preparation of aminoacylated target compounds

[0153] 11-1. Preparation of Compound Ij, Compound Il and Compound Ir

[0154]

[0155] Where R b The compounds Ij, Il and Ir were prepared by using propionyl, cyclopropionyl and deuterated acetyl groups respectively.

[0156] Referring to Example 6, Ic was used as the substrate, acetyl chloride was replaced by propionyl chloride, and the preparation method was the same as compound Id to obtain compound Ij with a yield of 57.7%. HPLC: 95.10%, MS [M+1]: 521.

[0157] Referring to Example 6, Ic was used as the substrate, acetyl chloride was replaced by cyclopropionyl chloride, and the preparation method was the same as compound Id to obtain compound Il with a yield of 54.4%. HPLC: 96.32%, 1 H NMR (400MHz, Acetone-d6)8.54(s,1H),8.36(s,1H),7.95(m,1H),7.35(m,1H),7.21( m, 1H), 3.85 (s, 3H), 2.82 (s, 3H), 2.39 (m, 1H), 1.30 (m, 1H), 1.00 (m, 2H), 0.91 (m, 2H). MS[M+1]:533.

[0158] Referring to Example 6, Ic was used as the substrate, acetyl chloride was replaced by deuterated acetyl chloride, and the preparation method was the same as compound Id to obtain compound Ir with a yield of 55.6%. HPLC: 95.83%, MS [M+1]: 510.

[0159] 11-2. Preparation of Compound Ib, Compound Ii, Compound Ik and Compound Iq

[0160] Referring to Example 6, Ia was used as a substrate and reacted with acetyl chloride, propionyl chloride, cyclopropionyl chloride, and deuterated acetyl chloride, respectively. The preparation method was the same as compound Id to obtain compound Ib (yield 50.8%, HPLC: 95.33%, MS [M + 1]: 409), compound Ii (yield 55.3%, HPLC: 96.26%, MS [M + 1]: 423), compound Ik (yield 46.5%, HPLC: 96.11%, 1HNMR (400 MHz, Acetone-d6) δ9.93 (s, 1H), 8.59 (s, 1H), 8.45 (s, 1H), 4.05 (s, 3H), 3.16 (s, 3H), 2.81 (s, 3H), 2.41 (m, 1H), 1.30 (m, 1H), 1.00 (m, 2H), 0.90 (m, 2H). MS [M+1]: 435), compound Iq (yield 48.3%, HPLC: 94.88%, MS [M+1]: 412).

[0161] 11-3. Preparation of Compound Ig and Compound Ih

[0162] Referring to Example 6, Ie and If were used as substrates and reacted with acetyl chloride respectively. The preparation method was the same as compound Id to obtain compounds Ig (yield 52.7%, HPLC: 96.37%, MS [M+1]: 435), Ih (yield 48.8%, HPLC: 95.89%, 1 HNMR (400MHz, Acetone-d6) δ 8.56 (s, 1H), 8.43 (s, 1H), 7.88 (m, 1H), 7.64 (m, 1H), 7.15 (m, 1H), 6.28 (s, 1H), 3.82 (s, 3H), 2.73 (s, 3H), 2.37 (s, 3H). MS[M+1]:477).

[0163] Example 12: Preparation of Alkyl-Substituted Amino Target Compounds

[0164] 12-1. Preparation of Compounds In and Ip

[0165]

[0166] Wherein Ra = methyl, cyclopropyl, and compounds In and Ip are prepared respectively.

[0167] Compound 10a (4.83 g, 10 mmol) was dissolved in NMP (50 ml), and methylamine aqueous solution (10 ml) was added, and the temperature was raised to 70°C, and the reaction was carried out for 3 h. The precipitated solid was filtered, washed with water, and purified by column chromatography to obtain compound In, with a yield of 60.2%, HPLC: 96.53%. MS [M+1]: 479.

[0168] Compound Ip was prepared in the same manner by replacing methylamine with cyclopropylamine, with a yield of 57.8%, HPLC: 95.76%, MS[M+1]: 505.

[0169] 12-2. Preparation of Compounds Im and Io

[0170]

[0171] Where Ra = methyl, cyclopropyl, to prepare compounds Im and Io respectively

[0172] Compound 10b (3.85 g, 10 mmol) was dissolved in NMP (50 ml), and methylamine aqueous solution (10 ml) was added, and the temperature was raised to 70°C, and the reaction was carried out for 3 h. The precipitated solid was filtered, washed with water, and purified by column chromatography to obtain compound Im, with a yield of 55.1%, HPLC: 95.74%. MS [M+1]: 381.

[0173] Compound Io was prepared in the same manner by replacing methylamine with cyclopropylamine, with a yield of 53.8%, HPLC: 94.92%, MS[M+1]: 407.

[0174] Example 13, Kinase Activity Test

[0175] Compounds were screened against kinases by ADP-Glo ​​fluorescence assay with ATP concentration of 25 μM. Compounds were screened against mTOR by Lance UltraAssay assay with ATP concentration of Km, and BKM120 and PI103 were used as control compounds.

[0176] PI3Ks

[0177] 1. Compound serial dilution and preparation of source plates: 1) Dilute the compound to 100x the final desired maximum inhibitor concentration in the reaction with 100% DMSO. Transfer 100μl of this compound dilution to the wells of a 384-well plate. For example, if the highest inhibitor concentration required is 10μM, prepare a 1000μM composite DMSO solution in this step. 2) For all compounds, transfer the compound in the tube to one well on a 96-well storage plate and serially dilute the compound by transferring 10μl to 30μl of 100% DMSO to the next well, and so on, for a total of 10 concentrations. 3) In the same 384-well plate, add 50μl of 100% DMSO to two empty wells of the no compound control and no enzyme control. Label this plate as the source plate.

[0178] 2. Prepare assay plates: Transfer 50 nl of compound in 100% DMSO to the assay plate by Echo.

[0179] 3. Prepare 2x Kinase Solution: 1) Prepare PI3Kα and PI3Kγ solutions in 1x Kinase Buffer at a concentration of 2x the final concentration of each reagent in the assay. 2) Add 2.5 μl of Kinase Solution to each well of the assay plate, except for the control wells without enzyme (add 2.5 μl of 1x Kinase Buffer). 3) Shake the plate.

[0180] 4. Prepare 2x substrate solution: 1) Prepare substrate solution of PIP2 substrate and ATP in 1x kinase reaction buffer at a concentration of 2x the final concentration of each reagent required in the assay. 2) Add 2.5 μl of substrate solution to each well of the assay plate to start the reaction. 3) Shake the plate.

[0181] 5. Kinase reaction: Cover the assay plate and incubate at room temperature for 1 hour.

[0182] 6. Kinase assay: 1) Equilibrate ADP-Glo ​​reagent to room temperature. 2) Add 5 μl ADP-Glo ​​reagent to each well to terminate the reaction. 3) Mix briefly in a centrifuge, shake slowly on a shaker, and equilibrate for 120 minutes. 4) Add 10 μl kinase assay reagent to each well, shake for 1 minute, equilibrate for 30 minutes, and then read the luminescence on a plate reader.

[0183] 7. Collect data on Envision and calculate IC50 values.

[0184] Kinase mTOR response

[0185] 1. Prepare 1x kinase buffer for testing kinase mTOR

[0186] 2. Preparation of compounds for testing kinase mTOR:

[0187] 1) Compound serial dilution

[0188] a) Dilute the compound to 100x the final desired maximum inhibitor concentration in the reaction with 100 DMSO. Transfer 100 μl of this compound dilution to a well of a 384-well plate. For example, if the desired maximum inhibitor concentration is 10 μM, prepare a 1000 μM compound DMSO solution in this step.

[0189] b) For all compounds, serially dilute the compound by transferring 10 μl to 30 μl of 100 DMSO in the next well, and so on, for a total of 10 concentrations.

[0190] c) Add 50 μl of 100% DMSO to two empty wells in the same 384-well plate for no compound control and no enzyme control. Label this plate as the source plate.

[0191] 2) Prepare the test plate

[0192] 100 nl of compound was transferred from the source plate to the detection plate by Echo.

[0193] 3. Kinase mTOR response

[0194] 1) Prepare 2x Kinase Solution: Prepare mTOR solution in 1x Kinase Buffer at 2x the final concentration of each reagent in the assay. Add 5 μl of Kinase Solution to each well of the assay plate, except for the control wells without enzyme (add 5 μl of 1x Kinase Buffer). Shake the plate.

[0195] 2) Prepare 2x substrate solution: Prepare substrate solution of ULight-4E-BP1 peptide substrate and ATP in 1x kinase reaction buffer at 2x the final concentration of each reagent required in the assay. Start the reaction by adding 5 μl of substrate solution to each well of the assay plate. Shake the plate.

[0196] 3) Kinase mTOR reaction: Incubate on assay plate and at room temperature for 30 minutes.

[0197] 4. Kinase assay

[0198] 1) Prepare a detection solution of kinase quenching buffer (EDTA) and Eu-anti-phospho-4E-BP1 antibody in Lance detection buffer at 2 times the desired final concentration of each reagent.

[0199] 2) Add 10 μl of detection solution buffer to each well of the detection plate.

[0200] 3) Mix briefly by centrifuging. Cover the assay plate and allow the plate to equilibrate at room temperature for 60 minutes before reading on a plate reader.

[0201] 5. Collect data on Envision and calculate IC50 values.

[0202] The results of the kinase activity test are shown in Table 3 below:

[0203] Table 3

[0204]

[0205]

[0206] Example 14: In vivo tumor inhibition effect

[0207] method:

[0208] 1×10 7 Human gastric cancer HGC27 cells were injected into the armpits of nude mice. After three generations, the HGC27 tumor was removed and placed in a glass dish filled with saline. The surface blood vessels were removed, the necrotic area was removed, and the tumor was cut into 1-2 mm 3 The tumor was inserted into the left armpit of the nude mouse using a trocar. When the tumor grew to an average volume of 100-300 mm 3Afterwards, the animals were randomly divided into groups according to tumor volume and given Ic and Id by gavage every day. During the administration process, the tumor volume was measured twice a week, and the body weight and tumor volume were measured on the 18th day after administration for 17 days. The nude mice were sacrificed and the tumor was weighed to calculate the relative tumor volume (RTV) and relative tumor proliferation rate (T / C), and statistical analysis was performed using SPSS 19.0.

[0209] The calculation formula is as follows:

[0210] (1)TV(tumor volume)=1 / 2×a×b 2 , where a and b represent the length and width of the tumor, respectively;

[0211] (2)RTV(relative tumor volume)=V t / V0, where V0 is the tumor volume measured at the time of group administration (i.e., d0), and Vt is the tumor volume at each measurement;

[0212] (3) T / C (%) = T RTV / C RTV × 100%, where T RTV is the RTV of the treatment group, C RTV is the RTV of the solvent control group;

[0213] (4) IR (%) = (1-TWt / TWc) × 100%, where TWt is the tumor weight of the treatment group and TWc is the tumor weight of the solvent control group.

[0214] result:

[0215] Under the experimental conditions, compound Ic and Id 10 mg / kg intragastrically administered can significantly inhibit the growth of human gastric cancer HGC27 nude mouse transplanted tumors, and no obvious toxicity was observed. The body weight changes of nude mice were as follows: Figure 1 The effects of the two on the volume of human gastric cancer HGC27 nude mice transplanted tumors and tumor volume are shown in Table 4, and the effects on tumor weight are shown in Table 5 and Figure 2 As shown:

[0216] Table 4 Experimental therapeutic effects of compounds Ic and Id on human gastric cancer HGC27 nude mouse transplanted tumors

[0217]

[0218]

[0219] t-test, compared with Vehicle, ***: P < 0.001, **: P < 0.01, *: P < 0.05.

[0220] Table 5: Effects of Compounds Ic and Id on Tumor Weight of Human Gastric Cancer HGC27 Xenografts in Nude Mice

[0221]

[0222] t-test, compared with Vehicle, ***: P < 0.001, **: P < 0.01, *: P < 0.05.

[0223] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A 7-methylthiazolo[5,4-d]pyrimidine compound, comprising a compound represented by formula (I) or a physiologically acceptable salt thereof: in, R1 is hydrogen or acetyl; R2 is 2,4-difluoro-substituted benzene ring group.

2. A method for preparing a 7-methylthiazolo[5,4-d]pyrimidine compound according to claim 1, characterized in that: The method comprises the following steps: S1. Compound With compound Coupling reaction to obtain amino target compounds Or S2, amino compounds With R b X undergoes acylation reaction to obtain the acylated target compound R b is an acetyl group, and X is a halogen atom.

3. The method for preparing 7-methylthiazolo[5,4-d]pyrimidine compounds according to claim 1, characterized in that: Compound With compound The coupling reaction gave the compound Further amination reaction gave the compound or compound With R b X undergoes acylation to give compound Among them, R b is an acetyl group, and X is a halogen atom.

4. The method for preparing 7-methylthiazolo[5,4-d]pyrimidine compounds according to claim 2, characterized in that: Compound It is prepared by the following steps: A1. 5-Bromo-2-methoxypyridin-3-amine and compounds The compound was obtained by reaction in the presence of DMAP and pyridine. A2. Compounds In the presence of potassium acetate, PdCl2 and dioxane, it reacts with diboric acid pinacol ester to obtain the compound 5. The method for preparing 7-methylthiazolo[5,4-d]pyrimidine compounds according to claim 2, characterized in that: Compound It is prepared by the following steps: B1. Compounds The compound is obtained by reacting in the presence of iron powder and acetic acid. B2. Compounds The compound is obtained by reacting in the presence of potassium thiocyanate and acetic acid. B3, compound The compound was prepared by reaction with isoamyl nitrite B4. Compounds The compound was obtained by reaction in the presence of benzophenone imine, BINAP, Pd2(dba)3 and Cs2CO3. B5. Compounds Reaction with citric acid to obtain compound B6. Compounds Bromination to give compound 6. Use of the 7-methylthiazolo[5,4-d]pyrimidine compound according to claim 1 in the preparation of a drug for treating PI3 kinase-related diseases.

7. Use of the 7-methylthiazolo[5,4-d]pyrimidine compound according to claim 1 in the preparation of anti-tumor drugs.

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