CN116262758A

CN116262758A - 7-methylthiazolo [5,4-d ] pyrimidine compound, preparation method and application thereof

Info

Publication number: CN116262758A
Application number: CN202111534335.8A
Authority: CN
Inventors: 谢军; 姜春阳; 李惠; 李红昌; 周小群
Original assignee: Shanghai Scienpharm Biotechnology Co ltd
Current assignee: Shanghai Scienpharm Biotechnology Co ltd
Priority date: 2021-12-15
Filing date: 2021-12-15
Publication date: 2023-06-16
Also published as: WO2023109158A1

Abstract

The invention discloses a 7-methylthiazolo [5,4-d ]]Pyrimidine compounds, a preparation method and application thereof; comprising a compound of formula (I) or a physiologically acceptable salt thereof:

wherein R is ₁ Is hydrogen, substituted or unsubstituted alkyl, cycloalkyl, substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted cycloalkylcarbonyl; r is R ₂ Is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aromatic heterocyclic group. The PI3K inhibitor compound has novel structure, excellent inhibition activity to PI3K kinase, obviously improved selectivity of PI3K alpha/mTOR, reduced safety risk caused by strong inhibition of mTOR kinase through regulation of mTOR enzyme activity, and reduced toxicity to partial RTK enzymeThe induced risk has better drug effect and lower toxicity.

Description

7-methylthiazolo [5,4-d ] pyrimidine compound, preparation method and application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a 7-methylthiazolo [5,4-d ] pyrimidine compound, a preparation method and application thereof.

Background

The phosphoinositide-3-kinase (pi 3 k) family is a class of kinases that specifically catalyze the phosphorylation of the hydroxyl group at the 3-position of phosphoinositides and produce inositol lipids with a second messenger effect. The signaling pathway formed by PI3K and its downstream molecules, signaling protein kinase B (Akt)/rapamycin target protein (mTOR), is one of the most important intracellular signaling pathways in mammals, critical for a variety of important physiological functions including cell cycle, cell survival, protein synthesis and growth, metabolism, motility, and angiogenesis. PI3ks, as a family of lipid kinases, can be divided into three main classes (I, II and class III) based on their structure and substrate specificity. Class I PI3ks are in turn divided into two major subclasses, class IA and class IB, wherein class IA PI3ks include pi3kα, pi3kβ, pi3kδ, consisting of their corresponding catalytic subunits p110 (p110α, p110β, p110δ) and regulatory subunit p 85; class IB (PI 3kγ) then consists of catalytic subunit p110γ and regulatory subunit p101 or p 87.

U.S. FDA approval idelania was developed by Gileyd Sciences (Gilead Sciences) on 7.23.2014, and European EMA was approved by 18.2014 on 9.trade name

Edranin is a PI3K delta kinase inhibitor, and can induce apoptosis and inhibit cell proliferation. It is used for treating recurrent Chronic Lymphocytic Leukemia (CLL), recurrent follicular B cell non-hodgkin lymphoma (FL) and recurrent Small Lymphocytic Lymphoma (SLL).

On day 14 of 9 in 2017, the U.S. FDA accelerated approval of Bayer's Copanlisib to the market under the trade name

For use in treating adult patients suffering from recurrent follicular lymphoma and having received at least two systemic therapies, copanlisib is a PI3K inhibitor that inhibits both PI3K- α and PI3K- δ kinase subtypes.

Buparlisib (BKM 120), developed by North China, is a pan-class I PI3K inhibitor acting on p110α/β/δ/γ. For use in clinical studies in the treatment of locally advanced or metastatic breast cancer that is positive for postmenopausal estrogen receptors, negative for HER 2. The company is also conducting a second-stage clinical study for the treatment of follicular lymphoma, gastrointestinal stromal tumor, 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 glioblastoma. In addition, the obtained PI3K inhibitor approved by FDA and marketed by duvelisib and alpelisib has wide application prospect as phosphatidylinositol-3-kinase inhibitor (PI 3K). Notably, the PI3K inhibitors currently on the market have seen significant toxic side effects in clinical applications, such as autoimmune dysfunction, opportunistic infections, skin toxicity, hypertension and hyperglycemia, etc., both idelalisib and duvelisib being alerted by the FDA to the black frame. In addition, inhibition of PI3K signaling mediates the negative feedback loop and/or induces activation of other proliferative signaling pathways, thereby attenuating the antitumor efficacy of PI3K inhibitors. To overcome these major obstacles, it is highly desirable to develop novel PI3K inhibitor compounds that are structurally different, have better potency, and lower toxicity.

Disclosure of Invention

The invention aims to develop a 7-methylthiazolo [5,4-d ] pyrimidine compound, a preparation method and application thereof. The 7-methylthiazolo [5,4-d ] pyrimidine PI3K inhibitor compound has a novel structure different from that of the existing compounds, shows antiproliferative activity, and is therefore useful for the prevention or treatment of diseases associated with hyper-proliferation, particularly for the preparation of a medicament for inhibiting PI3 kinase.

The aim of the invention is realized by the following technical scheme:

the invention provides a 7-methylthiazolo [5,4-d ] pyrimidine compound which comprises a compound shown in a formula (I) or physiologically acceptable salt thereof:

wherein R is ₁ Is hydrogen, substituted or unsubstituted alkyl, cycloalkyl, substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted cycloalkylcarbonyl;

R ₂ is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aromatic heterocyclic group.

As one embodiment of the present invention, R ₁ Any one selected from hydrogen, acetyl, propionyl, cyclopropylcarbonyl, 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, thiophene-2-group and cyclopropyl.

As an embodiment of the present invention, any one selected from the following compounds:

the invention also provides a preparation method of the 7-methylthiazolo [5,4-d ] pyrimidine compound, which comprises the following steps:

s1, compounds

And the compound->

Coupling reaction to obtain amino target compound->

Or S2, amino compounds

And R is R _b X is subjected to acylation reaction to obtain an acylated target compound +.>

R _b Is an alkyl acyl group, X is a halogen atom;

or S3, compound

And the compound->

Coupling reaction to obtain compound

Further with R _a -NH ₂ Amination reaction is carried out to obtain the target compound of alkyl or cycloalkyl substituted amino>

Wherein R is _a Is alkyl or cycloalkyl.

As one embodiment of the invention, the preparation method of the 7-methylthiazolo [5,4-d ] pyrimidine compound comprises the following steps:

compounds of formula (I)

And the compound->

Coupling reaction to obtain compound

Further amination to give the compound->

Or a compound

And R is R _b X is subjected to acylation reaction to obtain a compound

Wherein R is _b Is an alkanoyl group, and X is a halogen atom.

As one embodiment of the invention, the coupling reaction is carried out in potassium carbonate, dioxane and PdCl ₂ In the presence of a catalyst.

As one embodiment of the invention, the acylation reaction is carried out in the presence of pyridine in DCM as a solvent.

As one embodiment of the present invention, the amination reaction is carried out by using NMP as a solvent and ammonia water.

As one embodiment of the present invention, the compounds

Is prepared by the following steps:

a1, 2-bromo-4-amino-5-methoxypyridine and compounds

Reacting in the presence of DMAP and pyridine to obtain the compound +.>

A2, compound

In potassium acetate, pdCl ₂ And a dioxane in the presence of a bisboronic acid pinacol ester to give the compound +.>

As one embodiment of the present invention, the compounds

Is prepared by the following steps:

b1, compound

Reacting in the presence of iron powder and acetic acid to obtain the compound +.>

B2, compound

Reacting in the presence of potassium thiocyanate and acetic acid to obtain the compound

B3, compound

Reacting with isoamyl nitrite to obtain the compound ∈K>

B4, compound

In benzhydrylamine, BINAP, pd ₂ (dba) ₃ And Cs ₂ CO ₃ Reacting in the presence of a compound +.>

B5, compound

Reacting with citric acid to obtain compound->

B6, compound

Re-bromination to give the compound->

As one embodiment of the present invention, the compounds

Is prepared by the following steps:

c1, compounds

C2, compounds

C3, compounds

Bromination reaction is carried out in the presence of tert-butyl nitrite and acetonitrile to obtain the compound +.>

The invention also provides 7-methylthiazolo [5,4-d ]]Use of pyrimidine PI3K inhibitor compounds in the manufacture of a medicament for the treatment of PI3 kinase related diseases. In particular to the application in preparing the medicine for treating tumor diseases. The compounds of the present invention show excellent inhibition in various tumor cell linesEffect, in particular Compound Id IC in lymphoma cell line DOHH2 ₅₀ The value is 0.063 μm, which is greatly better than 0.53 μm of the control BKM 120.

Compared with the prior art, the invention has the following beneficial effects:

1) The 7-methylthiazolo [5,4-d ] pyrimidine PI3K inhibitor compound has novel structure, excellent inhibition activity on PI3K kinase, remarkably improved selectivity of PI3K alpha/mTOR, and can weaken safety risks caused by strong inhibition of mTOR kinase, such as hyperglycemia, and risks caused by induction of partial RTK enzyme through regulation of mTOR enzyme activity, and has better drug effect and lower toxicity.

2) The tumor inhibition experiment in animals shows that the compound of the invention can achieve excellent tumor inhibition effect at a lower dosage, the weight of animals is basically unchanged, the compound has good safety, and meanwhile, the compound shows good metabolic performance.

3) The invention also provides a simple preparation method of the 7-methylthiazolo [5,4-d ] pyrimidine PI3K inhibitor compound.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 is the effect of compounds Ic and Id on weight change in human gastric cancer HGC27 nude mice;

fig. 2 is an experimental therapeutic effect of compounds Ic and Id on human gastric cancer HGC27 nude mice transplantation tumor.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that several modifications and improvements can be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention. All publications mentioned are incorporated by reference in their entirety.

One embodiment of the invention includes a compound of formula (I):

or a physiologically acceptable salt thereof, wherein:

R ₁ is hydrogen, substituted or unsubstituted alkyl, cycloalkyl, substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted cycloalkylcarbonyl;

The alkyl is C1-C8 alkyl, the cycloalkyl is C3-C8 cycloalkyl, the alkylcarbonyl is C2-C8 alkylcarbonyl, and the cycloalkylcarbonyl is C4-C8 cycloalkylcarbonyl; aryl is phenyl and naphthyl, aromatic heterocyclic groups are thienyl, thiazolyl, pyrazolyl, pyridyl and furyl, and substituent groups are halogen and alkyl.

In a preferred embodiment, the invention includes compounds of formula (I) wherein R ₁ Any one of hydrogen, acetyl, propionyl, cyclopropylformyl, deuterated acetyl, methyl and cyclopropyl.

In a further preferred embodiment, the invention comprises 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, cyclopropyl.

In a further preferred embodiment, the invention includes any one of the compounds of the following formulas or a physiologically acceptable salt thereof.

TABLE 1

/>

If there is a difference between the chemical nomenclature and the illustrated chemical structure, the illustrated chemical structure is preferred over the chemical nomenclature given.

Without being limited to theory or mechanism, the compounds of the present invention exhibit unexpected phosphatidylinositol-3-kinase inhibitory activity as well as chemical and structural stability. This unexpected activity is believed to be based on the structure of the compounds, in particular the parent nucleus of these compounds (7-methylthiazolo [5, 4-d)]Pyrimidine compounds). In addition, R is appropriately selected in consideration of the activity in vivo ₁ And R is ₂ Providing the necessary activity for the appropriate isoform.

The invention also provides a preparation method of the 7-methylthiazolo [5,4-d ] pyrimidine PI3K inhibitor compound.

Preparation of Compounds Ic and Id: preparing an intermediate 3 by taking the compound 1 as a starting material, then brominating to prepare an intermediate 8, coupling to prepare an intermediate 10, and aminating to prepare a target compound Ic; the target compound Id was obtained from Ic acetylation. Specific reaction scheme 1:

/>

scheme 1

Intermediate 3 was prepared using compound 1 as starting material, and key intermediate 7 was further prepared, see scheme 2:

scheme 2

Amino-based target compounds: the 5-bromo-2-methoxypyridine-3-amine is used for preparing a borate intermediate 9, and then the borate intermediate 9 is coupled with the intermediate 7 to prepare the amino target compound, and the specific reaction scheme is shown in a reaction scheme 3.

Scheme 3

Wherein R2 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aromatic heterocyclic group, preferably a2, 4-difluoro-substituted benzene ring group, a 4-fluoro-substituted benzene ring group, a methyl group, a thiophen-2-yl group, a cyclopropyl group;

the amino group target compounds are compounds such as compound Ic, compound Ia, compound Ie and compound If.

Preparation of alkyl-substituted amino-based target compounds: in the reaction scheme 4, 5-bromo-2-methoxypyridine-3-amine is used for preparing a borate intermediate 9, and then coupled with the intermediate 8 to prepare a target compound 10, and then further prepared into an alkyl substituted amino compound.

Scheme 4

Wherein Ra is alkyl, cycloalkyl, preferably methyl, cyclopropyl;

R ₂ is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aromatic heterocyclic group, preferably a2, 4-difluoro-substituted benzene ring group, a 4-fluoro-substituted benzene ring group, a methyl group, a thiophen-2-yl group, a cyclopropyl group;

the alkyl substituted amino compounds are compounds In, ip, im, io and the like.

Preparation of acylated target compound: the amino target compound is taken as a substrate to carry out acylation reaction to prepare the acylated target compound, and the specific reaction scheme is shown in a reaction scheme 5.

Scheme 5

Wherein R is _b Is an alkanoyl group, preferably acetyl, propionyl, cyclopropoyl, deuterated acetyl;

x is chlorine, bromine or fluorine,

R ₂ is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aromatic heterocyclic group, preferably a2, 4-difluoro substituted benzene ring group, a 4-fluoro substituted benzene ring group, a methyl group, a thiophen-2-yl group, a cyclopropyl group or the like.

The acylated target compounds such as compound Ij, compound Il, compound Ir, compound Ib, compound Ii, compound Ik, compound Iq, compound Ig, compound Ih and the like.

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

Compound 1 (4.16 g,1 eq) was dissolved in 100ml acetic acid and cooled to 5-10 ℃ in an ice-water bath, iron powder (5 g,4.5 eq) was added in portions and the reaction was continued at room temperature for 2h. Post-treatment: the reaction solution was filtered, and the filtrate was concentrated. The residual liquid is dissolved in water, extracted for 3 times by ethyl acetate, washed once by saturated sodium chloride aqueous solution, and the organic phase is decompressed and concentrated to obtain the compound 2, and the yield is 85.2%. HPLC purity 99.56%; MS [ M+1]:178.

compound 2 (3.56 g,1 eq) was dissolved in 30ml of acetic acid, potassium thiocyanate (1.94 g,1 eq) was added, and the mixture was heated under reflux for 3h. After the reaction is finished, the reaction solution is cooled to room temperature, water is added dropwise, and solids are gradually separated out; the filtration gave an earthy solid, compound 3, 90.6% yield. HPLC purity 94.27%; MS [ M+1]:201.

example 2 preparation of 2-bromo-5-chloro-7-methylthiazolo [5,4-d ] pyrimidine

Compound 3 (4.0 g,1 eq) was dissolved in 10ml acetonitrile and cooled to 0deg.C in an ice-water bath followed by the slow addition of tert-butyl nitrite (3.0 g,1.5 eq) followed by the addition of copper bromide (5.9 g,1.25 eq). Keeping at 0 ℃ for 30min, naturally heating to room temperature, and continuing to react for 2.5h. The reaction mixture was added with water, and extracted 3 times with ethyl acetate. The organic layer was washed 2 times with saturated aqueous sodium chloride solution. The organic phase was concentrated under reduced pressure to give compound 8 in 88.5% yield. HPLC purity 87.54%; MS [ M+1]:264.

EXAMPLE 3 preparation of N- (2-methoxy-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-3-yl) sulfonamide (Compound 9)

The following borate compounds are prepared by using sulfonic anhydride or sulfonyl chloride and adopting a similar method:

wherein R is ₂ Compound 9 corresponds to 9a, 9b, 9c and 9d, respectively, as 2, 4-difluoro-group, methyl, cyclopropyl, thiophen-2-yl, table 2 below:

TABLE 2

9a: preparation of 2, 4-difluoro-N- (2-methoxy-5- (4, 5-tetramethyl-1, 3, 2-dioxabor-2-yl) pyridin-3-yl) benzenesulfonamide

Step 1: three-port flask was filled with 2-bromo-4-amino-5-methoxypyridine (10.15 g,0.05 mol), DMAP (1.22 g), pyridine (7.12 g,0.09 mol) in DCM (61 ml), and the temperature was lowered to-10 to 0℃N ₂ 3 substitutions. 2, 4-difluorobenzenesulfonyl chloride (13.8 g,0.065 mol) in DCM (61 mL) was added dropwise and the reaction monitored by TLC until complete; the reaction solution was poured into 100ml of water,and (5) stirring and separating the liquid. The organic phase was washed once with water (100 ml), saturated sodium bicarbonate (100 ml), water (100 ml). The organic phase was concentrated to dryness under reduced pressure and the concentrated residue was recrystallized from ethyl acetate. Intermediate S9a was obtained in 82.6% yield. 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)。

Step 2: in a three-necked flask, the above-mentioned product S9a (18.9 g), potassium acetate (14.7 g), pinacol biborate (15.6 g), pdCl were introduced ₂ (dppf) (1.24 g), dioxane (330 ml), and stirring. Nitrogen was purged 3 times. The temperature was raised to 90℃and the reaction was carried out overnight.

After completion of the reaction, ethyl acetate (300 ml) and 300ml of water were added to the reaction mixture, the mixture was stirred and separated, and the aqueous layer was extracted once with 150ml of ethyl acetate. The organic phases were combined, filtered through celite, and the filtrate was washed once with saturated brine. Adding active carbon, heating and stirring for 1h; filtering, concentrating under reduced pressure, purifying by silica gel column chromatography, and recrystallizing with acetonitrile-water to obtain compound 9a with 77.5% yield. 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)。

9b: preparation of N- (2-methoxy-5- (4, 5-tetramethyl-1, 3, 2-dioxaboran-2-yl) pyridin-3-yl) methanesulfonamide

Step 1: 2-bromo-4-amino-5-methoxypyridine (10.15 g,0.05 mol), DMAP (1.22 g), pyridine (7.12 g,0.09 mol) were dissolved in DCM (61 ml), cooled to-10-0℃and nitrogen purged 3 times. A solution of methanesulfonic anhydride (11.37 g,0.065 mol) in DCM (61 mL) was added dropwise and the reaction was followed by TLC until complete.

After the completion of the reaction, the reaction mixture was poured into 100ml of water, and the mixture was separated by stirring. The organic phase was washed successively with water (100 ml), saturated sodium bicarbonate (100 ml), water (100 ml). The organic phase was concentrated to dryness under reduced pressure and recrystallized from ethyl acetate to give compound S9b in 81.3% yield. 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)。

Step 2: three-necked flask was charged with S9b (28.1 g), potassium acetate (29.4 g),Pinacol biborate (31.2 g), pdCl ₂ (dppf) (2.5 g) was dissolved in dioxane (660 ml). Nitrogen was purged 3 times. The temperature was raised to 90℃and the reaction was carried out overnight.

At the end of the reaction, ethyl acetate (600 ml) and 600ml of water were added, the layers were separated by stirring, and the aqueous layer was extracted with 300ml of ethyl acetate. The organic phase was filtered through celite, and the filtrate was washed with saturated brine. Adding active carbon, heating and stirring for 1h; filtering and concentrating. The residue was passed through a column and recrystallized from acetonitrile-water. Drying at 60 ℃. Compound 9b was obtained in 75.8% yield. 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)。

9c: preparation of N- (2-methoxy-5- (4, 5-tetramethyl-1, 3, 2-dioxaboran-2-yl) pyridin-3-yl) cyclopropanesulfonamide

The procedure of steps 1 and 2 of example 9a was followed except that cyclopropanesulfonyl chloride was used instead of 2, 4-difluorobenzenesulfonyl chloride to give compound 9c in 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)。

9d: preparation of N- (2-methoxy-5- (4, 5-tetramethyl-1, 3, 2-dioxaboran-2-yl) pyridin-3-yl) thiophene-2-sulfonamide

Thiophene-2-sulfonyl chloride was used in place of 2, 4-difluorobenzenesulfonyl chloride, and the remainder was the same as in steps 1 and 2 of example 9a to give compound 9d in 58.8% two-step yield. 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)。

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

Preparation of 4-1 Compound 10a

Adding water (2 ml) to compound 8 (0.265 g,1 eq), compound 9a (0.511g, 1.2 eq), potassium carbonate (0.553 g,4 eq)Six rings (6 ml) and the solid was dissolved with stirring and replaced 3 times with nitrogen. Adding PdCl ₂ (dppf) (0.074 g,0.1 eq) and nitrogen was replaced 3 times. Heating to 80 ℃, and preserving heat for reaction for 8 hours. After the completion of the reaction, the reaction was quenched with water, extracted 3 times with ethyl acetate, 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 give solid compound 10a in 45.3% yield. HPLC purity 95.62%;1H NMR (400 MHz, DMSO-d 6) δ10.63 (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。

Preparation of 4-2 Compound 10b

Compound 8 (0.265 g,1 eq), compound 9b (0.393 g,1.2 eq), potassium carbonate (0.553 g,4 eq) were added with water (2 ml), dioxane (6 ml), stirred, and purged 3 times with nitrogen. Adding PdCl ₂ (dppf) (0.074 g,0.1 eq) and nitrogen was replaced 3 times. Heating to 80 ℃, and preserving heat for reaction for 8 hours. After the reaction, water was added to quench the reaction, extraction was performed 3 times with ethyl acetate, the organic layer was washed once with saturated brine, and the organic phase was concentrated to dryness under reduced pressure, followed by purification by silica gel column chromatography to give solid compound 10b in a yield of 52.1%. HPLC purity 96.44%; MS [ M+1]]：386。

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)

Compound 10a (7.02 g,14.5 mmol) was dissolved in NMP (50 ml), 28% ammonia (10 ml) was added, and the temperature was raised to 70℃for 3h. After the reaction, cooling to room temperature, precipitating solid, filtering, washing a filter cake with water, and using PE: EA (2:1) was the mobile phase and was chromatographed on silica gel to give compound Ic (yield 85%). ¹ H 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。

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)

Compound Ic (0.929 g,1 eq) and pyridine (0.470 g,3 eq) were dissolved in DCM (5 ml), cooled to 0deg.C, and a solution of acetyl chloride (0.172 g,1.2 eq) in DCM (2 ml) was added dropwise. The temperature is kept between 0 and 5 ℃ and the reaction is carried out for 6 to 8 hours. After the completion of the reaction, the reaction mixture was washed once with 10% hydrochloric acid, saturated sodium bicarbonate and water. The organic phase was concentrated to dryness under reduced pressure, and the organic phase was dried over PE: EA (2:1) was the mobile phase and purified by silica gel column chromatography to give compound Id in 60.5% yield. 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。

EXAMPLE 7 preparation of Compound 4

A mixture of compound 3 (4 g,20mmol,1.0 eq) in acetonitrile (30 mL) was added to isoamyl nitrite (7.0 g,60mmol,3.0 eq) and stirred at room temperature for 3h. LCMS showed complete reaction. The mixture was diluted with ethyl acetate, washed with water and brine, and dried over Na ₂ SO ₄ Drying above, and concentrating. The residue was purified by column chromatography to give compound 4 (2 g,50% yield) as a yellow solid.

Example 8 preparation of Compound 5

Compound 4 (2 g,10.8mmol,1.0 eq)) A mixture of benzhydrylamine (2.4 g,12.9mmol,1.2 eq) in toluene (20.0 ml) was added to BINAP (684 mg,1.1mmol,0.1 eq), pd ₂ (dba) ₃ (1 g,1.1mmol,0.1 eq) and Cs ₂ CO ₃ (7 g,21.6mmol,2.0 eq). The mixture was stirred at 120℃for 16 hours. LCMS showed complete reaction. The mixture was diluted with EA, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by prep TLC to give compound 5 (1 g,36% yield) as a yellow solid.

Example 9 preparation of 2-bromo-7-methylthiazolo [5,4-d ] pyrimidin-5-amine (compound 7)

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

A mixture of Compound 6 (400 mg,2.4mmol,1.0 eq) and NBS (254 mg,4.8mmol,2.0 eq) in DMF (15 ml) was stirred at room temperature for 5h. LCMS showed complete reaction. The resulting product was diluted with ethyl acetate, washed with water and brine, and dried over Na ₂ SO ₄ Drying above, and concentrating. The residue was purified by prep TLC to give compound 7 (165 mg,35% yield) as a yellow solid. ¹ HNMR (400 mhz, dmso): 2.55 (s, 3H), 7.06 (s, 2H). LCMS: rt:1.287 minutes; MS [ M+1]]：245，247。

EXAMPLE 10 preparation of amino-based Compounds of interest

Wherein R is ₂ 2, 4-difluorophenyl, methyl, cyclopropyl and 2-thienyl, compound 9 corresponds to 9a, 9b, 9c and9d, respectively reacting with the compound 7 to obtain the compound Ic, ia, ie, if.

Preparation of Compound Ic

Three-port bottles were filled with 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), dioxane (5 ml) and nitrogen replaced 3 times. PdCl2 (dppf) (0.09 g) was added. N2 was replaced 3 times. Reacting for 4-6h at 85-95 ℃.

Pouring the reaction solution into water, and extracting with ethyl acetate for 3 times; washing with saturated saline for one time; the organic phase was dried over a suitable amount of anhydrous sodium sulfate, filtered, and concentrated. Purifying the crude product by column to obtain the product compound Ic with the yield of 50.7%, HPLC:97.16%, MS [ M+1]:465.

preparation of Compound Ia

The compound Ia was obtained in 43.2% yield by the same method as that of Ic in this example using the compound 9b as a reactant. HPLC:95.65%, MS [ M+1]:367.

preparation of Compound Ie

The compound Ie was obtained in 45.1% yield by the same method as that of Ic in this example using compound 9c as a reactant. HPLC:95.10%,1H NMR (400 MHz, acetone-d 6) delta 7.57 (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.

preparation of Compound If

The compound If was obtained in 39.8% yield by using the compound 9d as a reactant in the same manner as the Ic in this example. HPLC:96.59%,1H NMR (400 MHz, acetone-d 6) 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.

EXAMPLE 11 preparation of aminoacylation Compounds of interest

Preparation of 11-1, compound Ij, compound Il and Compound Ir

Wherein R is _b Compounds Ij, il and Ir are prepared respectively for propionyl, cyclopropoyl and deuterated acetyl.

Referring to example 6, using Ic as the substrate, acetyl chloride was substituted for Cheng Bingxian chloride and the same procedure was used to prepare compound Id, compound Ij was obtained in 57.7% yield. HPLC:95.10%, MS [ M+1]:521.

referring to example 6, using Ic as substrate, acetyl chloride was replaced with cyclopropylchloride, which was prepared in the same manner as compound Id to give compound Il in 54.4% yield. 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。

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

preparation of 11-2, compound Ib, compound Ii, compound Ik and Compound Iq

Reference example 6, reaction with acetyl chloride, propionyl chloride, cyclopropoyl chloride, deuterated acetyl chloride, respectively, with Ia as substrate, preparation method was the same as for Compound Id, compound Ib (yield 50.8%, HPLC:95.33%, MS [ M+1 ]) was obtained in this order]:409 Compound Ii (yield 55.3%, HPLC:96.26%, MS [ M+1]]:423 Compound Ik (yield 46.5%, HPLC:96.11%, ¹ H NMR(400MHz,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)。

Preparation of 11-3, compound Ig and Compound Ih

Reference example 6, reaction with acetyl chloride respectively using Ie and If as substrates, preparation method and compound Id, compound Ig (yield 52.7%, HPLC:96.37%, MS [ M+1 ]) was obtained in this order]:435 Ih (yield 48.8%, HPLC:95.89 percent, ¹ H NMR(400MHz,Acetone-d6)δ8.56(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)。

EXAMPLE 12 preparation of alkyl-substituted amino class of target Compounds

Preparation of 12-1, compounds In, ip

Wherein ra=methyl, cyclopropyl, compounds In and Ip were prepared, respectively.

Compound 10a (4.83 g,10 mmol) was dissolved in NMP (50 ml), aqueous methylamine (10 ml) was added, and the temperature was raised to 70℃and reacted for 3 hours. The precipitated solid was filtered, washed with water, and purified by column chromatography to give compound In, yield 60.2%, HPLC:96.53%. MS [ M+1]:479.

the same procedure as for the conversion of methylamine to cyclopropylamine gives compound Ip in 57.8% yield by HPLC:95.76%. MS [ M+1]:505.

preparation of 12-2, compounds Im, io

Wherein Ra=methyl, cyclopropyl, compounds Im and Io are prepared respectively

Compound 10b (3.85 g,10 mmol) was dissolved in NMP (50 ml), aqueous methylamine (10 ml) was added, and the temperature was raised to 70℃and reacted for 3 hours. The precipitated solid was filtered, washed with water, and purified by column chromatography to give compound Im in 55.1% yield, HPLC:95.74%. MS [ M+1]:381.

the same procedure was used to prepare compound Io in 53.8% yield by HPLC:94.92%. MS [ M+1]:407.

example 13 kinase Activity test

Compounds were screened for kinase by ADP-Glo fluorescence assay at an ATP concentration of 25. Mu.M. Compounds were screened for mTOR by the Lance Ultra Assay assay, ATP concentration of Km, and BKM120 and PI103 were used as control compounds.

Kinase PI3Ks

1. Compound serial dilution and preparation of source plate: 1) The compound was diluted with 100% dmso to the highest inhibitor concentration 100x ultimately required in the reaction. 100 μl of this compound dilution was transferred to the wells of 384 well plates. For example, if the highest inhibitor concentration required is 10. Mu.M, a 1000. Mu.M complex DMSO solution is prepared in this step. 2) For all compounds, compounds in the tube were transferred to one well on a 96-well storage plate and serially diluted by transferring 10 μl to 30 μl of 100% dmso to the next well, and so on, for a total of 10 concentrations. 3) In the same 384 well plate, 50 μl of 100% dmso was added to both wells of the compound-free control and the enzyme-free control. This plate is denoted as source plate.

2. Preparing a detection plate: 50nl of compound was transferred to the assay plate by Echo in 100% DMSO.

3. Preparing a 2x kinase solution: 1) PI3kα and PI3kγ solutions were prepared in 1x kinase buffer at a concentration 2 times the final concentration of each reagent in the assay. 2) To each well of the assay plate was added 2.5 μl of kinase solution, control Kong Chuwai without enzyme (2.5 μl of 1x kinase buffer was added). 3) The tray is rocked.

4. Preparing a 2x substrate solution: 1) A substrate solution of PIP2 substrate and ATP was prepared in 1x kinase reaction buffer at a concentration of 2 times the final concentration of each reagent required in the assay. 2) The reaction was started by adding 2.5. Mu.l of substrate solution to each well of the assay plate. 3) The tray is rocked.

5. Kinase reaction: the assay plate was covered and incubated for 1 hour at room temperature.

6. Kinase detection: 1) The ADP-Glo reagent was equilibrated to room temperature. 2) The reaction was stopped by adding 5. Mu.l ADP-Glo reagent per well 3) briefly mixed with a centrifuge, slowly shaken on a shaker, equilibrated for 120 min 4) 10ul kinase detection reagent per well, shaken for 1 min, equilibrated for 30min, and then read luminescence on a plate reader.

7. Data were collected on Envision and IC50 values were calculated.

Kinase mTOR response

1. Preparation of 1x kinase buffer for testing kinase mTOR

2. Preparation of compounds for testing the kinase mTOR:

1) Serial dilution of compounds

a) The compound was diluted with 100DMSO to the final desired highest inhibitor concentration in the reaction of 100x. 100 μl of this compound dilution was transferred to the wells of 384 well plates. For example, if the highest inhibitor concentration required is 10. Mu.M, a 1000. Mu.M complex DMSO solution is prepared in this step.

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

c) Mu.l of 100% DMSO was added to two wells in the same 384 well plate for the compound-free control and the enzyme-free control. This plate is denoted as source plate.

2) Preparing a detection plate

100nl of compound was transferred from the source plate to the assay plate by Echo.

3. Kinase mTOR response

1) Preparing a 2x kinase solution: mTOR solution was prepared in 1x kinase buffer at a concentration 2 times the final concentration of each reagent in the assay. To each well of the assay plate 5 μl of kinase solution, enzyme free control Kong Chuwai (5 μl of 1x kinase buffer was added). The tray is rocked.

2) Preparing a 2x substrate solution: a substrate solution of ULIGHT-4E-BP1 peptide substrate and ATP was prepared in 1 Xkinase reaction buffer at a concentration of 2 times the final concentration of each reagent required in the assay. The reaction was started by adding 5. Mu.l of substrate solution to each well of the assay plate. The tray is rocked.

3) Kinase mTOR response: incubate on assay plate and at room temperature for 30 min.

4. Kinase assay

1) A detection solution of kinase quenching buffer (EDTA) and Eu-anti-phospho-4E-BP1 antibody was prepared in Lance detection buffer at 2 times the required final concentration of each reagent.

2) Mu.l of assay solution buffer was added to each well of the assay plate.

3) Mix briefly with a centrifuge. The assay plates were capped, allowed to equilibrate for 60 minutes at room temperature, and then read on a plate reader.

5. Data were collected on Envision and IC50 values were calculated.

The kinase activity test results are shown in Table 3 below:

TABLE 3 Table 3

Example 14 in vivo tumor inhibiting Effect

The method comprises the following steps:

will be 1X 10 ⁷ Injecting human gastric cancer HGC27 cells into armpit of nude mouse, transferring to three generations, dissecting HGC27 mouse tumor blocks, placing into a glass dish containing normal saline, removing surface blood vessel, cutting to remove necrotic region, and cutting tumor blocks into 1-2 mm ³ Tumor mass was accessed with a trocar under the left armpit of a nude mouse. When the tumor grows to an average volume of 100-300 mm ³ Animals were then randomly grouped by tumor volume and given Ic and Id by intragastric administration daily. During dosing, tumor volumes were weighed and measured 2 times per week, dosing was performed for 17 days, body weights were measured on day 18, tumor volumes were measured, nude mice were sacrificed for tumor mass weighing, relative Tumor Volumes (RTV), relative tumor proliferation rates (T/C) were calculated, and statistical analysis was performed using SPSS 19.0.

The calculation formula is as follows:

(1)TV(tumor volume)＝1/2×a×b ² wherein a and b respectively represent the length and width of the tumor;

(2)RTV(relative tumor volume)＝V _t /V ₀ where V0 is the tumor volume measured at the time of group administration (i.e., d 0), and Vt is the tumor volume at each measurement;

(3)T/C(％)＝T _RTV /C _RTV x 100%, where T _RTV RTV, C for treatment group _RTV RTV as solvent control;

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

Results:

under the experimental conditions, the compound Ic and Id 10mg/kg can obviously inhibit the growth of human gastric cancer HGC27 nude mice transplanted tumor by gastric lavage administration, and no obvious toxicity is seen, and the weight change of the nude mice is shown in figure 1. The effect of the two on the tumor volume and the transplanted tumor volume of the human gastric cancer HGC27 nude mice is shown in Table 4, and the effect on the tumor weight is shown in Table 5 and FIG. 2:

table 4 Experimental therapeutic Effect of Compounds Ic and Id on human gastric cancer HGC27 nude mice transplantable tumor

t-test, compared to Vehicle: p <0.001,: p <0.01,: p <0.05.

Table 5: effect of Compounds Ic and Id on tumor weight of human gastric cancer HGC27 nude mice transplantable tumor

t-test, compared to Vehicle: p <0.001,: p <0.01,: p <0.05.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims

1. A 7-methylthiazolo [5,4-d ] pyrimidine compound comprising a compound of formula (I):

2. 7-methylthiazolo [5,4-d ] according to claim 1]Pyrimidine compound, characterized in that R ₁ Any one selected from hydrogen, acetyl, propionyl, cyclopropylcarbonyl, deuterated acetyl, methyl and cyclopropyl; r is R ₂ Any one of 2, 4-difluoro substituted benzene ring group, 4-fluoro substituted benzene ring group, methyl, thiophene-2-group and cyclopropyl.

3. The 7-methylthiazolo [5,4-d ] pyrimidines according to claim 1, wherein any one of the following compounds is selected from:

4. a process for the preparation of 7-methylthiazolo [5,4-d ] pyrimidines according to claim 1, comprising the steps of:

s1, compounds

And the compound->

Coupling reaction to obtain amino target compound

Or S2, amino compounds

And R is R _b X undergoes acylation reaction to obtain an acylated target compound

R _b Is an alkyl acyl group, X is a halogen atom;

or S3, compound

And the compound->

Coupling reaction to obtain compound

Wherein R is _a Is alkyl or cycloalkyl.

5. The method for preparing 7-methylthiazolo [5,4-d ] pyrimidine compounds according to claim 4, wherein the method comprises the following steps:

compounds of formula (I)

And the compound->

Coupling reaction to obtain compound

Further amination to give the compound->

Or a compound

And R is R _b X is subjected to acylation reaction to obtain a compound

Wherein R is _b Is an alkanoyl group, and X is a halogen atom.

6. The 7-methylthiazolo [5,4-d ] as claimed in claim 4]The preparation method of pyrimidine compounds is characterized in that the compounds

Is prepared by the following steps:

a1, 2-bromo-4-amino-5-methoxypyridine and compounds

Reacting in the presence of DMAP and pyridine to obtain the compound +.>

A2, compound

In potassium acetate, pdCl ₂ And reacting with pinacol ester of bisboronic acid in the presence of dioxane to give the compound/>

7. The 7-methylthiazolo [5,4-d ] as claimed in claim 4]The preparation method of pyrimidine compounds is characterized in that the compounds

Is prepared by the following steps:

b1, compound

B2, compound

B3, compound

Reacting with isoamyl nitrite to obtain the compound ∈K>

B4, compound

In benzhydrylamine, BINAP, pd ₂ (dba) ₃ And Cs ₂ CO ₃ The reaction is carried out in the presence of the catalyst to obtainCompound->

B5, compound

Reacting with citric acid to obtain compound->

B6, compound

Re-bromination to give the compound->

8. The process for preparing 7-methylthiazolo [5,4-d ] pyrimidines according to claim 4, wherein,

compounds of formula (I)

Is prepared by the following steps:

c1, compounds

C2, compounds

C3, compounds

9. Use of a 7-methylthiazolo [5,4-d ] pyrimidine compound according to claim 1, 2 or 3, or a 7-methylthiazolo [5,4-d ] pyrimidine compound prepared by the method according to any one of claims 4 to 8, in the preparation of a medicament for treating PI3 kinase-related diseases.

10. Use of 7-methylthiazolo [5,4-d ] pyrimidine compounds according to claim 1, 2 or 3 or 7-methylthiazolo [5,4-d ] pyrimidine compounds prepared by the method according to any one of claims 4-8 in the preparation of antitumor drugs.