阅读说明：本技术 噻唑并嘧啶类化合物及其制备方法、用途和药物组合物 (Thiazolopyrimidine compound and preparation method, application and pharmaceutical composition thereof ) 是由 许恒 陈晓光 林松文 金晶 季鸣 吴德雨 张敬博 伏蓉 张妍 张明祎 于 2020-05-09 设计创作，主要内容包括：本发明属医药技术领域,具体涉及噻唑并嘧啶类化合物及其制备方法、用途和药物组合物。所述噻唑并嘧啶类化合物由式(I)所示,其为PI3K抑制剂,可以用于制备预防和/或治疗与PI3K活性相关的疾病的药物,如肿瘤、自身免疫性疾病、肾脏疾病、心血管疾病、炎症、代谢功能障碍、内分泌功能障碍或神经疾病。(The invention belongs to the technical field of medicines, and particularly relates to a thiazolopyrimidine compound, and a preparation method, application and a pharmaceutical composition thereof. The thiazolopyrimidine compound is shown as a formula (I), is a PI3K inhibitor, and can be used for preparing medicines for preventing and/or treating diseases related to the activity of PI3K, such as tumors, autoimmune diseases, kidney diseases, cardiovascular diseases, inflammation, metabolic dysfunction, endocrine dysfunction or neurological diseases.)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

R₁selected from 3-9 membered heterocycloalkyl containing 1-3 heteroatoms selected from nitrogen, oxygen or sulfur; the R is₁Optionally substituted with m Ra;

each Ra is independently selected from C1-3 alkyl, C1-3 alkoxy;

m is 0, 1,2,3 or 4;

R₂selected from 6-10 membered aryl or 5-10 membered heteroaryl containing 1-3 heteroatoms selected from nitrogen, oxygen or sulfur; wherein the 6-10 membered aryl or 5-10 membered heteroaryl is optionally substituted with n Rb;

each Rb is independently selected from the group consisting of: fluoro, chloro, cyano, difluoromethyl, trifluoromethyl;

n is 0, 1,2,3, 4 or 5.

2. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R is₁Selected from:

the R is₁Optionally substituted with m Ra, each Ra being independently selected from methyl, methoxy;

m is 0, 1 or 2.

3. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 or 2, wherein R is₂Selected from:

4. the compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

5. a pharmaceutical composition comprising at least one compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof and optionally a pharmaceutically acceptable carrier and/or excipient.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition further comprises a pharmaceutically active ingredient other than the compound or a pharmaceutically acceptable salt thereof.

7. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 5 or 6, for the manufacture of a medicament for the prevention and/or treatment of a PI 3K-mediated disease.

8. The use of claim 7, wherein the PI3K mediated disease is selected from the group consisting of a tumor, an autoimmune disease, a renal disease, a cardiovascular disease, an inflammatory disease, a metabolic dysfunction, an endocrine dysfunction, and a neurological disease.

Technical Field

The invention belongs to the technical field of medicines, and relates to a thiazolopyrimidine compound, and a preparation method, application and a pharmaceutical composition thereof.

Background

Phosphatidylinositol 3-kinases (PI 3K) belong to the lipid kinase family and are classified into three major groups (I, II and III) according to their structure, regulation and specificity of lipid substrates. The most intensively studied at present are class I PI3K, such PI3K being a heterodimer consisting of a regulatory subunit (p85) and a catalytic subunit (p 110). Class I PI3K contains 4 subtypes, two of which, PI 3K. alpha. and PI 3K. beta. are widely present in all classes of cells, while the other two subtypes, PI 3K. delta. and PI 3K. gamma. are predominantly distributed in leukocytes (Vanhaaesebroeck et al, Trends Biochem Sci., 2005, 30 (4): 194-204). As the major downstream effector of Receptor Tyrosine Kinases (RTKs) and G-protein coupled receptors (GPCRs), PI3K mediates the signaling of various growth factors and cytokines into cells by catalyzing the production of phosphatidylinositol-3, 4, 5-triphosphate (PIP3) from phosphatidylinositol-4, 5-diphosphate (PIP 2). PIP3 acts as a second messenger within the cell that activates serine/threonine protein kinase (AKT) and downstream effectors, including the mammalian target of rapamycin (mTOR), thereby regulating a variety of cellular functions.

The PI3K signal pathway is one of the most common abnormal signal pathways in tumor cells and has a critical influence on the generation and development processes of tumors. In particular, PIK3CA gene encoding p110 alpha has a large number of amplifications and variations in most tumors, such as breast, lung, intestine, ovary, head and neck, stomach, prostate, brain, liver, digestive tract, leukemia, etc. (ZHao et al, nat. drug Discov. Rev.2009, 8:627 644). In recent years, PI3K and other related nodes on its pathway such as AKT and mTOR have become popular targets for targeting antitumor drugs. Several structural backbone types of PI3K inhibitors have been reported in succession, which show superior antitumor effects in both cellular and animal models, and several compounds have been tested in clinical trials against solid and hematological tumors, such as BKM120(Novartis, phase III), BEZ235(Novartis, phase II), PF-05212384(Pfizer, phase II), BAY 80-6946(Bayer, phase III), XL147(Exelixis, phase I/II), etc., in single or combined administration.

PI3K has become a very attractive drug target, and the development of safer and more effective PI3K inhibitors for preventing and/or treating PI 3K-related diseases has been a problem to be solved in the art.

CN105693744A discloses a substituted thienopyrimidine compound which has a good PI3K α inhibitory activity at the kinase level, however, it has a poor activity at the cellular level and is difficult to successfully act as a drug in vivo.

Therefore, there remains a need in the art to develop new more active PI3K inhibitors that can function in vivo.

Disclosure of Invention

The technical problem solved by the invention is to provide a novel PI3K inhibitor, a preparation method, a pharmaceutical composition and an application thereof, wherein the PI3K inhibitor has stronger inhibitory activity on class I PI3K, including PI3K alpha, PI3K beta, PI3K gamma and/or PI3K delta, especially on PI3K alpha, so that the PI3K inhibitor has better prevention and/or treatment effects on diseases mediated by PI3K, such as cancer, immune diseases, cardiovascular diseases, viral infection, inflammation, metabolic dysfunction, endocrine dysfunction or neurological diseases.

In order to solve the technical problem, the invention provides the following technical scheme:

in a first aspect of the technical scheme of the invention, a compound shown as a formula (I) or a pharmaceutically acceptable salt thereof is provided:

wherein

R₁Selected from 3-9 membered heterocycloalkyl containing 1-3 heteroatoms selected from nitrogen, oxygen or sulfur; preferably, R is₁Selected from:

wherein, R is₁Optionally substituted with m Ra;

each Ra is independently selected from C1-3 alkyl, C1-3 alkoxy; preferably, each Ra is independently selected from methyl, methoxy;

m is 0, 1,2,3 or 4; preferably, m is 0, 1 or 2;

R₂selected from 6-10 membered aryl or 5-10 membered heteroaryl containing 1-3 heteroatoms selected from nitrogen, oxygen or sulfur; preferably, R is₂Selected from:

wherein the 6-10 membered aryl or 5-10 membered heteroaryl is optionally substituted with n Rb;

each Rb is independently selected from the group consisting of: fluoro, chloro, cyano, difluoromethyl, trifluoromethyl;

n is 0, 1,2,3, 4 or 5.

Preferably, the compound is selected from:

in a second aspect of the present invention, there is provided a pharmaceutical composition, wherein the pharmaceutical composition comprises at least one compound as described above or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier and/or excipient.

Preferably, the pharmaceutical composition further comprises a pharmaceutically active ingredient other than the compound or a pharmaceutically acceptable salt thereof.

A third aspect of the present invention provides a use of the compound or its pharmaceutically acceptable salt described above, or the pharmaceutical composition described above, in the preparation of a medicament for preventing and/or treating a PI 3K-mediated disease.

Preferably, the PI 3K-mediated disease comprises a tumor, an autoimmune disease, a renal disease, a cardiovascular disease, an inflammation, a metabolic dysfunction, an endocrine dysfunction, or a neurological disease.

In a fourth aspect of the present invention, there is provided a method for preparing the compound, comprising the steps of:

(1) taking the compound A as an initial material, and preparing a compound B through nucleophilic attack reaction;

(2) reacting the compound B with KSCN to prepare a compound C;

(3) reacting the compound C with iron powder/acetic acid to prepare a compound D

(4) Reacting the compound D with tert-butyl nitrite and cupric bromide to prepare a compound E

(5) Coupling compound E with an arylboronic acid ester to produce a compound of formula (I) or a pharmaceutically acceptable salt thereof.

R₁And R₂The definition of (a) is the same as that of the first aspect of the present invention.

Some of the terms used in the present invention are defined below, and other undefined terms have meanings well known to those skilled in the art.

Halogen means fluorine, chlorine, bromine or iodine.

C_1-3Alkyl refers to straight and branched chain saturated aliphatic hydrocarbon groups having 1 to 3 carbon atoms. Examples of such groups include, but are not limited to: methyl, ethyl, propyl, isopropyl.

C_1-3Alkoxy means an-O-alkyl group, wherein the alkyl group contains 1 to 3 carbon atoms and is straight, branched or cyclic. Examples of such groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy or cyclopropoxy.

3-9 membered heterocycloalkyl refers to a saturated monocyclic, fused, spiro or polycyclic structure having 3 to 9 ring atoms. Examples of such groups include, but are not limited to: azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, piperazin-1-yl, homopiperazin-1-yl, 4-morpholinyl, 4-thiomorpholinyl, 1, 4-oxazepan-4-yl, hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl and tetrahydro-1H-furo [3,4-c ] pyrrol-5 (3H) -yl.

Aryl means a monocyclic or bicyclic aromatic carbocyclic group, typically having 6 to 10 carbon atoms; such as phenyl or naphthyl; phenyl is preferred.

Heteroaryl refers to 5-10 membered aromatic heterocyclic groups including, but not limited to: 5-membered heteroaryl: furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, triazolyl (1,2, 4-triazolyl, 1,3, 4-triazolyl or 1,2, 3-triazolyl), thiadiazolyl (1,3, 4-thiadiazolyl, 1,2, 5-thiadiazolyl, 1,2, 3-thiadiazolyl or 1,2, 4-thiadiazolyl), and oxadiazolyl (1,3, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,2, 3-oxadiazolyl or 1,2, 4-oxadiazolyl); and 6-membered heteroaryl: pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl; and bicyclic groups such as benzofuranyl, benzothienyl, indazolyl, purinyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, indolizinyl, indolyl, isoindolyl. Preferred heteroaryl groups are thienyl, thiazolyl, pyridyl, pyrimidinyl.

"optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "alkyl optionally substituted with halogen" means that halogen may, but need not, be present, and the description includes the case where alkyl is substituted with halogen and the case where alkyl is not substituted with halogen.

The compounds of the invention may contain one or more chiral centers, which exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, and atropisomers, and mixtures thereof (e.g., racemic mixtures), are within the scope of the present invention.

The compounds of the present invention include geometric isomers thereof. For example, if the compounds of the present invention contain double bonds or fused rings, these compounds may exist as geometric isomers, and their cis, trans forms and mixtures of cis and trans forms are included in the scope of the present invention.

The compounds of the present invention include tautomers thereof. Tautomers refer to structural isomers of different energies that interconvert via a low energy barrier, such as keto-enol and imine-enamine tautomerism.

The compounds of the present invention also include isotopically-labeled compounds thereof, in which one or more atoms are replaced by atoms naturally found to have the same atomic number, but a different atomic mass or mass number. Examples include, but are not limited to: hydrogen isotope²H and³h; carbon isotope¹¹C、¹³C and¹⁴c; isotope of chlorine³⁶Cl; isotopes of fluorine¹⁸F; iodine isotopologueVegetable extract¹²³I and¹²⁵i; isotope of nitrogen¹³N and¹⁵n; oxygen isotope¹⁵O、¹⁷O and¹⁸o; isotope of phosphorus³²Isotopes of P and sulfur³⁵S。

Various hydrates and solvates of the compound or salt thereof of the present invention and polymorphic forms thereof (polymorphisms) are also included in the scope of the present invention.

Prodrugs of the compounds described herein are also included within the scope of the invention. Some derivatives of the compounds described in the present invention have weak pharmacological activity or no pharmacological activity themselves, but when these derivatives are administered into or onto the body, they may be converted into the compounds described in the present invention having pharmacological activity by means of, for example, hydrolytic cleavage, and the like, and these derivatives are referred to as "prodrugs". Further information on the use of prodrugs can be found in Pro-drugs as Novel Delivery Systems, vol.14, ACS Symposium Series (t.higuchi and w.stella) and Bioreversible Carriers in Drug Design, Pergamon Press,1987(ed.e.b.roche, American Pharmaceutical Association).

The compounds of the present invention include pharmaceutically acceptable salts thereof. Pharmaceutically acceptable salts are salts that are pharmaceutically acceptable and possess the desired pharmacological activity of the parent compound. Pharmaceutically acceptable salts are described in detail in j.pharma.sci.,1977,66,1-19 by Berge et al, which is incorporated herein by reference. The compounds of the present invention may contain sufficient acidic groups, sufficient basic groups, or both types of functional groups, and accordingly react with some inorganic or organic bases, or inorganic and organic acids, to form pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, dihydrogenphosphate, metaphosphate, hydrochloride, hydrobromide, hydroiodide, acetate, propionate, caprylate, or a mixture thereof, Citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate.

When used as a medicament, the compounds of the present invention are generally administered in the form of a pharmaceutical composition. Accordingly, pharmaceutical compositions of the compounds of the present invention and a pharmaceutically acceptable carrier, diluent or excipient are also included within the scope of the present invention. As used herein, carriers, adjuvants, excipients include any and all solvents, diluents or other liquid excipients, dispersing or suspending agents, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like as appropriate for the particular dosage form desired. In Remington: the Science and Practice of Pharmacy, 21^stedition，2005，ed.D.B.Troy，Lippincott Williams&Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds.J.Swarbrick and J.C.Boylan, 1988. 1999, Marcel Dekker, New York, disclose various carriers for the formulation of pharmaceutically acceptable compositions and known techniques for their preparation, the contents of which are incorporated herein by reference.

The compositions of the present invention may be administered by any route suitable for the condition to be treated. In particular by administration in the form: parenterally, e.g., in the form of injectable solutions or suspensions; enterally, e.g., orally, e.g., in tablet or capsule form; topically, for example in the form of a lotion, gel, ointment or cream or in the form of a nasal or suppository. Topical application is, for example, application to the skin. Another form of topical administration is to the eye.

The pharmaceutical compositions may be administered in solid, semi-solid, liquid or gaseous form, or may be in a dry powder, such as lyophilized form. Pharmaceutical compositions can be packaged in a form convenient for delivery, including, for example, solid dosage forms such as capsules, sachets, cachets, gelatin, paper, tablets, suppositories, pellets, pills, troches, and lozenges. The type of packaging will generally depend on the route of administration. Implantable sustained release formulations, as well as transdermal formulations, are also contemplated.

Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block copolymers, lanolin, sugars (e.g., lactose, glucose and sucrose), starches (e.g., corn starch and potato starch), cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols, such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; and phosphate buffers, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate. Coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preserving and anti-oxidizing agents may also be present in the composition, according to the judgment of the person skilled in the art.

The compounds of the present invention may be used alone or in combination with other therapeutic agents for the treatment of the diseases or conditions described herein, such as cancer. In certain embodiments, the compounds of the present invention are combined in a pharmaceutical combination formulation, or as a combination therapy in a dosing regimen, with a second compound having anti-hyperproliferative properties or for the treatment of a hyperproliferative disease, such as cancer. The second compound of the pharmaceutical combination or dosing regimen preferably has complementary activities to the compounds of the present invention such that they do not adversely affect each other. Such compounds are suitably present in the combination in an amount effective for the intended purpose. In one embodiment, the compounds of the present invention are combined with other anti-tumor agents. The anti-tumor medicine comprises: alkylating agents including, but not limited to, cyclophosphamide, mechlorethamine, melphalan, cinchonine, carmustine; platinoids including but not limited to carboplatin, cisplatin, oxaliplatin; topoisomerase inhibitors including, but not limited to, topotecan, camptothecin, topotecan, irinotecan; antibiotics, including but not limited to, anthracyclines, actinomycin D, daunorubicin, doxorubicin, mitoxantrone, bleomycin, and plicamycin; anti-microtubule or anti-mitotic agents including, but not limited to, paclitaxel, vinorelbine, docetaxel, doxorubicin; antimetabolites including, but not limited to, fluorouracil, methotrexate, cytarabine, mercaptopurine (mecaptoprine), thioguanine, and gemcitabine; antibodies, including but not limited to herceptin, bevacizumab; hormones, including but not limited to letrozole (letrozole), vorozole (vorazole), tamoxifen, toremifene, fulvestrant, flutamide, nilutamide, triptorelin; kinase inhibitors, EGFR kinase inhibitors, including but not limited to gefitinib (gefitinib), erlotinib (erlotinib), lapatinib (lapatinib), afatinib (afatinib); VEGFR inhibitors including, but not limited to, Sorafenib (Sorafenib), regrafenib (Regorafenib), Sunitinib (Sunitinib), Cabozantinib (Cabozantinib), Pazopanib (Pazopanib), vandetanib (vandetanib), axitinib (axitinib); ALK inhibitors including, but not limited to, Crizotinib (Crizotinib), ceritinib (ceritinib), Alectinib; Bcr-Abl inhibitors, including but not limited to Imatinib (Imatinib), panatinib (Ponatinib), Nilotinib (Nilotinib), Dasatinib (Dasatinib); BTK inhibitors, including but not limited to Ibrutinib (Ibrutinib); B-RAF inhibitors, including but not limited to Vemurafenib (Vemurafenib); cyclin-dependent kinase CDK4/6 inhibitors, Palbociclib (Palbociclib); mTOR inhibitors, including but not limited to rapamycin (rapamycin), everolimus (everolimus); sirtuin inhibitors, including but not limited to vorinostat (vorinostat); PD1/PDL1 antibody, Keytruda (Pembrolizumab), Opdivo (Nivolumab).

In a fourth aspect of the present invention, there is provided a use of the compound of the first aspect, a stereoisomer, a geometric isomer, a tautomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the third aspect, in the preparation of a medicament for preventing and/or treating a PI 3K-mediated disease, wherein the PI 3K-mediated disease includes cancer, an immune disease, a cardiovascular disease, a viral infection, inflammation, a metabolic dysfunction, an endocrine dysfunction or a neurological disease.

The beneficial technical effects are as follows:

in comparison with the comparative example, in the case of having the same R₁Radicals and the same R₂When the compounds are combined, the example compounds with the thiazolopyrimidine structure have stronger in-vitro PI3K alpha kinase inhibitory activity and human HGC-27 gastric cancer cell proliferation inhibitory activity than the corresponding comparative example compounds with the thienopyrimidine structure. These comparative data illustrate the importance of thiazolopyrimidine structures for the in vitro PI3K α kinase inhibitory activity and tumor cell proliferation inhibitory activity of the example compounds.

In a nude mouse xenograft tumor model, the tumor growth inhibition rates at 20mg/kg and 40mg/kg of the dose of example 1 having a thiazolopyrimidine structure in the present invention were both stronger than those at 40mg/kg of the corresponding comparative example 1 having a thienopyrimidine structure.

Drawings

FIG. 1 is a tumor growth curve showing the growth inhibitory effect of example 1 and comparative example 1 on a nude mouse xenograft tumor model of human gastric cancer HGC-27.

Detailed Description

The following are specific examples of the present invention, which further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

In the following examples, molecules with a single chiral center exist as a racemic mixture unless otherwise noted by structural formula or chemical name. Unless otherwise noted by structural formula or chemical name, those molecules having two or more chiral centers exist as racemic mixtures of diastereomers. The single enantiomers/diastereomers may be obtained by methods known to those skilled in the art.

Preparation method

The compounds of the present invention may be synthesized according to the synthetic schemes herein and/or by techniques well known in the art. For example, the compounds provided by the present invention can be prepared according to the following general synthetic methods.

In one general synthetic approach, compounds of formula (I) were prepared according to method-1.

Synthesis method-1

Specifically, in Process-1, the thiazolopyrimidine compound of the present invention can be prepared by a 5-step reaction. For example, compound A (4, 6-dichloro-5-nitropyrimidine) reacts with various 3-to 9-membered heterocyclic alkanes to give compound B; reacting the compound B with KSCN to obtain a compound C; reacting the compound C with iron powder/acetic acid to obtain a compound D; reacting the compound D with tert-butyl nitrite and copper bromide to obtain a compound E; and (3) coupling the compound E with arylboronic acid ester to prepare the thiazolopyrimidine compound.

The compounds of the present invention may be synthesized according to one or more of the synthetic schemes herein and/or by techniques well known in the art. One skilled in the art will recognize that the synthetic methods of certain embodiments described in detail herein can be readily adapted to synthesize other embodiments. In some embodiments, the compounds described herein may be prepared by an appropriate combination of synthetic methods well known in the art. Many starting materials and other reagents are available from commercial suppliers, such as alfa aesar (china) chemical limited, or are readily prepared using synthetic methods commonly used in the art.

¹H NMR spectra were recorded on instruments operated at 400MHz or 500 MHz. H NMR spectra were obtained as a solution (reported in ppm) using CDCl₃(7.26ppm) or DMSO-d₆(2.50ppm) or internal tetramethylsilane (0.00ppm) as reference standard. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), dd (doublet of doublets), dt (doublet of triplets). Coupling constants are given in hertz (Hz).

If desired, the (R) -and (S) -isomers of the non-limiting exemplary compounds, if present, can be resolved by methods known to those skilled in the art, e.g., by forming diastereomeric salts or complexes, which can be separated, e.g., by crystallization; by forming diastereomeric derivatives, which can be separated, for example, by crystallization or chromatography; selectively reacting one enantiomer with an enantiomer-specific reagent, followed by separation of the modified and unmodified enantiomers; or chromatographic separation in a chiral environment such as a chiral chromatography column. Alternatively, specific enantiomers may be prepared by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to another by asymmetric conversion.

In the following preparations and examples, "Me" means methyl, "Et" means ethyl, "PE" means petroleum ether, "EtOAc" means ethyl acetate, "MeOH" means methanol, "DMSO-d" means₆"means deuterated dimethyl sulfoxide," DCM "means dichloromethane," PTLC "means preparative thin layer chromatography," KSCN "means potassium thiocyanide," PdCl₂(dppf) "means (1,1' -bis (diphenylphosphino) ferrocene palladium chloride)," mL "means mL," mmol "means millimole," μ M "means micromole," nM "means nanomole""C" means degrees Celsius.

Example 1: 2, 4-difluoro-N- (2-methoxy-5- (7-morpholinothiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of 4- (6-chloro-5-nitropyrimidin-4-yl) morpholine

To a mixture of 4, 6-dichloro-5-nitropyrimidine (970mg, 5.0mmol) and triethylamine (506mg,5.0mmol,1.0equiv) in tetrahydrofuran (20mL) was added morpholine (436mg,5.0mmol) at 0 ℃. The resulting mixture was stirred at 0 ℃ for 30 min, then diluted with water (100mL) and extracted with EtOAc (50 mL. times.3). The combined organic layers were washed with water (50 mL. times.2) and brine (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash column chromatography (silica gel, PE/EtOAc ═ 4:1) to give the product as a light yellow solid (1.064g, 87% yield).¹H NMR(400MHz,DMSO-d₆)δ8.53(s,1H),3.68(t,J＝4.8Hz,4H),3.54(t,J＝4.8Hz,4H).

Step 2: synthesis of 4- (5-nitro-6-thiocyanopyrimidin-4-yl) morpholine

A mixture of 4- (6-chloro-5-nitropyrimidin-4-yl) morpholine (1.064g, 4.35mmol) and KSCN (0.549g, 5.65mmol) in HOAc (20mL) was stirred at room temperature overnight. Water (100mL) was added and extracted with EtOAc (50 mL. times.3). The combined organic layers were washed with water (50 mL. times.2) and brine (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash column chromatography (silica gel, PE/EtOAc ═ 3:1) to give the product as a yellow solid (1.067g, 92% yield).¹H NMR(500MHz,DMSO-d₆)δ8.59(s,1H),3.68(t,J＝4.8Hz,4H),3.60(br s,4H).

And step 3: synthesis of 7-morpholinyl thiazolo [5,4-d ] pyrimidin-2-amine

A mixture of 4- (5-nitro-6-thiocyanopyrimidin-4-yl) morpholine (1.067g, 4.0mmol) and iron powder (0.894g, 16mmol) in HOAc (20mL) was stirred at 60 ℃ for 1.5 h. After removal of HOAc in vacuo, the resulting residue was diluted with water (100mL) and neutralized with saturated aqueous sodium bicarbonate. The resulting mixture was extracted with EtOAc (50 mL. times.3). The combined organic layers were washed with water (50 mL. times.2) and brine (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. DCM (20mL) was added and the resulting suspension was stirred at 50 ℃ for 30 min. After cooling to room temperature, the resulting solid was collected by suction filtration. The filter cake was washed with DCM and dried to give the product as a yellow solid (0.642g, 68% yield).¹H NMR(500MHz,DMSO-d₆)δ8.15(s,1H),7.67(s,2H),4.08(t,J＝4.8Hz,4H),3.69(t,J＝4.8Hz,4H).

And 4, step 4: synthesis of 4- (2-bromothiazolo [5,4-d ] pyrimidin-7-yl) morpholine

Reacting 7-morpholinyl thiazolo [5,4-d ]]Pyrimidin-2-amine (0.536g, 2.26mmol), tert-butyl nitrite (0.466g, 4.52mmol) and CuBr₂A mixture of (0.758g, 3.39mmol) in acetonitrile (10mL) was stirred at room temperature for 1.5 h. The residue was diluted with water (50mL) and EtOAc (50mL) and insoluble material was removed by suction filtration through celite. The filtrate separated into an aqueous layer and an ethyl acetate layer. The aqueous layer was extracted with EtOAc (30 mL. times.2). The combined organic layers were washed with water (50mL) and brine (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash column chromatography (silica gel, PE/EtOAc ═ 8:1) to give the product as a white solid (0.120g, 18%).¹H NMR(400MHz,DMSO-d₆)δ8.42(s,1H),4.20(br s,4H),3.74(t,J＝5.0Hz,4H).

And 5: synthesis of 2, 4-difluoro-N- (2-methoxy-5- (7-morpholinothiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Reacting 4- (2-bromothiazolo [5,4-d ]]A mixture of pyrimidin-7-yl) morpholine (60mg,0.2mmol), N- (2-methoxy-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-3-yl) -2, 4-difluorobenzenesulfonamide (0.103g,0.24mmol) and 2M potassium carbonate (0.3mL,0.6mmol) in dioxane (7mL) was degassed and PdCl was added₂(dppf) (15mg,0.02 mmol). The resulting reaction mixture was degassed and backfilled with argon (three cycles), then stirred at 100 ℃ for 4 hours under an argon atmosphere. The reaction mixture was concentrated in vacuo, diluted with water (30mL) and extracted with EtOAc (30 mL. times.3). The combined organic layers were washed with water (30mL) and brine (30mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by PTLC (silica gel, DCM/MeOH ═ 20:1) to give the product as a light yellow solid (59mg, 57% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.54(s,1H),8.66(d,J＝2.2Hz,1H),8.43(s,1H),8.10(d,J＝2.2Hz,1H),7.80(td,J＝8.6,6.4Hz,1H),7.67–7.54(m,1H),7.26(td,J＝8.6,2.0Hz,1H),4.32(br s,4H),3.86–3.76(m,4H),3.75(s,3H).

Example 2: 2, 4-difluoro-N- (2-methoxy-5- (7- (4-methoxypiperidin-1-yl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of 4-chloro-6- (4-methoxypiperidin-1-yl) -5-nitropyrimidine

The title compound was prepared from 4-methoxypiperidine following the procedure of step 1 in example 1The title compound (pale yellow solid, 78% yield).¹H NMR(400MHz,DMSO-d₆)δ8.49(s,1H),3.72–3.61(m,2H),3.54–3.45(m,1H),3.43–3.33(m,2H),3.26(s,3H),1.95–1.84(m,2H),1.61–1.48(m,2H).

Step 2: synthesis of 4- (4-methoxypiperidin-1-yl) -5-nitro-6-thiocyanopyrimidine

The title compound was prepared from 4-chloro-6- (4-methoxypiperidin-1-yl) -5-nitropyrimidine following the procedure of step 2 in example 1 (yellow solid, 87% yield).¹H NMR(400MHz,DMSO-d₆)δ8.55(s,1H),3.66(br s,2H),3.58–3.49(m,1H),3.44(br s,2H),3.28(s,3H),1.97–1.87(m,2H),1.65–1.54(m,2H).

And step 3: 7- (4-methoxypiperidin-1-yl) thiazolo [5,4-d ] pyrimidin-2-amine

The title compound was prepared from 4- (4-methoxypiperidin-1-yl) -5-nitro-6-thiocyanopyrimidine (white solid, 39% yield) according to the method of step 3 in example 1.¹H NMR(400MHz,DMSO-d₆)δ8.12(s,1H),7.62(s,2H),4.69–4.52(m,2H),3.65–3.55(m,2H),3.51–3.38(m,1H),3.28(s,3H),1.96–1.83(m,2H),1.50–1.36(m,2H).

And 4, step 4: synthesis of 2-bromo-7- (4-methoxypiperidin-1-yl) thiazolo [5,4-d ] pyrimidine

Following the procedure of step 4 in example 1, starting from 7- (4-methoxypiperidin-1-yl) thiazolo [5, 4-d)]Pyrimidin-2-amine the title compound was prepared as a white solid in 29% yield.¹H NMR(400MHz,DMSO-d₆)δ8.38(s,1H),4.55(br s,2H),3.84(br s,2H),3.56–3.48(m,1H),3.30(s,3H),2.01–1.90(m,2H),1.61–1.45(m,2H).

And 5: synthesis of 2, 4-difluoro-N- (2-methoxy-5- (7- (4-methoxypiperidin-1-yl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Following the procedure of step 5 in example 1, starting from 2-bromo-7- (4-methoxypiperidin-1-yl) thiazolo [5,4-d]Pyrimidine the title compound was prepared (light yellow solid, 72% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.55(s,1H),8.61(d,J＝2.2Hz,1H),8.38(s,1H),8.11(d,J＝2.2Hz,1H),7.82(td,J＝8.6,6.4Hz,1H),7.66–7.53(m,1H),7.26(td,J＝8.4,2.1Hz,1H),4.70(br s,2H),3.92(br s,2H),3.77(s,3H),3.61–3.50(m,1H),3.32(s,3H),2.08–1.88(m,2H),1.65–1.46(m,2H).

Example 3: n- (5- (7- (2, 6-dimethylmorpholinyl) thiazolo [5,4-d ] pyrimidin-2-yl) -2-methoxypyridin-3-yl) -2, 4-difluorobenzenesulfonamide

Step 1: synthesis of 4- (6-chloro-5-nitropyrimidin-4-yl) -2, 6-dimethylmorpholine

The title compound was prepared from 2, 6-dimethylmorpholine as a pale yellow solid in 69% yield following the procedure of step 1 in example 1.¹H NMR(400MHz,DMSO-d₆)δ8.53(s,1H),3.82(d,J＝13.0Hz,2H),3.68–3.56(m,2H),2.80(dd,J＝13.1,10.6Hz,2H),1.10(d,J＝6.2Hz,3H).

Step 2: synthesis of 2, 6-dimethyl-4- (5-nitro-6-thiocyanopyrimidin-4-yl) morpholine

The title compound was prepared from 4- (6-chloro-5-nitropyrimidin-4-yl) -2, 6-dimethylmorpholine by the method of step 2 in example 1 (yellow solid, 86% yield).¹H NMR(400MHz,DMSO-d₆)δ8.59(s,1H),3.87(br s,2H),3.68–3.56(m,2H),2.88(dd,J＝13.0,10.5Hz,2H),1.12(d,J＝6.2Hz,3H).

And step 3: synthesis of 7- (2, 6-dimethylmorpholinyl) thiazolo [5,4-d ] pyrimidin-2-amine

The title compound was prepared as a yellow solid in 32% yield from 2, 6-dimethyl-4- (5-nitro-6-thiocyanopyrimidin-4-yl) morpholine according to the method of step 3 in example 1.¹H NMR(400MHz,DMSO-d₆)δ8.14(s,1H),7.65(br s,2H),5.13(d,J＝12.7Hz,2H),3.61(br s,2H),2.62(t,J＝11.9Hz,2H),1.14(d,J＝5.8Hz,6H).

And 4, step 4: synthesis of 4- (2-bromothiazolo [5,4-d ] pyrimidin-7-yl) -2, 6-dimethylmorpholine

By the method of step 4 in example 1, starting from 7- (2, 6-dimethylmorpholinyl) thiazolo [5,4-d ]]Pyrimidin-2-amine the title compound was prepared as a white solid in 81% yield.¹H NMR(400MHz,DMSO-d₆)δ8.41(s,1H),5.16(br s,2H),3.68–3.56(m,2H),2.80(br s,2H),1.17(d,J＝6.2Hz,6H).

And 5: synthesis of N- (5- (7- (2, 6-dimethylmorpholinyl) thiazolo [5,4-d ] pyrimidin-2-yl) -2-methoxypyridin-3-yl) -2, 4-difluorobenzenesulfonamide

By the method of step 5 in example 1, from 4- (2-bromothiazolo [5,4-d ]]Pyrimidin-7-yl) -2, 6-dimethylmorpholine the title compound was prepared as a light yellow solid in 57% yield.¹H NMR(400MH_Z,DMSO-d₆):δ8.37(s,1H),7.95(d,J＝1.9Hz,1H),7.87–7.77(m,2H),7.21(td,J＝9.8,2.1Hz,1H),7.16–7.04(m,1H),5.42(br s,2H),3.85(s,3H),3.75–3.62(m,2H),2.84(br s,2H),1.22(d,J＝6.1Hz,6H).

Example 4: (S) -2, 4-difluoro-N- (2-methoxy-5- (7- (3-methylmorpholinyl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of (S) -4- (6-chloro-5-nitropyrimidin-4-yl) -3-methylmorpholine

The title compound was prepared from (S) -3-methylmorpholine by the method of step 1 in example 1 (light yellow solid, 77% yield).¹H NMR(400MHz,DMSO-d₆)δ8.54(s,1H),4.51(d,J＝5.2Hz,1H),3.92–3.80(m,1H),3.71(d,J＝11.7Hz,1H),3.65–3.57(m,1H),3.49–3.34(m,2H),3.31–3.22(m,1H),1.28(d,J＝6.8Hz,3H).

Step 2: synthesis of (S) -3-methyl-4- (5-nitro-6-thiocyanopyrimidin-4-yl) morpholine

The title compound was prepared from (S) -4- (6-chloro-5-nitropyrimidin-4-yl) -3-methylmorpholine by the method of step 2 in example 1 (yellow solid, 90% yield).¹H NMR(400MHz,DMSO-d₆)δ8.59(s,1H),3.85(dd,J＝10.9,2.9Hz,1H),3.74(d,J＝11.8Hz,1H),3.62(dd,J＝11.7,3.0Hz,1H),3.57–3.47(m,1H),3.47–3.26(m,2H),1.36(d,J＝6.9Hz,3H).

And step 3: synthesis of (S) -7- (3-methylmorpholinyl) thiazolo [5,4-d ] pyrimidin-2-amine

The title compound was prepared from (S) -3-methyl-4- (5-nitro-6-thiocyanopyrimidin-4-yl) morpholine by the method of step 3 in example 1 (yellow solid, 52% yield).¹H NMR(400MHz,DMSO-d₆)δ8.15(s,1H),7.65(s,2H),5.30(d,J＝4.9Hz,1H),4.88(d,J＝13.6Hz,1H),3.91(dd,J＝11.3,3.1Hz,1H),3.70(d,J＝11.3Hz,1H),3.65(dd,J＝11.4,2.9Hz,1H),3.50(td,J＝11.8,2.8Hz,1H),3.28(dd,J＝12.5,3.5Hz,1H),1.23(d,J＝6.8Hz,3H).

And 4, step 4: synthesis of (S) -4- (2-bromothiazolo [5,4-d ] pyrimidin-7-yl) -3-methylmorpholine

By the method of step 4 in example 1, from (S) -7- (3-methylmorpholinyl) thiazolo [5,4-d]Pyrimidin-2-amine the title compound was prepared (white solid, 31%).¹H NMR(400MHz,DMSO-d₆)δ8.43(s,1H),5.27(br s,1H),4.96(br s,1H),3.99(dd,J＝11.1,3.1Hz,1H),3.77(d,J＝11.6Hz,1H),3.68(dd,J＝11.6,3.0Hz,1H),3.59–3.37(m,2H),1.32(d,J＝6.8Hz,3H).

And 5: synthesis of (S) -2, 4-difluoro-N- (2-methoxy-5- (7- (3-methylmorpholinyl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

By the method of step 5 in example 1, from (S) -4- (2-bromothiazolo [5, 4-d)]Pyrimidin-7-yl) -3-methylmorpholine the title compound was prepared as a light yellow solid in 61% yield.¹H NMR(400MH_Z,DMSO-d₆):δ10.53(s,1H),8.63(d,J＝2.1Hz,1H),8.41(s,1H),8.10(d,J＝2.1Hz,1H),7.80(td,J＝8.6,6.3Hz,1H),7.67–7.52(m,1H),7.25(td,J＝8.5,2.3Hz,1H),5.43(br s,1H),5.10(br s,1H),4.00(d,J＝8.8Hz,1H),3.90–3.65(m,5H),3.64–3.38(m,2H),1.36(d,J＝6.7Hz,3H).

Example 5: (R) -2, 4-difluoro-N- (2-methoxy-5- (7- (3-methylmorpholinyl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of (R) -4- (6-chloro-5-nitropyrimidin-4-yl) -3-methylmorpholine

The title compound was prepared from (R) -3-methylmorpholine by the method of step 1 in example 1 (light yellow solid, 75% yield).¹H NMR(400MHz,DMSO-d₆)δ8.54(s,1H),4.51(q,J＝5.1Hz,1H),3.92–3.80(m,1H),3.71(d,J＝11.7Hz,1H),3.61(dd,J＝11.7,3.0Hz,1H),3.48–3.35(m,2H),3.31–3.22(m,1H),1.28(d,J＝6.8Hz,3H).

Step 2: synthesis of (R) -3-methyl-4- (5-nitro-6-thiocyanopyrimidin-4-yl) morpholine

The title compound was prepared from (R) -4- (6-chloro-5-nitropyrimidin-4-yl) -3-methylmorpholine by the method of step 2 in example 1 (yellow solid, 79% yield).¹H NMR(400MHz,DMSO-d₆)δ8.59(s,1H),4.87(br s,1H),3.90–3.80(m,1H),3.74(d,J＝11.6Hz,1H),3.62(dd,J＝11.7,2.9Hz,1H),3.58–3.47(m,1H),3.47–3.36(m,1H),2.93(br s,1H),1.36(d,J＝6.9Hz,3H).

And step 3: synthesis of (R) -7- (3-methylmorpholinyl) thiazolo [5,4-d ] pyrimidin-2-amine

The title compound was prepared from (R) -3-methyl-4- (5-nitro-6-thiocyanopyrimidin-4-yl) morpholine by the method of step 3 in example 1 (yellow solid, 80% yield).¹H NMR(400MHz,DMSO-d₆)δ8.15(s,1H),7.65(s,2H),5.30(d,J＝5.0Hz,1H),4.88(d,J＝12.9Hz,1H),3.91(dd,J＝11.3,3.1Hz,1H),3.70(d,J＝11.3Hz,1H),3.65(dd,J＝11.4,2.9Hz,1H),3.50(td,J＝11.9,2.8Hz,1H),3.29(dt,J＝12.7,3.3Hz,1H),1.23(d,J＝6.8Hz,3H).

And 4, step 4: synthesis of (R) -4- (2-bromothiazolo [5,4-d ] pyrimidin-7-yl) -3-methylmorpholine

By the method of step 4 in example 1, from (R) -7- (3-methylmorpholinyl) thiazolo [5,4-d]Pyrimidin-2-amine the title compound was prepared (white solid, 27%).¹H NMR(400MHz,DMSO-d₆)δ8.43(s,1H),5.27(br s,1H),4.96(br s,1H),3.99(dd,J＝11.1,3.0Hz,1H),3.77(d,J＝11.6Hz,1H),3.68(dd,J＝11.6,3.1Hz,1H),3.59–3.37(m,2H),1.32(d,J＝6.8Hz,3H).

And 5: synthesis of (R) -2, 4-difluoro-N- (2-methoxy-5- (7- (3-methylmorpholinyl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Following the procedure of step 5 in example 1, from (R) -4- (2-bromothiazolo [5, 4-d)]Pyrimidin-7-yl) -3-methylmorpholine the title compound was prepared as a light yellow solid in 42% yield.¹H NMR(400MHz,DMSO-d₆)δ10.54(s,1H),8.63(d,J＝1.9Hz,1H),8.42(s,1H),8.11(d,J＝1.8Hz,1H),7.87–7.76(m,1H),7.68–7.53(m,1H),7.32–7.21(m,1H),5.44(br s,1H),5.11(br s,1H),4.01(d,J＝8.9Hz,1H),3.89–3.67(m,5H),3.65–3.41(m,2H),1.37(d,J＝6.7Hz,3H).

Example 6: 2, 4-difluoro-N- (2-methoxy-5- (7- (piperidin-1-yl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of 4-chloro-5-nitro-6- (piperidin-1-yl) pyrimidine

The title compound was prepared from piperidine according to the procedure for step 1 of example 1 (light yellow solid, 55% yield).¹H NMR(400MHz,DMSO-d₆)δ8.47(s,1H),3.59–3.46(m,4H),1.70–1.53(m,6H).

Step 2: synthesis of 5-nitro-4- (piperidin-1-yl) -6-thiocyanopyrimidine

The title compound was prepared from 4-chloro-5-nitro-6- (piperidin-1-yl) pyrimidine according to the procedure of step 2 in example 1 (yellow solid, 68% yield).¹H NMR(400MHz,DMSO-d₆)δ8.54(s,1H),3.52(br s,4H),1.71–1.57(m,6H).

And step 3: synthesis of 7- (piperidin-1-yl) thiazolo [5,4-d ] pyrimidin-2-amines

The title compound was prepared from 5-nitro-4- (piperidin-1-yl) -6-thiocyanopyrimidine (yellow solid, 40% yield) by the method of step 3 in example 1.¹H NMR(400MHz,DMSO-d₆)δ8.10(s,1H),7.59(br s,2H),4.13–4.02(m,4H),1.70–1.60(m,2H),1.60–1.50(m,4H).

And 4, step 4: synthesis of 2-bromo-7- (piperidin-1-yl) thiazolo [5,4-d ] pyrimidine

Following the procedure of step 4 in example 1, starting from 7- (piperidin-1-yl) thiazolo [5,4-d]Pyrimidin-2-amine the title compound was prepared as a white solid in 56% yield.¹H NMR(400MHz,DMSO-d₆)δ8.36(s,1H),4.17(br s,4H),1.74–1.55(m,6H).

And 5: synthesis of 2, 4-difluoro-N- (2-methoxy-5- (7- (piperidin-1-yl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Following the procedure of step 5 in example 1, starting from 2-bromo-7- (piperidin-1-yl) thiazolo [5,4-d]Pyrimidine the title compound was prepared (pale yellow solid, 56% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.55(s,1H),8.62(d,J＝2.2Hz,1H),8.37(s,1H),8.11(d,J＝2.2Hz,1H),7.81(td,J＝8.6,6.3Hz,1H),7.64–7.56(m,1H),7.26(td,J＝8.5,2.5Hz,1H),4.28(br s,4H),3.77(s,3H),1.79–1.60(m,6H).

Example 7: 2, 4-difluoro-N- (2-methoxy-5- (7- (pyrrolidin-1-yl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of 4-chloro-5-nitro-6- (pyrrolidin-1-yl) pyrimidine

The title compound was prepared from pyrrolidine according to the method of step 1 in example 1 (yellow solid, yield: n)52％)。¹H NMR(400MHz,DMSO-d₆)δ8.48(s,1H),3.38(br s,4H),1.99–1.84(m,4H).

Step 2: synthesis of 5-nitro-4- (pyrrolidin-1-yl) -6-thiocyanopyrimidine

The title compound was prepared from 4-chloro-5-nitro-6- (pyrrolidin-1-yl) pyrimidine according to the procedure of step 2 in example 1 (yellow solid, 78% yield).¹H NMR(400MHz,DMSO-d₆)δ8.58(s,1H),3.78(br s,2H),3.05(br s,2H),1.95(br s,4H).

And step 3: synthesis of 7- (pyrrolidin-1-yl) thiazolo [5,4-d ] pyrimidin-2-amine

The title compound was prepared from 5-nitro-4- (pyrrolidin-1-yl) -6-thiocyanopyrimidine (white solid, 55% yield) according to the method of step 3 in example 1.¹H NMR(400MHz,DMSO-d₆)δ8.07(s,1H),7.42(br s,2H),3.79(br s,4H),1.93–1.86(m,4H).

And 4, step 4: synthesis of 2-bromo-7- (pyrrolidin-1-yl) thiazolo [5,4-d ] pyrimidine

Following the procedure of step 4 in example 1, starting from 7- (pyrrolidin-1-yl) thiazolo [5,4-d]Pyrimidin-2-amine the title compound was prepared as a white solid in 18% yield.¹H NMR(400MHz,DMSO-d₆)δ8.36(s,1H),4.00(br s,2H),3.66(br s,2H),2.12–1.77(m,4H).

And 5: synthesis of 2, 4-difluoro-N- (2-methoxy-5- (7- (pyrrolidin-1-yl) thiazolo [5,4-d ] pyrimidin-2-yl) pyridin-3-yl) benzenesulfonamide

Following the procedure of step 5 in example 1, starting from 2-bromo-7- (pyrrolidin-1-yl) thiazolo [5,4-d]Pyrimidine the title compound was prepared (light yellow solid, 19% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.53(s,1H),8.60(d,J＝1.6Hz,1H),8.36(s,1H),8.10(d,J＝1.9Hz,1H),7.87–7.76(m,1H),7.68–7.53(m,1H),7.33–7.18(m,1H),4.15(br s,2H),3.76(s,3H),3.70(br s,2H),2.08(br s,2H),1.96(br s,2H).

Example 8: 2, 4-difluoro-N- (5- (7- (hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl) thiazolo [5,4-d ] pyrimidin-2-yl) -2-methoxypyridin-3-yl) benzenesulfonamide

Step 1: 2- (6-chloro-5-nitropyrimidin-4-yl) octahydrocyclopenta [ c ] pyrrole

The procedure is as in step 1 of example 1, starting from octahydrocyclopenta [ c]Pyrrole the title compound was prepared (yellow solid, 66% yield).¹H NMR(400MHz,DMSO-d₆)δ8.47(s,1H),3.68(dd,J＝11.0,7.6Hz,2H),3.23(br d,J＝9.9Hz,2H),2.79–2.64(m,2H),1.85–1.67(m,3H),1.65–1.53(m,1H),1.51–1.39(m,2H).

Step 2: synthesis of 2- (5-nitro-6-thiocyanopyrimidin-4-yl) octahydrocyclopenta [ c ] pyrrole

Following the procedure of step 2 of example 1, starting from 2- (6-chloro-5-nitropyrimidin-4-yl) octahydrocyclopenta [ c]Pyrrole the title compound was prepared (yellow solid, 73% yield).¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),3.99(br s,1H),3.66(br s,3H),2.84–2.65(m,2H),1.92–1.66(m,3H),1.66–1.53(m,1H),1.52–1.39(m,2H).

And step 3: synthesis of 7- (hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl) thiazolo [5,4-d ] pyrimidin-2-amine

Following the procedure of step 3 of example 1, starting from 2- (5-nitro-6-thiocyanopyrimidin-4-yl) octahydrocyclopenta [ c]Pyrrole the title compound was prepared (yellow solid, 66% yield).¹H NMR(400MHz,DMSO-d₆)δ8.07(s,1H),7.48(br s,2H),4.06–3.92(m,2H),3.66(br d,J＝11.2Hz,2H),2.76–2.63(m,2H),1.88–1.76(m,2H),1.75–1.64(m,1H),1.62–1.51(m,1H),1.49–1.39(m,2H).

And 4, step 4: synthesis of 2-bromo-7- (hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl) thiazolo [5,4-d ] pyrimidine

By the method of step 4 in example 1, starting from 7- (hexahydrocyclopenta [ c ]]Pyrrol-2 (1H) -yl) thiazolo [5,4-d]Pyrimidin-2-amine the title compound was prepared as a white solid in 33% yield.¹H NMR(400MHz,DMSO-d₆)δ8.35(s,1H),4.21(br s,1H),3.87(br s,2H),3.52(br s,1H),2.77(br s,2H),1.90–1.78(m,2H),1.78–1.69(m,1H),1.66–1.56(m,1H),1.56–1.44(m,2H).

And 5: synthesis of 2, 4-difluoro-N- (5- (7- (hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl) thiazolo [5,4-d ] pyrimidin-2-yl) -2-methoxypyridin-3-yl) benzenesulfonamide

By the method according to step 5 of example 1, from 2-bromo-7- (hexahydrocyclopenta [2 ]c]Pyrrol-2 (1H) -yl) thiazolo [5,4-d]Pyrimidine the title compound was prepared (light yellow solid, 19% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.54(s,1H),8.61(d,J＝2.2Hz,1H),8.35(s,1H),8.12(d,J＝2.2Hz,1H),7.82(td,J＝8.6,6.3Hz,1H),7.66–7.52(m,1H),7.26(td,J＝8.5,2.2Hz,1H),4.36(br s,1H),3.99(br d,2H),3.75(s,3H),3.56(br s,1H),2.82(br d,2H),1.96–1.83(m,2H),1.82–1.71(m,1H),1.69–1.59(m,1H),1.59–1.48(m,2H).

Comparative example 1: 2, 4-difluoro-N- (2-methoxy-5- (4-morpholinyl thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of 4- (6-bromothieno [2,3-d ] pyrimidin-4 yl) morpholine

Reacting 6-bromo-4-chlorothieno [2,3-d ]]Pyrimidine (0.749g,3.0mmol), morpholine (0.392g,4.5mmol) and triethylamine (0.607g, 6.0mmol) were in dioxane (15mL) and then stirred at 100 deg.C overnight. The reaction mixture was concentrated in vacuo, diluted with water (30mL) and extracted with ethyl acetate (30 mL. times.3). The combined organic layers were washed with water (50mL) and brine (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash column chromatography (silica gel, petroleum ether/ethyl acetate 7:1) to give the product as a pale yellow solid (0.791g, 88%).¹H NMR(500MHz,DMSO-d₆)δ8.40(s,1H),7.87(s,1H),3.82(t,J＝4.8Hz,4H),3.72(t,J＝4.8Hz,4H).

Step 2: synthesis of 2, 4-difluoro-N- (2-methoxy-5- (4-morpholinothieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

Reacting 4- (6-bromothieno [2,3-d ]]A mixture of pyrimidin-4-yl) morpholine (0.150g, 0.5mmol), N- (2-methoxy-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-3-yl) -2, 4-difluorobenzenesulfonamide (0.256g, 0.6mmol) and 2M potassium carbonate (0.77mL,1.5mmol) in dioxane (10mL) was degassed and PdCl was added₂(dppf) (0.037g,0.05 mmol). The resulting reaction mixture was degassed and backfilled with argon (three cycles), then stirred at 100 ℃ for 8 hours under an argon atmosphere. The reaction mixture was concentrated in vacuo, diluted with water (30mL) and extracted with ethyl acetate (30 mL. times.3). The combined organic layers were washed with water (30mL) and brine (30mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (silica gel, DCM/methanol 100:1) to give the product as a yellow solid (0.203g, 39% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.37(s,1H),8.48(d,J＝2.5Hz,1H),8.43(s,1H),8.04(d,J＝2.5Hz,1H),7.95(s,1H),7.76(td,J＝8.5,6.3Hz,1H),7.63–7.55(m,1H),7.22(td,J＝8.5,2.3Hz,1H),3.91(t,J＝4.8Hz,4H),3.77(t,J＝4.8Hz,4H),3.63(s,3H).

Comparative example 2: 2, 4-difluoro-N- (2-methoxy-5- (4- (4-methoxypiperidin-1-yl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of 6-bromo-4- (4-methoxypiperidin-1-yl) thieno [2,3-d ] pyrimidine

The title compound was prepared from 4-methoxypiperidine (light yellow solid, 91% yield) according to the procedure of step 1 in comparative example 1.¹H NMR(500MHz,DMSO-d₆)δ8.43(s,1H),7.32(s,1H),4.17–4.04(m,2H),3.68–3.59(m,2H),3.59–3.51(m,1H),3.41(s,3H),2.08–1.96(m,2H),1.80–1.69(m,2H).

Step 2: synthesis of 2, 4-difluoro-N- (2-methoxy-5- (4- (4-methoxypiperidin-1-yl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

By the method of step 2 in comparative example 1, starting from 6-bromo-4- (4-methoxypiperidin-1-yl) thieno [2,3-d]Pyrimidine the title compound was prepared (yellow solid, 75% yield).¹H NMR(400MHz,DMSO-d₆)δ10.37(s,1H),8.47(d,J＝2.4Hz,1H),8.39(s,1H),8.03(d,J＝2.4Hz,1H),7.87(s,1H),7.77(td,J＝8.6,6.4Hz,1H),7.64–7.55(m,1H),7.23(td,J＝8.4,2.3Hz,1H),4.26–4.15(m,2H),3.69–3.58(m,5H),3.58–3.50(m,1H),3.31(s,3H),2.04–1.93(m,2H),1.64–1.50(m,2H).

Comparative example 3: n- (5- (4- (2, 6-dimethylmorpholinyl) thieno 2,3-d ] pyrimidin-6-yl) -2-methoxypyridin-3-yl) -2, 4-difluorobenzenesulfonamide

Step 1: synthesis of 4- (6-bromothieno [2,3-d ] pyrimidin-4-yl) -2, 6-dimethylmorpholine

The title compound was prepared from 2, 6-dimethylmorpholine as a pale yellow solid in 86% yield following the procedure of step 1 of comparative example 1.¹H NMR(500MHz,DMSO-d₆)δ8.40(s,1H),7.89(s,1H),4.41(d,J＝11.9Hz,2H),3.70–3.59(m,2H),2.81(dd,J＝13.2,10.6Hz,2H),1.17(d,J＝6.2Hz,6H).

Step 2: synthesis of N- (5- (4- (2, 6-dimethylmorpholinyl) thieno 2,3-d ] pyrimidin-6-yl) -2-methoxypyridin-3-yl) -2, 4-difluorobenzenesulfonamide

4- (6-Bromomthieno [2,3-d ] according to the method of step 2 in comparative example 1]Pyrimidin-4-yl) -2, 6-dimethylmorpholine the title compound was prepared as a yellow solid in 76% yield.¹H NMR(400MH_Z,DMSO-d₆):δ10.42(s,1H),8.51(d,J＝2.3Hz,1H),8.43(s,1H),8.01(d,J＝1.9Hz,1H),7.93(s,1H),7.76(td,J＝8.6,6.6Hz,1H),7.65–7.55(m,1H),7.23(td,J＝8.6,2.0Hz,1H),4.51(d,J＝12.9Hz,2H),3.73–3.65(m,2H),3.64(s,3H),2.87(dd,J＝12.9,10.7Hz,2H),1.20(d,J＝6.2Hz,6H).

Comparative example 4: (S) -2, 4-difluoro-N- (2-methoxy-5- (4- (3-methylmorpholinyl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of (S) -4- (6-bromothieno [2,3-d ] pyrimidin-4-yl) -3-methylmorpholine

The title compound was prepared from (S) -3-methylmorpholine as a pale yellow solid in 75% yield following the procedure of step 1 in comparative example 1.¹H NMR(500MHz,DMSO-d₆)δ8.43(s,1H),7.32(s,1H),4.65(q,J＝6.5Hz,1H),4.31(d,J＝12.0Hz,1H),4.03(dd,J＝11.5,3.0Hz,1H),3.84–3.74(m,2H),3.68–3.52(m,2H),1.46(d,J＝6.9Hz,3H).

Step 2: synthesis of (S) -2, 4-difluoro-N- (2-methoxy-5- (4- (3-methylmorpholinyl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

By the method of step 2 in comparative example 1, from (S) -44- (6-bromothieno [2,3-d]Pyrimidin-4-yl) -3-methylmorpholine the title compound was prepared (yellow solid, 67% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.37(s,1H),8.44(d,J＝2.4Hz,1H),8.40(s,1H),8.00(d,J＝2.4Hz,1H),7.87(s,1H),7.76(td,J＝8.6,6.3Hz,1H),7.63–7.52(m,1H),7.21(td,J＝8.4,2.2Hz,1H),4.84(q,J＝6.6Hz,1H),4.40–4.28(m,1H),4.04–3.90(m,1H),3.81–3.66(m,2H),3.63(s,3H),3.60–3.46(m,2H),1.31(d,J＝6.8Hz,3H).

Comparative example 5: (R) -2, 4-difluoro-N- (2-m-methoxy-5- (4- (3-methylmorpholinyl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of (R) -4- (6-bromothieno [2,3-d ] pyrimidin-4-yl) -3-methylmorpholine

The title compound was prepared from (R) -3-methylmorpholine by the method of step 1 in comparative example 1 (light yellow solid, 88% yield).¹H NMR(500MHz,DMSO-d₆)δ8.43(s,1H),7.32(s,1H),4.65(q,J＝6.3Hz,1H),4.30(d,J＝12.0Hz,1H),4.03(d,J＝10.9Hz,1H),3.86–3.72(m,2H),3.69–3.50(m,2H),1.46(d,J＝6.9Hz,3H).

Step 2: synthesis of (R) -2, 4-difluoro-N- (2-m-methoxy-5- (4- (3-methylmorpholinyl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

The title compound was prepared according to the procedure of step 2 of comparative example 1 (yellow solid, 39% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.37(s,1H),8.45(d,J＝2.4Hz,1H),8.40(s,1H),8.01(d,J＝2.4Hz,1H),7.87(s,1H),7.76(td,J＝8.6,6.3Hz,1H),7.62–7.53(m,1H),7.22(td,J＝8.4,2.1Hz,1H),4.84(q,J＝6.4Hz,1H),4.44–4.24(m,1H),4.06–3.89(m,1H),3.79–3.67(m,2H),3.64(s,3H),3.60–3.46(m,2H),1.31(d,J＝6.8Hz,3H).

Comparative example 6: 2, 4-difluoro-N- (2-methoxy-5- (4- (piperidin-1-yl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of 6-bromo-4- (piperidin-1-yl) thieno [2,3-d ] pyrimidine

The title compound was prepared from piperidine as in step 1 of comparative example 1 (light yellow solid, 85% yield).¹H NMR(400MHz,DMSO-d₆)δ8.35(s,1H),7.75(s,1H),3.84–3.78(m,4H),1.74–1.56(m,6H).

Step 2: synthesis of 2, 4-difluoro-N- (2-methoxy-5- (4- (piperidin-1-yl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

By the method of step 2 of comparative example 1, starting from 6-bromo-4- (piperidin-1-yl) thieno [2,3-d]Pyrimidine the title compound was prepared (yellow solid, 46% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.37(s,1H),8.46(d,J＝2.4Hz,1H),8.37(s,1H),8.01(d,J＝2.4Hz,1H),7.84(s,1H),7.77(td,J＝8.6,6.3Hz,1H),7.64–7.54(m,1H),7.23(td,J＝8.4,2.2Hz,1H),3.96–3.84(m,4H),3.65(s,3H),1.77–1.58(m,6H).

Comparative example 7: 2, 4-difluoro-N- (2-methoxy-5- (4- (pyrrolidin-1-yl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

Step 1: synthesis of 6-bromo-4- (pyrrolidin-1-yl) thieno [2,3-d ] pyrimidine

The title compound was prepared from pyrrolidine (light yellow solid, 71% yield) following the procedure of step 1 in comparative example 1.¹H NMR(400MHz,DMSO-d₆)δ8.30(s,1H),7.78(s,1H),3.73(br s,4H),1.97(br s,4H).

Step 2: synthesis of 2, 4-difluoro-N- (2-methoxy-5- (4- (pyrrolidin-1-yl) thieno [2,3-d ] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide

By the method of step 2 in comparative example 1, starting from 6-bromo-4- (pyrrolidin-1-yl) thieno [2,3-d]Pyrimidine the title compound was prepared (yellow solid, 20% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.35(s,1H),8.42(d,J＝1.9Hz,1H),8.31(s,1H),7.99(d,J＝2.0Hz,1H),7.89(s,1H),7.77(td,J＝8.5,6.3Hz,1H),7.66–7.50(m,1H),7.23(td,J＝8.4,2.3Hz,1H),3.82(br s,4H),3.64(s,3H),2.01(br s,4H).

Comparative example 8: 2, 4-difluoro-N- (5- (4- (hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl) thieno [2,3-d ] pyrimidin-6-yl) -2-methoxypyridin-3-yl) benzenesulfonamide

Step 1: synthesis of 6-bromo-4- (hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl) thieno [2,3-d ] pyrimidine

According to comparative example1, step 1, from octahydrocyclopenta [ c]Pyrrole the title compound was prepared as a light yellow solid in 98% yield.¹H NMR(400MHz,DMSO-d₆)δ8.29(s,1H),7.81(s,1H),3.96(dd,J＝11.2,8.0Hz,2H),3.57(dd,J＝11.4,3.7Hz,2H),2.84–2.69(m,2H),1.91–1.70(m,3H),1.69–1.44(m,3H).

Step 2: synthesis of 2, 4-difluoro-N- (5- (4- (hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl) thieno [2,3-d ] pyrimidin-6-yl) -2-methoxypyridin-3-yl) benzenesulfonamide

By the method of step 2 in comparative example 1, from 6-bromo-4- (hexahydrocyclopenta [ c ]]Pyrrol-2 (1H) -yl) thieno [2,3-d]Pyrimidine the title compound was prepared (yellow solid, 64% yield).¹H NMR(400MH_Z,DMSO-d₆):δ10.35(s,1H),8.44(d,J＝2.3Hz,1H),8.31(s,1H),8.00(d,J＝2.3Hz,1H),7.92(s,1H),7.77(td,J＝8.5,6.5Hz,1H),7.65–7.53(m,1H),7.23(td,J＝8.5,2.2Hz,1H),4.05(br s,2H),3.71–3.60(m,5H),2.80(br s,2H),1.97–1.72(m,3H),1.72–1.49(m,3H).

Evaluation of pharmacological Activity

Experimental example 1: kinase activity assay for PI3K alpha

The inhibitory activity of the compounds of the invention on PI3K alpha kinase was assessed using an in vitro kinase activity assay. The ADP-Glo kinase assay kit was purchased from Promega corporation. PI3K alpha kinase was purchased from Invitrogen. All assays were performed at room temperature. Briefly, compounds, PI3K α kinase, PIP2 and ATP were dissolved in kinase buffer and then added to each well of a 384-well plate. The 384 well plates were incubated at room temperature for 1 hour, followed by the addition of ADP-Glo reagent and incubation at room temperature for 2 hours. After addition of the kinase detection reagent to each well, the 384 well plates were incubated at room temperature for 30 minutes, followed by reading the luminescence on an EnVision plate reader.

Percent inhibition was calculated based on the following equation:

inhibition [% 100- (max-sample RLU)/(max-min) [% 100 ].

Where sample RLU is the luminescence reading at a given compound concentration, max is the reading for the DMSO control, and min is the reading for the no enzyme live control. IC of compound was calculated by XLFit program in Excel₅₀The value is obtained.

The results are shown in Table 1. The experimental results show that the same R is contained in the mixture₁Radicals and the same R₂When the compounds are combined, the example compounds with the thiazolopyrimidine structure have stronger in vitro PI3K alpha kinase inhibitory activity than the corresponding comparative compounds with the thienopyrimidine structure.

Table 1: inhibitory Activity on PI3K alpha kinase

Experimental example 2: determination of tumor cell survival by MTT method

HGC-27 cells (human gastric cancer cells) in the logarithmic growth phase were digested with 0.25% trypsin-EDTA until the cells became round, then terminated with complete medium and made into single cell suspensions. Cells were seeded at 2500/100. mu.L/well in 96-well plates and placed at 37 ℃ in 5% CO₂Was incubated overnight in the cell incubator of (1). The next day 100. mu.L of complete medium (DMSO final concentration 0.1%) containing different concentrations of test compound and corresponding solvent control were added to the corresponding wells and incubation continued in the cell culture chamber for 72 h. Then 20. mu.L of freshly prepared MTT solution (50mg/mL in PBS) was added to each well to give a final MTT concentration of 5 mg/mL. After the cells are placed in a cell culture box for further incubation for 4 hours, the supernatant is carefully discarded, 180 mu L of DMSO is added into each hole to dissolve the MTT formazan sediment, and the MTT formazan sediment is uniformly mixed by shaking through a micro oscillator. The Optical Density (OD) was measured at a detection wavelength of 570 nm. To be provided withTumor cells treated with 0.1% DMSO were used as control group, and the inhibition rate of the test compound on the growth of tumor cells was calculated by the following formula, and IC was calculated according to the middle effect equation₅₀：

Inhibition (%) - (control mean OD value-dosing mean OD value)/control mean OD value × 100%

The results are shown in Table 2. The experimental results show that the same R is contained in the mixture₁Radicals and the same R₂When the compounds are used as the active ingredient, the compound of the embodiment with the thiazolopyrimidine structure has stronger in vitro tumor cell proliferation inhibition activity than the corresponding compound of the comparative example with the thienopyrimidine structure.

TABLE 2 proliferation inhibitory Activity on human gastric carcinoma HGC27 cells

Experimental example 3: study on xenograft efficacy of nude mice

Collecting human gastric cancer HGC-27 tumor cells under aseptic condition, adjusting cell density to 1 × 10 with sterilized normal saline⁶And (2) inoculating 0.2mL of the cells per liter to the axillary back subcutaneous part of the nude mouse, taking out the cells under aseptic conditions when the tumor grows to the size of 1cm in diameter, cutting the cells into tumor blocks with the size of 1mm multiplied by 1mm, and evenly inoculating the tumor blocks to the axillary back subcutaneous part of the nude mouse. After two weeks, the tumor grows to 100-200 mm³Thereafter, animals were randomized and dosing was initiated (day 0). The test compound was orally administered once daily, and the control group was orally administered with the solvent control. Body weights were weighed twice weekly and tumor lengths and widths were measured with a vernier caliper. After the experiment, the nude mice were dislocated and sacrificed, the tumor tissue was peeled off, weighed and photographed. Finally, the tumor inhibition rate is calculated to obtain the tumorThe inhibition rate was evaluated for the intensity of antitumor effect, and the results are shown in Table 3 and FIG. 1.

Tumor volume was calculated according to the following formula:

tumor volume ═ a × b²) And/2, a and b represent the maximum length and the maximum width of the tumor body respectively.

The percent tumor growth inhibition was calculated according to the following formula: tumor growth inhibition (%) - (1-T/C) × 100, T is the tumor volume of the test compound group, and C is the tumor volume of the solvent control group.

The results are shown in Table 3 and FIG. 1. The experimental results showed that the tumor growth inhibition rates at 20mg/kg and 40mg/kg doses of example 1 having a thiazolopyrimidine structure were higher than the tumor growth inhibition rate at 40mg/kg dose of the corresponding comparative example 1 having a thienopyrimidine in a nude mouse xenograft tumor model.

TABLE 3 growth inhibitory Effect of example 1 and comparative example 1 on human gastric carcinoma HGC-27 in subcutaneous xenograft tumors of nude mice

**p<0.01，***p<0.001

Summary of pharmacological activity:

the experimental results show that the same R is contained in the mixture₁Radicals and the same R₂When the compounds are combined, the example compound with the thiazolopyrimidine structure has stronger in vitro PI3K alpha kinase inhibitory activity and human HGC-27 gastric cancer cell proliferation inhibitory activity than the corresponding comparative compound with thienopyrimidine.

In the nude mouse xenograft tumor model, the tumor growth inhibition rates at 20mg/kg and 40mg/kg of the dose of example 1 having a thiazolopyrimidine structure were both stronger than those at 40mg/kg of the corresponding comparative example 1 having a thienopyrimidine structure.