Thienopyrimidine compounds, processes for their preparation, uses and pharmaceutical compositions

Abstract

The present application belongs to the technical field of pharmacy, and relates to thienopyrimidine compounds, a preparation method, purposes and a pharmaceutical composition thereof.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to a thienopyrimidine compound and a preparation method, use and pharmaceutical composition thereof. Background Art

[0002] Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that play important regulatory roles in cell survival, growth, proliferation, metabolism, and migration. PI3Ks are activated by cell surface receptors such as tyrosine kinase receptors (RTKs) or G protein-coupled receptors (GPCRs). They convert phosphatidylinositol 4,5-bisphosphate (PIP2) to the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3) at the plasma membrane. PI3Ks are divided into three classes (class I, class II, and class III). Class I PI3Ks are composed of four isoforms of catalytic subunits (α, β, δ, and γ). PI3Kα and PI3Kβ isoforms are ubiquitously expressed in all cells, while PI3Kδ and PI3Kγ isoforms are primarily restricted to leukocytes. Overactivated PI3K signaling has been found in many cancer types.

[0003] Among them, PI3Kδ has shown clinical relevance in B-cell malignancies. Furthermore, activating mutations in PIK3CD encoding p110δ lead to activated PI3Kδ syndrome (APDS), a primary immunodeficiency. Recent studies have also shown that inactivation of PI3Kδ stimulates anti-tumor immune responses by impairing the function of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Therefore, PI3Kδ has become one of the most popular targets for the treatment of B-cell malignancies and other B-cell-related diseases.

[0004] Conpanlisib, a novel pan-PI3K inhibitor, received accelerated FDA approval in 2017 for relapsed follicular lymphoma. While conpanlisib exhibits selectivity across different isoforms, this selectivity is not clear. Therefore, further development is needed for agents with greater selectivity for PI3K isoforms, particularly the δ isoform. Beyond B-cell malignancies, PI3Kδ inhibitors are also being actively developed for inflammatory and autoimmune diseases. Two other PI3Kδ inhibitors, nemiralisib and GSK-2292767, are being developed in inhaled forms and are in clinical trials for the treatment of asthma and COPD.

[0005] Although PI3Kδ inhibitors have made significant progress in the field of new drug research and development, there is still a need to develop novel skeleton structures to improve the selectivity and activity of PI3Kδ in order to explore therapies with higher safety and efficacy. Summary of the Invention

[0006] The technical problem solved by the present invention is to provide a novel selective PI3Kδ inhibitor, its preparation method, pharmaceutical composition and use. Such selective PI3Kδ inhibitor has a strong inhibitory effect on PI3Kδ isoforms and has better preventive and / or therapeutic effects on lymphoma.

[0007] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0008] The first aspect of the technical solution of the present invention is to provide a compound represented by formula (I), its tautomers or pharmaceutically acceptable salts:

[0009]

[0010] in

[0011] R1 is selected from the group consisting of hydrogen, cyano, trifluoromethyl, halogen, C 1-4 Alkyl, C 1-4 alkoxy;

[0012] R2 is selected from C 1-4 Alkyl; or phenyl or 3-7 membered cycloalkyl;

[0013] X is selected from nitrogen and oxygen.

[0014] In another preferred embodiment, the present invention provides a compound represented by formula (I), a tautomer or a pharmaceutically acceptable salt thereof:

[0015] in

[0016] R1 is selected from the group consisting of hydrogen, cyano, trifluoromethyl, halogen, C 1-4 Alkyl, C 1-4 Alkoxy; preferably selected from fluorine, methoxy, ethoxy, propoxy.

[0017] R2 is selected from C 1-4 Alkyl; or phenyl or 3-7 membered cycloalkyl;

[0018] X is selected from nitrogen and oxygen.

[0019] Further preferably, the present invention provides a compound represented by formula (I), its tautomers or pharmaceutically acceptable salts:

[0020] in

[0021] R1 is selected from the group consisting of hydrogen, cyano, trifluoromethyl, halogen, C 1-4 Alkyl, C 1-4 Alkoxy

[0022] R2 is selected from C 1-4Alkyl; or phenyl or 3-7 membered cycloalkyl; preferably selected from ethyl, propyl, isopropyl, n-butyl, cyclopropyl, cyclobutyl, cyclopentyl;

[0023] X is selected from nitrogen and oxygen.

[0024] Specifically, the preferred compounds according to the present invention are as follows:

[0025]

[0026] The second aspect of the technical solution of the present invention is to provide a method for preparing the compound and its tautomers, which comprises the following steps:

[0027]

[0028] (1) Using compound A as the starting material, compound B is prepared through a substitution reaction;

[0029] (2) Compound B is subjected to a deprotection reaction to prepare compound C;

[0030] (3) Compound C is subjected to an acylation reaction to prepare Compound D;

[0031] (4) Compound D is subjected to a coupling reaction to obtain a compound represented by formula (I), a tautomer or a pharmaceutically acceptable salt thereof.

[0032] The third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which comprises the compound, its tautomer or pharmaceutically acceptable salt, and optionally a pharmaceutically acceptable carrier and / or excipient; preferably, the pharmaceutical composition further comprises one or more active pharmaceutical ingredients for preventing and / or treating tumors in addition to the compound, its tautomer or pharmaceutically acceptable salt; preferably, the pharmaceutical composition is a pharmaceutically acceptable pharmaceutical preparation for preventing and / or treating PI3Kδ-related tumors.

[0033] In another aspect, the present invention also provides a pharmaceutical preparation comprising at least one of the compounds, tautomers or pharmaceutically acceptable salts thereof and optionally a pharmaceutically acceptable carrier or / and excipient; preferably, the pharmaceutical preparation is selected from the following pharmaceutical dosage forms: parenteral preparations, such as injection solutions or suspensions; enteral preparations, such as oral preparations, such as tablets or capsules; topical preparations, such as lotions, gels, ointments, emulsions, nasal preparations, suppositories, transdermal preparations or ophthalmic preparations.

[0034] In yet another aspect, the present invention further provides the use of the compound, its tautomer or pharmaceutically acceptable salt, or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating PI3Kδ-associated tumors. In other words, the present invention provides a method for preventing and / or treating tumors, comprising administering a preventively and / or therapeutically effective amount of the compound, its tautomer or pharmaceutically acceptable salt, or the pharmaceutical composition to a subject in need thereof.

[0035] Some of the terms used in the present invention are defined below. Other undefined terms have meanings commonly known to those skilled in the art.

[0036] Halogen refers to fluorine, chlorine, bromine or iodine.

[0037] Alkyl refers to a straight-chain or branched saturated aliphatic hydrocarbon group having 1 to 4 carbon atoms. Examples of such groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, and tert-butyl.

[0038] C 1-4 Alkoxy refers to an -O-alkyl group, wherein the alkyl group contains 1 to 4 carbon atoms and is linear, branched or cyclic. Examples of such groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropyloxy, n-butoxy, isobutoxy, and tert-butoxy.

[0039] C 3-7 Cycloalkyl refers to a saturated monocyclic, fused, spirocyclic or polycyclic structure having 3 to 7 carbon ring atoms. Examples of such groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cycloheptyl.

[0040] The compounds of the present invention include their tautomers. Tautomers refer to structural isomers of different energies that can interconvert via a low energy barrier, such as keto-enol and imine-enamine tautomerization.

[0041] The compounds of the present invention also include isotopically labeled compounds thereof, wherein one or more atoms are replaced by atoms with the same atomic number but a different atomic mass or mass number found in nature. Examples include, but are not limited to, hydrogen isotopes. 2 H and 3 H; carbon isotope 11 C. 13 C and 14 C; chlorine isotope 36 Cl; fluorine isotopes 18 F; iodine isotope 123 I and 125 I; Nitrogen isotopes 13 N and 15 N; oxygen isotope 15 O.17 O and 18 O; phosphorus isotope 32 P and sulfur isotopes 35 S.

[0042] Various hydrates and solvates of the compounds or salts thereof described in the present invention, as well as polymorphisms thereof, are also included in the scope of the present invention.

[0043] Prodrugs of the compounds of the present invention are also included within the scope of the present invention. Certain derivatives of the compounds of the present invention have weak or no pharmacological activity themselves, but when these derivatives are administered into or onto the body, they can be converted into pharmacologically active compounds of the present invention by, for example, hydrolytic cleavage. 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).

[0044] The compounds of the present invention include pharmaceutically acceptable salts thereof. A pharmaceutically acceptable salt is a salt that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharma. Sci., 1977, 66, 1-19, 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 react accordingly with some inorganic or organic bases, or inorganic and organic acids to form pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromides, hydroiodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioate, hexyne-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, Tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate.

[0045] When the compounds of the present invention are used as medicines, they are usually administered in the form of pharmaceutical compositions. Therefore, pharmaceutical compositions of the compounds of the present invention and pharmaceutically acceptable carriers, diluents or excipients are also included in the scope of the present invention. The carriers, adjuvants, and excipients used herein include any and all solvents, diluents or other liquid excipients, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. that are suitable for the desired specific dosage form. Various carriers for preparing pharmaceutically acceptable compositions and known techniques for their preparation are disclosed in Remington: The Science and Practice of Pharmacy, 21st edition, 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, the contents of which are incorporated herein by reference.

[0046] The compositions of the present invention can be administered by any route appropriate to the condition to be treated. In particular, they can be administered parenterally, for example, as an injectable solution or suspension; enterally, for example, orally, for example, as a tablet or capsule; or topically, for example, as a lotion, gel, ointment, or cream, or as a nasal or suppository. Topical administration is, for example, application to the skin. Another form of topical administration is administration to the eye.

[0047] The pharmaceutical composition can be administered in solid, semisolid, liquid, or gaseous form, or in a dried powder, such as a lyophilized form. The pharmaceutical composition 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, lozenges, 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.

[0048] Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, 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 silicon dioxide, 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; Stone powder; excipients such as cocoa butter and suppository wax; 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; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; and phosphate buffered saline, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate. Colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants may also be present in the composition according to the judgment of the formulator.

[0049] The compound of the present invention can be used alone or in combination with other therapeutic agents for treating diseases or conditions (such as cancer) of the present invention. In certain embodiments, the compound of the present invention is combined with a second compound having anti-hyperproliferative properties or for treating hyperproliferative diseases (such as cancer) in a pharmaceutical combination formulation, or combined in a dosage regimen as a combined therapy. The second compound of the pharmaceutical combination formulation or the quantitative dosing regimen preferably has an activity complementary to the compound of the present invention so that they do not adversely affect each other. Such a compound is suitably present in the combination in an amount effective for the purpose of the plan. In one embodiment, the compound of the present invention is combined with other anti-tumor drugs.The anti-tumor drugs include: alkylating agents, including but not limited to cyclophosphamide, nitrogen mustard, mafalan, myleran, carmustine; metal platinum, 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, dactinomycin, daunorubicin, doxorubicin, mitoxantrone, bleomycin, 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, mecaptopurine, thioguanine, and gemcitabine; antibodies, including but not limited to herceptin, bevacizumab; hormones, including but not limited to letrozole, vorazole, tamoxifen, toremifene, fulvestrant, flutamide, nilutamide, triptorelin; kinase inhibitors, EGFR kinase inhibitors, including but not limited to gefitinib, erlotinib, lapatinib, afatinib; VEGFR inhibitors, including but not limited to sorafenib, regorafenib, sunitinib, cabozantinib, pazopanib, vandetanib, axitinib; ALK inhibitors, including but not limited to crizotinib, ceritinib, alectinib; Bcr-Abl inhibitors, including but not limited to imatinib, ponatinib, nilotinib, dasatinib; BTK inhibitors, including but not limited to ibrutinib; B-RAF inhibitors, including but not limited to vemurafenib; cyclin-dependent kinase CDK4 / 6 inhibitors, palbociclib; mTOR inhibitors, including but not limited to rapamycin, everolimus; deacetylase inhibitors, including but not limited to vorinostat; PD1 / PDL1 antibodies, Keytruda (pembrolizumab), Opdivo (nivolumab).

[0050] The fourth aspect of the technical solution of the present invention provides the use of the compound described in the first aspect, its tautomer or pharmaceutically acceptable salt, or the pharmaceutical composition described in the third aspect in the preparation of a selective PI3Kδ inhibitor for the prevention and / or treatment of tumor diseases.

[0051] Beneficial technical effects: The compounds of the present invention have strong kinase inhibitory activity against PI3Kδ isoforms and high selectivity against PI3Kα. The compounds of the present invention have strong in vivo anti-tumor proliferative activity against Pfeiffer lymphoma cell xenografts in nude mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : is the growth inhibition curve of the compound on tumor cells, which shows the growth inhibition effect of Example Compound 6 on lymphoma cells Pfeiffer. DETAILED DESCRIPTION

[0053] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the scope of protection of the present invention is not limited to these embodiments. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0054] In the following examples, unless otherwise noted by structural formula or chemical name, molecules with a single chiral center exist as racemic mixtures. Unless otherwise noted by structural formula or chemical name, molecules with two or more chiral centers exist as racemic mixtures of diastereomers. Single enantiomers / diastereomers can be obtained by methods known to those skilled in the art.

[0055] Preparation method

[0056] The compounds of the present invention can be synthesized according to the synthetic schemes herein and / or 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.

[0057] In a general synthetic method, the compound represented by formula (I) is prepared according to Method-1.

[0058] Method-1

[0059]

[0060] Specifically, in Method-1, the thienopyrimidine compounds described herein can be prepared by a 4-step reaction. For example, starting from compound A, compound B is prepared by substitution reaction; compound C is prepared by deprotection reaction from compound B; compound D is prepared by acylation reaction from compound C; and compound D is subjected to coupling reaction to obtain the thienopyrimidine compound of formula (I).

[0061] The compounds described herein can be synthesized according to one or more of the synthetic schemes described herein and / or techniques well known in the art. One skilled in the art will recognize that the synthetic methods described in detail for certain embodiments herein can be readily adapted for the synthesis of other embodiments. In some embodiments, the compounds described herein can be prepared by appropriate combinations of synthetic methods well known in the art. Many of the starting materials and other reagents used in synthesizing the compounds described herein are commercially available from commercial vendors such as Alfa Aesar (China) Chemical Co., Ltd. or are readily prepared using synthetic methods commonly employed in the art.

[0062] 1 H NMR spectra were recorded on instruments operating at 400 MHz or 500 MHz. H NMR spectra were obtained in solution form (reported in ppm) using CDCl3(7.26 ppm) or DMSO-d6(2.50 ppm) or internal standard tetramethylsilane (0.00 ppm) as the reference standard. When reporting peak multiplicities, the following abbreviations were used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), dd (doublet of doublets), dt (doublet of triplets). Coupling constants given in Hertz (Hz).

[0063] When desired, (R)- and (S)-isomers, if present, of the non-limiting exemplary compounds can be resolved by methods known to those skilled in the art, for example, by forming diastereomeric salts or complexes, which can be separated by, for example, crystallization; by forming diastereomeric derivatives, which can be separated by, for example, crystallization or chromatography; by selective reaction of one enantiomer with an enantiomer-specific reagent followed by separation of the modified and unmodified enantiomers; or by chromatographic separation in a chiral environment. Alternatively, a particular enantiomer can be prepared by asymmetric synthesis using an optically active reagent, substrate, catalyst, or solvent, or by asymmetric transformation of one enantiomer into the other.

[0064] In the following preparation methods and examples, "Me" refers to methyl, "Et" refers to ethyl, "EA" refers to ethyl acetate, "MeOH" refers to methanol, "DMSO-d6" refers to deuterated dimethyl sulfoxide, "DCM" refers to dichloromethane, "PdCl2(dppf)" refers to 1,1'-bis(diphenylphosphinoferrocenepalladium dichloride), "HCl" refers to hydrochloric acid, "THF" refers to tetrahydrofuran, "NaH" refers to sodium hydride, "rt" refers to room temperature, "mL" refers to milliliter, "mmol" refers to millimole, "μM" refers to micromole, "nM" refers to nanomole, and "°C" refers to degrees Celsius.

[0065] Example 1: Synthesis of (S)-1-(3-((6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)thiophene[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one (Compound 1)

[0066]

[0067] Step 1: Synthesis of tert-butyl (S)-3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidine-1-carboxylate

[0068]

[0069] 6-Bromo-4-chlorothienyl[2,3-d]pyrimidine (12.476 g, 50 mmol), (S)-tert-butyl 3-aminopyrrolidine-1-carboxylate (11.176 g, 60 mmol), and triethylamine (10.435 ml, 75 mmol) were refluxed in 1,4-dioxane (200 ml) overnight. After cooling to room temperature, the volatiles were removed under reduced pressure. The residue was washed with water (200 ml) and EA (100 ml), and the insoluble solid was removed by filtration. The solid was dried under reduced pressure for 5 hours to obtain the product as a white solid (16.439 g, 82% yield).

[0070] 1 H NMR (400MHz, DMSO-d6) δ8.37 (s, 1H), 7.96 (d, J = 6.2Hz, 1H), 7.92(s,1H),4.76-4.55(m,1H),3.63-3.62(m,1H),3.49-3.33(m,2H),3.24-3.25(m,1H),2.19-2.17(m,1H),1.94-1.91(m,1H),1.42 –1.40(m,9H).

[0071] MS(ESI+)m / z:399.0,401.0[M+H]+.

[0072] Step 2: Synthesis of (S)-6-bromo-N-(pyrrolidin-3-yl)thiophene[2,3-d]pyrimidin-4-amine

[0073]

[0074] To a solution of (S)-tert-butyl 3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidine-1-carboxylate (20 mmol) in DCM (50 mL) was added trifluoroacetic acid (15 mL). The reaction mixture was stirred at room temperature for 1.5 hours. Volatiles were removed under reduced pressure. The residue was used in the next step without further purification.

[0075] 1 H NMR (400MHz, DMSO-d6) δ8.33(s,1H),7.89(s,1H),7.80(d,J= 6.7Hz,1H),4.57-4.47(m,1H),3.31(s,1H),3.07-3.03(m,1H),2.98-2.87(m,1H),2.83-2.77(m,1H),2.74-2.67(m,1H),2.09-2.00(m, 1H),1.73-1.65(m,1H).

[0076] MS(ESI+)m / z:299.0,301.0[M+H]+.

[0077] Step 3: Synthesis of (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one

[0078]

[0079] A mixture of (S)-6-bromo-N-(pyrrolidin-3-yl)thiophene[2,3-d]pyrimidin-4-amine (3 g, 10 mmol), propionic anhydride (2.56 mL, 20 mmol), and triethylamine (8.34 mL, 60 mmol) in DCM (50 mL) was stirred overnight at room temperature. Water (300 mL) was added, and the resulting mixture was extracted with DCM (100 mL x 3). The combined organic layers were washed with water (150 mL x 3) and brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (silica gel, DCM / MeOH = 12:1, v / v) to afford the product as a white solid (3.2 g, 90% yield).

[0080] 1H NMR (400MHz, DMSO-d6) δ8.38 (d, J = 2.8Hz, 1H), 7.94 (dd, J = 6.2, 2.6Hz, 1H), 7.90 (d, J = 2.6Hz, 1H), 4.74-4.61 (m, 1H), 3.84- 3.63(m,1H),3.60–3.35(m,3H),2.31–2.11(m,3H),2.08–1.89(m,1H),1.04–0.93(m,3H).

[0081] MS(ESI+)m / z:355.0,357.0[M+H]+.

[0082] Step 4: Synthesis of (S)-1-(3-((6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one

[0083]

[0084] A mixture of (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one (178 mg, 0.5 mmol), 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridine (182 mg, 0.6 mmol), and 2M aqueous KCO solution (750 μl) in dioxane (10 ml) was degassed and filled with argon, followed by the addition of PdCl(dppf) (37 mg, 0.05 mmol). The mixture was degassed and filled with argon three times, then stirred at 100°C under argon for 5 hours. The reaction mixture was cooled to room temperature, diluted with water (20 ml) and DCM (20 ml), and acidified with 2M HCl solution to a pH of 5-6. The resulting mixture was extracted with DCM (30 mL x 2). The combined organic layers were washed with water (50 ml x 2) and brine (50 ml), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (silica gel, DCM / MeOH = 30:1, v / v) to give the product as a white solid (169 mg, 75% yield).

[0085] 1H NMR (400MHz, DMSO-d6) δ8.72(s,1H),8.40(d,J=2.8Hz,1H), 8.28(t,J=2.8Hz,1H),8.06(d,J=2.9Hz,1H),8.00(dd,J=5.9,3.2 Hz,1H),4.80–4.65(m,1H),4.05(s,3H),3.88–3.69(m,1H),3.65–3.38(m,3H),2.36–2.13(m,3H),2.13–1.92(m,1H),1.03–0.97 (m,3H).

[0086] 13 C NMR(151MHz,DMSO-d6)δ171.2,165.4,159.6,156.1,154.0,147.8, 133.8,132.7,123.0,122.7(q,J C-F =274.8Hz),117.2,116.7,112.0(q,J C-F =32.7Hz),54.5,51.0,50.7,50.3,49.2,44.0,43.6,31.2,29.5,26.8, 26.5,8.8.

[0087] HRMS (ESI) m / z: 452.1353 [M+H] +.

[0088] Example 2: Synthesis of (S)-1-(3-((6-(6-methoxy-5-(cyano)pyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one (Compound 2)

[0089]

[0090] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-cyanopyridine.

[0091] 1H NMR(400MHz, DMSO-d6)δ8.76(s,1H),8.57–8.50(m,1H),8.41 (d,J=1.8Hz,1H),8.06(d,J=2.2Hz,1H),7.99(s,1H),4.80–4.65 (m,1H),4.06(s,3H),3.87–3.68(m,1H),3.63–3.37(m,3H),2.37–2.15(m,3H),2.12–1.89(m,1H),1.03–0.96(m,3H).

[0092] 13 C NMR(101MHz,DMSO-d6)δ171.7,166.1,163.5,156.7,154.6,148.8, 141.4,133.0,124.0,117.6,117.5,115.2,96.7,55.4,51.6,51.3,50.9,49.8,44.6,44.1,31.8,30.0,27.4,27.1,9.4.

[0093] HRMS (ESI) m / z: 409.1430 [M+H] +.

[0094] Example 3: Synthesis of (S)-1-(3-((6-(6-methoxy-5-fluoropyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one (Compound 3)

[0095]

[0096] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-fluoropyridine.

[0097] 1H NMR (400MHz, DMSO-d6) δ8.31(d,J=3.0Hz,1H),8.18(t,J=2.2 Hz,1H),7.89(d,J=4.8Hz,1H),7.86(d,J=5.6Hz,1H),7.82(ddd, J=11.3,4.5,2.1Hz,1H),4.72–4.56(m,1H),3.93(s,3H),3.81–3.62(m,1H),3.58–3.35(m,3H),2.26–2.07(m,3H),2.05–1.89(m, 1H),0.97–0.91(m,3H).

[0098] 13 C NMR(101MHz,DMSO-d6)δ171.8,165.8,156.6,154.4,152.9,147.3 (d,J C-F =258.7Hz),138.8,133.7,124.7,121.4,117.8,116.9,54.5,51.7, 51.3,51.0,49.8,44.7,44.2,31.8,30.1,27.5,27.2,9.4.

[0099] HRMS (ESI) m / z: 402.1385 [M+H] +.

[0100] Example 4: Synthesis of (S)-1-(3-((6-(6-methoxypyridin-3-yl)thiophene[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one (Compound 4)

[0101]

[0102] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0103] 1H NMR (400MHz, DMSO-d6) δ8.47(t,J=2.4Hz,1H),8.38(d,J=3.1 Hz,1H),8.01–7.92(m,3H),6.97(d,J=8.7Hz,1H),4.82–4.62(m, 1H),3.91(s,3H),3.88–3.67(m,1H),3.64–3.35(m,3H),2.35–2.15 (m,3H),2.12–1.93(m,1H),1.03–0.97(m,3H).

[0104] 13 C NMR(101MHz,DMSO-d6)δ171.8,165.6,164.1,156.5,154.2,144.4, 137.0,135.3,123.7,117.9,115.7,111.7,54.1,51.7,51.3,51.0,49.8,44.7,44.2,31.8,30.1,27.5,27.2,9.4.

[0105] HRMS (ESI) m / z: 384.1483 [M+H] +.

[0106] Example 5: Synthesis of (S)-1-(3-((6-(6-methoxy-5-methylpyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one (Compound 5)

[0107]

[0108] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-methylpyridine.

[0109] 1H NMR (400MHz, DMSO-d6) δ8.38(d,J=2.8Hz,1H),8.31(t,J=2.3Hz,1H),7.94(dd,J=8.3,3.6Hz,2H),7.83(t,J=2.6Hz,1H),4.80 –4.64(m,1H),3.93(s,3H),3.87–3.68(m,1H),3.63–3.38(m,3H), 2.33–2.24(m,2H),2.22(s,3H),2.20–1.94(m,2H),1.03–1.96(m, 3H).

[0110] 13 C NMR (101MHz, DMSO-d6) δ171.7,165.5,162.4,156.5,154.1,141.5, 136.5,135.5,123.6,121.3,117.9,115.5,54.1,51.6,51.2,50.9,49.8, 44.6,44.1,31.8,30.0,27.4,27.1,15.9,9.4.

[0111] HRMS (ESI) m / z: 398.1634 [M+H] +.

[0112] Example 6: Synthesis of (S)-1-(3-((6-(6-methoxy-5-methoxypyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one (Compound 6)

[0113]

[0114] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propyl-1-one and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0115] 1H NMR (400MHz, DMSO-d6) δ8.39 (d, J=2.9Hz, 1H), 8.03–8.00 (m, 1H), 7.98 (d, J=5.4Hz, 1H), 7.94 (d, J=3.8Hz, 1H), 7.49 (dd, J=3.6, 2.1Hz,1H),4.82–4.63(m,1H),3.92(s,3H),3.91(s,3H),3.88–3.69 (m,1H),3.66–3.37(m,3H),2.35–2.15(m,3H),2.14–1.93(m,1H), 1.04–0.96(m,3H).

[0116] 13 C NMR(101MHz,DMSO-d6)δ171.8,165.6,156.6,154.6,154.3,144.6, 135.5,134.2,124.1,117.9,115.9,115.6,56.2,54.0,51.7,51.3,50.9,49.8,44.7,44.2,31.8,30.1,27.5,27.1,25.5,9.4.

[0117] HRMS (ESI) m / z: 414.1590 [M+H] +.

[0118] Example 7: Synthesis of (S)-1-(3-((6-(5,6-dimethoxypyridin-3-yl)thiophene[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)butyl-1-one (Compound 7)

[0119]

[0120] Step 1: Synthesis of (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)butyl-1-one

[0121]

[0122] The title compound was prepared according to the procedure of Step 3 in Example 1 from (S)-6-bromo-N-(pyrrolidin-3-yl)thiophene[2,3-d]pyrimidin-4-amine and butyryl chloride.

[0123] 1H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J = 3.4 Hz, 1H), 7.93 (d, J = 5.6 Hz, 1H), 7.89 (d, J = 2.2 Hz, 1H), 4.75 - 4.60 (m, 1H), 3.85 - 3.64 (m, 1H), 3.62 - 3.32 (m, 3H), 2.31 - 2.11 (m, 3H), 2.09 - 1.88 (m, 1H), 1.60 - 1.46 (m, 2H), 0.93 - 0.86 (m, 3H).

[0124] MS (ESI+) m / z: 369.1, 371.1 [M+H]+.

[0125] Step 2: Synthesis of (S)-1-(3-((6-(5,6-dimethoxypyridin-3-yl)thieno[2,3- d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)butan-1-one (Compound 7)

[0126]

[0127] The title compound was prepared according to the procedure of Example 1, Step 4 from (S)-1-(3-((6-bromothieno[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)butan-1-one and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0128] 1 H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J = 3.4 Hz, 1H), 7.93 (d, J = 5.6 Hz, 1H), 7.89 (d, J = 2.2 Hz, 1H), 4.75 - 4.60 (m, 1H), 3.85 - 3.64 (m, 1H), 3.62 - 3.32 (m, 3H), 2.31 - 2.11 (m, 3H), 2.09 - 1.88 (m, 1H), 1.60 - 1.46 (m, 2H), 0.93 - 0.86 (m, 3H).

[0129] 13C NMR(101MHz,DMSO-d6)δ171.0,165.6,156.5,154.6,154.2,144.5, 135.5,134.2,124.1,117.8,115.8,115.6,56.2,53.9,51.8,51.2,50.8,49.8,44.8,44.1,36.2,35.8,31.8,30.0,18.2,14.3.

[0130] HRMS (ESI) m / z: 428.1746 [M+H] +.

[0131] Example 8: Synthesis of (S)-1-(3-((6-(5,6-dimethoxypyridin-3-yl)thiophene[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)butyl-1-one (Compound 8)

[0132]

[0133] Step 1: Synthesis of (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-2-methylpropyl-1-one

[0134]

[0135] The title compound was prepared according to the method of Step 3 in Example 1 from (S)-6-bromo-N-(pyrrolidin-3-yl)thienyl[2,3-d]pyrimidin-4-amine and isopropylcarbonyl chloride.

[0136] 1 H NMR (400MHz, DMSO-d6) δ8.38 (d, J=4.2Hz, 1H), 7.94 (s, 1H), 7.90(s,1H),4.81-4.56(m,1H),3.92-3.65(m,1H),3.63-3.34(m,3H),2.68-2.60(m,1H),2.33-2.11(m,1H),2.09-1.88(m,1H),1.05 –0.95(m,6H).

[0137] MS(ESI+)m / z:369.0,371.0[M+H]+.

[0138] Step 2: Synthesis of (S)-1-(3-((6-(5,6-dimethoxypyridin-3-yl)thiophene[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)butyl-1-one (Compound 8)

[0139]

[0140] The title compound was prepared according to the method of Example 1, Step 4 from (S)-1-(3-((6-bromothieno[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-2-methylpropyl-1- one and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0141] 1 H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 4.4 Hz, 1H), 8.01 (t, J = 1.9 Hz, 1H), 7.99 (d, J = 6.1 Hz, 1H), 7.95 (d, J = 2.9 Hz, 1H), 7.49 (dd, J = 5.5, 2.0 Hz, 1H), 4.84 - 4.62 (m, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 3.77 - 3.37 (m, 4H), 2.74 - 2.60 (m, 1H), 2.35 - 1.94 (m, 2H), 1.04 - 0.96 (m, 6H).

[0142] 13 C NMR (101 MHz, DMSO-d6) δ 174.9, 165.5, 156.6, 154.6, 154.2, 144.5, 135.5, 134.2, 124.1, 117.8, 115.8, 115.6, 56.2, 53.9, 51.5, 51.2, 51.0, 49.6, 44.7, 44.3, 31.9, 31.8, 31.5, 29.9, 19.6, 19.5, 19.4.

[0143] HRMS (ESI) m / z: 428.1750 [M+H]+.

[0144] Example 9: Synthesis of (S)-1-(3-((6-(5,6-dimethoxypyridin-3-yl)thieno[2,3-d]pyrimidin-4- yl)amino)pyrrolidin-1-yl)-2,2-dimethylpropyl-1-one (Compound 9)

[0145]

[0146] Step 1: Synthesis of (S)-1-(3-((6-bromothieno[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-2,2- dimethylpropyl-1-one

[0147]

[0148] The title compound was prepared according to the method of Step 3 in Example 1 from (S)-6-bromo-N-(pyrrolidin-3-yl)thiophene[2,3-d]pyrimidin-4-amine and tert-butylcarbonyl chloride.

[0149] 1 H NMR (400MHz, DMSO-d6) δ8.38(s,1H),7.92(s,1H),7.90(s,1H), 4.63(s,1H),3.83–3.51(m,3H),3.18–3.16(m,1H),2.18–1.97(m, 2H),1.17(s,9H).

[0150] MS(ESI+)m / z:383.0,385.0[M+H]+.

[0151] Step 2: Synthesis of (S)-1-(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-2,2-dimethylpropyl-1-one (Compound 9)

[0152]

[0153] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-2,2-dimethylpropyl-1-one and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0154] 1 H NMR (600MHz, DMSO-d6) δ8.39 (s, 1H), 8.01 (d, J = 2.0Hz, 1H), 7.95(s,1H),7.94(s,1H),7.49(d,J=1.9Hz,1H),4.68(s,1H),3.92(s,3H),3.91(s,3H),3.84–3.38(m,4H),2.29–1.87(m,2H),1.17(s,9H).

[0155] 13 C NMR(151MHz,DMSO-d6)δ174.98,164.99,156.03,154.03,153.65, 143.95,134.94,133.62,123.53,117.24,115.27,115.05,55.60,53.34,45.78,38.18,27.20.

[0156] HRMS (ESI) m / z: 442.1900 [M+H] +.

[0157] Example 10: Synthesis of (S)-cyclopropyl(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)methanone (Compound 10)

[0158]

[0159] Step 1: Synthesis of (S)-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(cyclopropyl)methanone

[0160]

[0161] The title compound was prepared according to the method of Step 3 in Example 1 from (S)-6-bromo-N-(pyrrolidin-3-yl)thiophene[2,3-d]pyrimidin-4-amine and cyclopropanecarbonyl chloride.

[0162] 1 H NMR(400MHz, DMSO-d6)δ8.38(d,J=5.4Hz,1H),7.99(dd,J=18.3,6.1Hz,1H),7.91(s,1H),4.84–4.58(m,1H),4.07–3.78(m, 1H),3.78–3.35(m,3H),2.38–2.13(m,1H),2.12–1.89(m,1H),1.84–1.68(m,1H),0.82–0.62(m,4H).

[0163] MS(ESI+)m / z:367.1,369.1[M+H]+.

[0164] Step 2: Synthesis of (S)-cyclopropyl(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)methanone (Compound 10)

[0165]

[0166] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(cyclopropyl)methanone and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0167] 1H NMR (400MHz, DMSO-d6) δ8.39(d,J=5.6Hz,1H),8.06–7.99(m, 2H),7.96(d,J=1.1Hz,1H),7.49(t,J=2.2Hz,1H),4.91–4.60(m, 1H),3.92(s,3H),3.91(s,3H),3.85–3.38(m,4H),2.39–1.94(m, 2H),1.87–1.68(m,1H),0.80–0.68(m,4H).

[0168] 13 C NMR (101MHz, DMSO-d6) δ171.4,165.6,156.5,154.6,154.2,144.5, 135.5,134.2,124.1,117.8,115.9,115.6,56.2,53.9,51.9,51.2,51.1 49.8,44.9,44.4,31.7,30.0,12.5,12.2,7.7,7.6,7.5.

[0169] HRMS (ESI) m / z: 426.1590 [M+H] +.

[0170] Example 11: Synthesis of (S)-cyclobutyl(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)methanone (Compound 11)

[0171]

[0172] Step 1: Synthesis of (S)-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(cyclobutyl)methanone

[0173]

[0174] The title compound was prepared according to the procedure of Step 3 in Example 1 from (S)-6-bromo-N-(pyrrolidin-3-yl)thienyl[2,3-d]pyrimidin-4-amine and cyclobutanecarbonyl chloride.

[0175] 1H NMR (400MHz, DMSO-d6) δ8.37(d,J=3.1Hz,1H),7.92(t,J=6.3Hz,1H),7.89(d,J=3.0Hz,1H),4.79–4.54(m,1H),3.74–3.63(m, 1H),3.56–3.34(m,3H),3.32–3.17(m,1H),2.27–1.84(m,7H),1.83 –1.64(m,1H).

[0176] MS(ESI+)m / z:381.0,383.0[M+H]+.

[0177] Step 2: Synthesis of (S)-cyclobutyl(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)methanone (Compound 11)

[0178]

[0179] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(cyclobutyl)methanone and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0180] 1 H NMR (400MHz, DMSO-d6) δ8.39(d,J=3.1Hz,1H),8.03–8.00(m, 1H),7.97(dd,J=6.0,4.2Hz,1H),7.94(d,J=4.6Hz,1H),7.49(dd, J=5.0,2.0Hz,1H),4.78–4.63(m,1H),3.92(s,3H),3.91(s,3H), 3.78–3.69(m,1H),3.62–3.42(m,2H),3.30–3.17(m,1H),2.33– 1.66(m,9H).

[0181] 13 C NMR (101MHz, DMSO-d6) δ172.6,165.6,156.6, 154.6,154.2,144.6, 135.5,134.2,124.1,117.9,115.9,115.6,56.2,54.0,51.2,51.1,51.0 ,49.7,44.2,38.0,37.8,31.8,29.9,24.8,24.7,24.5,24.4,18.0,17.9.

[0182] HRMS(ESI)m / z:440.1741[M+H]+.

[0183] Example 12: Synthesis of (S)-cyclopentyl(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)methanone (Compound 12)

[0184]

[0185] Step 1: Synthesis of (S)-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(cyclopentyl)methanone

[0186]

[0187] The title compound was prepared according to the method of Step 3 in Example 1 from (S)-6-bromo-N-(pyrrolidin-3-yl)thienyl[2,3-d]pyrimidin-4-amine and cyclopentylcarbonyl chloride.

[0188] 1 H NMR (400MHz, DMSO-d6) δ8.38(d,J=6.3Hz,1H),7.94(d,J=6.1Hz,1H),7.89(s,1H),4.77–4,59(m,1H),3.91–3.63(m,1H),3.61 –3.35(m,3H),2.42–1.87(m,3H),1.71–1.63(m,4H),1.40–1.09 (m,4H).

[0189] MS(ESI+)m / z:395.1,397.1[M+H]+.

[0190] Step 2: Synthesis of (S)-cyclopentyl(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)methanone (Compound 12)

[0191]

[0192] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(cyclopentyl)methanone and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0193] 1H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 4.5 Hz, 1H), 8.01 (t, J = 1.8 Hz, 1H), 7.98 (d, J = 6.2 Hz, 1H), 7.95 (d, J = 3.1 Hz, 1H), 7.49 (dd, J = 5.1, 2.0 Hz, 1H), 4.81 - 4.65 (m, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 3.77 - 3.34 (m, 4H), 2.91 - 2.75 (m, 1H), 2.35 - 1.93 (m, 2H), 1.85 - 1.46 (m, 8H).

[0194] 13 C NMR (101 MHz, DMSO-d6) δ 174.2, 165.6, 156.5, 154.6, 154.2, 144.5, 135.5, 134.2, 124.1, 117.8, 115.9, 115.6, 56.2, 53.9, 51.7, 51.2, 51.1, 49.7, 44.8, 44.4, 42.6, 42.2, 31.8, 30.0, 29.9, 29.8, 29.7, 29.6, 26.2, 26.1.

[0195] HRMS (ESI) m / z: 454.1896 [M+H]+.

[0196] Example 13: Synthesis of (S)-cyclohexyl(3-((6-(5,6-dimethoxypyridin-3-yl)thieno[2,3- d]pyrimidin-4-yl)amino)pyrrolidin-l-yl)methanone (Compound 13)

[0197]

[0198] Step 1: Synthesis of (S)-(3-((6-bromothieno[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-l- yl)(cyclopentyl)methanone

[0199]

[0200] The title compound was prepared from (S)-6-bromo-N-(pyrrolidin-3-yl)thieno[2,3- d]pyrimidin-4-amine and cyclohexanecarbonyl chloride according to the method of Example 1, Step 3.

[0201] 1H NMR (400 MHz, DMSO-d6) δ8.38 (d, J = 6.3 Hz, 1H), 7.93 (d, J = 6.2 Hz, 1H), 7.89 (s, 1H), 4.77–4.59 (m, 1H), 3.71–3.35 (m, 4H), 2.40 –1.86(m,3H),1.71–1.63(m,5H),1.37–1.14(m,5H).

[0202] MS(ESI+)m / z:409.1,411.1[M+H]+.

[0203] Step 2: Synthesis of (S)-cyclohexyl(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)methanone (Compound 13)

[0204]

[0205] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(cyclohexyl)methanone and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0206] 1 H NMR (400 MHz, DMSO-d6) δ8.39 (d, J = 6.5 Hz, 1H), 8.00 (t, J = 1.6 Hz, 1H), 7.97 (d, J = 6.0 Hz, 1H), 7.94 (d, J = 2.7 Hz, 1H), 7.47 (dd, J = 5.7, 1.8 Hz,1H),4.81–4.64(m,1H),3.92(s,3H),3.91(s,3H),3.77–3.35(m,4H),2.46–1.92(m,3H),1.71–1.63(m,5H),1.40–1.07 (m,5H).

[0207] 13 C NMR (101 MHz, DMSO-d6) δ174.0,165.5,156.5,154.6,154.2,144.5, 135.5,134.2,124.1,117.8,115.8,115.6,56.2,53.9,51.5,51.2,50. 9,49.6,44.7,44.3,42.1,41.8,31.8,29.9,29.2(2C),26.0,25.7(2C).

[0208] HRMS (ESI) m / z: 468.2054 [M+H] +.

[0209] Example 14: Synthesis of (S)-(3-((6-(5,6-dimethoxypyridin-3-yl)thiophene[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(phenyl)methanone (Compound 14)

[0210]

[0211] Step 1: Synthesis of (S)-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(phenyl)methanone

[0212]

[0213] The title compound was prepared according to the procedure of Step 3 in Example 1 from (S)-6-bromo-N-(pyrrolidin-3-yl)thiophene[2,3-d]pyrimidin-4-amine and phenylcarbonyl chloride.

[0214] 1 H NMR (400MHz, DMSO-d6) δ8.37(d,J=3.1Hz,1H),7.92(t,J=6.3Hz,1H),7.89(d,J=3.0Hz,1H),4.79–4.54(m,1H),3.74–3.63(m, 1H),3.56–3.34(m,3H),3.32–3.17(m,1H),2.27–1.84(m,7H),1.83 –1.64(m,1H).

[0215] MS(ESI+)m / z:381.0,383.0[M+H]+.

[0216] Step 2: Synthesis of (S)-(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(phenyl)methanone (Compound 14)

[0217]

[0218] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)(phenyl)methanone and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0219] 1H NMR(500MHz,DMSO-d6)δ8.36(d,J=48.1Hz,1H),8.10–7.99 (m,2H),7.95(d,J=27.7Hz,1H),7.55(dd,J=16.0,7.0Hz,2H),7.51 –7.41(m,4H),4.81–4.64(m,1H),3.95–3.89(m,6H),3.87–3.73 (m,1H),3.68–3.39(m,3H),2.38–1.97(m,2H).

[0220] 13 C NMR (126MHz, DMSO-d6) δ168.32,165.1,156.1,154.1,153.7,144.1, 136.8,135.1,133.7,129.9,128.3(2C),127.2(2C),123.6,117.3,115.4, 115.1,55.7,53.9,53.5,51.0,50.9,49.4,47.3,44.45,31.7,29.3.

[0221] HRMS (ESI) m / z: 428.1591 [M+H] +.

[0222] Example 15: Synthesis of (S)-1-(3-((6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)thiophene[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)propyl-1-one (Compound 15)

[0223]

[0224] Step 1: Synthesis of tert-butyl (S)-3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)oxy)pyrrolidine-1-carboxylate

[0225]

[0226] To a solution of (S)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (9.35 g, 50 mmol) in anhydrous THF was added NaH (60% in mineral oil, 4 g, 100 mmol) at 0 °C under argon atmosphere. The reaction mixture was stirred at room temperature for 30 min. Then 6-bromo-4-chlorothieno[2,3-d]pyrimidine was added and the reaction mixture was stirred at room temperature overnight. The mixture was quenched with water (50 ml) at 0 °C, diluted with water (500 ml) and extracted with DCM (200 mL x 3). The combined organic layers were washed with water (300 mL x 2) and brine (300 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was washed with EA and filtered. The product was dried to give a white solid (11.63 g, yield 58%).

[0227] 1 H NMR (400 MHz, DMSO-d6) δ 8.70 (s, 1H), 7.71 (s, 1H), 5.76 (s, 1H), 3.74-3.60 (m, 1H), 3.56-3.40 (m, 3H), 2.24-2.18 (m, 2H), 1.41-1.39 (m, 9H).

[0228] MS (ESI+) m / z: 400.0, 402.0 [M+H]+.

[0229] Step 2: Synthesis of (S)-6-bromo-4-(pyrrolidin-3-yloxy)thieno[2,3-d]pyrimidine

[0230]

[0231] The title compound was prepared from (S)-tert-butyl 3-((6-bromothieno[2,3- d]pyrimidin-4-yl)oxy)pyrrolidine-1-carboxylate according to the method of Step 2 in Example 1.

[0232] 1 H NMR (400 MHz, DMSO-d6) δ 8.66 (s, 1H), 7.63 (s, 1H), 5.64-5.60 (m, 1H), 3.15-3.04 (m, 2H), 3.00-2.93 (m, 2H), 2.84-2.78 (m, 1H), 2.17-2.01 (m, 1H), 1.94-1.83 (m, 1H).

[0233] MS (ESI+) m / z: 299.9, 301.9 [M+H]+.

[0234] Step 3: Synthesis of (S)-3-((6-bromothieno[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1- yl)propyl-1-en-1-one

[0235]

[0236] The title compound was prepared according to the method of Step 3 in Example 1 from (S)-6-bromo-4-(pyrrolidin-3-yloxy)thiophene[2,3-d]pyrimidine.

[0237] 1 H NMR (400MHz, DMSO-d6) δ8.70 (s, 1H), 7.69 (d, J = 5.0Hz, 1H), 5.88–5.69 (m, 1H), 3.94–3.37 (m, 4H), 2.42–2.14 (m, 4H), 1.06– 0.89 (m, 3H).

[0238] MS(ESI+)m / z 356.0,358.0[M+H]+.

[0239] Step 4: Synthesis of (S)-1-(3-((6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)propyl-1-one (Compound 15)

[0240]

[0241] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)propyl-1-one and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-trifluoromethylpyridine.

[0242] 1 H NMR (400MHz, DMSO-d6) δ8.85(s,1H),8.69(s,1H),8.55(d,J=2.2Hz,1H),8.02(d,J=3.9Hz,1H),5.93–5.76(m,1H),4.05(s,3H), 3.95–3.45(m,4H),2.51–2.22(m,4H),1.05–0.94(m,3H).

[0243] 13 C NMR(151MHz,DMSO-d6)δ171.2,167.3,162.2,159.9,153.3,148.5, 136.5,134.8,122.7(q,J C-F =271.8Hz),122.4,120.0,115.7,111.8(q,J C-F=32.7Hz),76.7,75.3,54.5,51.5,51.1,43.9,43.4,31.3,29.7,26.8, 26.7,8.7.

[0244] HRMS (ESI) m / z: 453.1191 [M+H] +.

[0245] Example 16: Synthesis of (S)-1-(3-((6-(6-methoxy-5-(cyano)pyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)propyl-1-one (Compound 16)

[0246]

[0247] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)propyl-1-one and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-cyanopyridine.

[0248] 1 H NMR (400MHz, DMSO-d6) δ8.86–8.82(m,2H),8.68(s,1H),7.93 (d,J=3.1Hz,1H),5.90–5.76(m,1H),4.04(d,J=7.2Hz,3H),3.95 –3.44(m,4H),2.40–2.18(m,4H),1.04–0.96(m,3H).

[0249] 13 C NMR (101MHz, DMSO) δ171.2,167.3,163.1,162.2,153.4,148.9, 141.4,136.0,122.8,119.9,115.7,114.6,96.1,76.7,75.2,54.9,51.4, 51.1,43.8,43.4,31.3,29.7,26.8,26.7,8.7.

[0250] HRMS (ESI) m / z: 410.1275 [M+H] +.

[0251] Example 17: Synthesis of (S)-1-(3-((6-(5-fluoro-6-methoxypyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)propyl-1-one (Compound 17)

[0252]

[0253] The title compound is prepared from (S)-3-((6-bromothieno[2,3-d]pyrimidin-4- yl)oxy)pyrrolidin-1-yl)propan-1-one and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-3-fluoropyridine according to the method of Example 1, Step 4.

[0254] 1 H NMR (400 MHz, DMSO-d6) δ 8.70 (s, 1H), 8.44 (d, J = 2.0 Hz, 1H), 8.39 - 8.33 (m, 1H), 7.92 (d, J = 3.6 Hz, 1H), 5.89 - 5.81 (m, 1H), 4.01 (s, 3H), 3.93 - 3.48 (m, 4H), 2.42 - 2.18 (m, 4H), 1.02 - 0.95 (m, 3H).

[0255] 13 C NMR (151 MHz, DMSO-d6) δ 171.2, 167.2, 162.1, 153.2, 152.7, 146.70 (d, J C-F = 258.2 Hz), 139.0, 136.8, 123.5, 121.4 (d, J C-F = 17.1 Hz), 119.9, 115.1, 76.7, 75.2, 53.9, 51.5, 51.1, 43.8, 43.4, 31.3, 29.7, 26.8, 26.7, 8.7.

[0256] HRMS (ESI) m / z: 403.1224 [M+H]+.

[0257] Example 18: Synthesis of (S)-1-(3-((6-(5,6-dimethoxypyridin-3-yl)thieno[2,3- d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)propan-1-one (Compound 18)

[0258]

[0259] The title compound is prepared from (S)-3-((6-bromothieno[2,3-d]pyrimidin-4- yl)oxy)pyrrolidin-1-yl)propan-1-one and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-3-fluoropyridine according to the method of Example 1, Step 4.

[0260] 1H NMR(400MHz, DMSO-d6)δ8.68(s,1H),8.10(d,J=1.7Hz,1H), 7.87(d,J=3.5Hz,1H),7.76(d,J=2.0Hz,1H),5.93–5.77(m,1H), 3.92(s,3H),3.92(s,3H),3.78–3.42(m,4H),2.40–2.20(m,4H), 1.06–0.92(m,3H).

[0261] 13 C NMR(101MHz,DMSO-d6)δ171.8,167.6,162.5,154.9,153.5,144.5, 139.0,134.8,123.5,120.5,116.0,114.7,77.2,75.8,56.4,53.9,52.0,51.7,44.4,44.0,31.9,30.3,27.3,9.3.

[0262] HRMS (ESI) m / z: 415.1433 [M+H] +.

[0263] Example 19: Synthesis of (S)-1-(3-((6-(5,6-dimethoxypyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)-2-methylpropyl-1-one (Compound 19)

[0264]

[0265] Step 1: Synthesis of (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)-2-methylpropyl-1-one

[0266]

[0267] The title compound was prepared according to the method of Step 3 in Example 1 from (S)-6-bromo-4-(pyrrolidin-3-yloxy)thiophene[2,3-d]pyrimidine and isopropylcarbonyl chloride.

[0268] 1 H NMR(400MHz, DMSO-d6)δ8.70(d,J=2.0Hz,1H),7.69(d,J=2.9 Hz,1H),5.91–5.71(m,1H),3.99–3.40(m,4H),2.78–2.56(m,1H), 2.41–2.15(m,2H),1.05–0.94(m,6H).

[0269] MS(ESI+)m / z:369.9,371.9[M+H]+.

[0270] Step 2: Synthesis of (S)-1-(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)-2-methylpropyl-1-one (Compound 19)

[0271]

[0272] The title compound was prepared according to the method of Step 4 in Example 1 from (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)-2-methylpropyl-1-one and 2,3-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.

[0273] 1 H NMR (400MHz, DMSO-d6) δ8.68 (d, J = 1.4 Hz, 1H), 8.10 (d, J = 2.0 Hz, 1H), 7.86 (d, J = 3.1 Hz, 1H), 7.75 (dd, J = 3.2, 2.2 Hz, 1H), 5.96 –5.70(m,1H),3.92(s,6H),3.85–3.44(m,4H),2.77–2.60(m,1H), 2.45–2.14(m,2H),1.06–0.94(m,6H).

[0274] 13 C NMR (101MHz, DMSO-d6) δ175.0,167.6,162.5,154.9,153.4,144.5, 139.0,134.8,123.5,120.5,116.0,114.7,77.2,75.6,56.4,53.9,52.0, 51.8,44.4,44.1,32.0,31.8,31.7,30.1,19.6,19.5,19.4.

[0275] HRMS (ESI) m / z: 429.1592 [M+H] +.

[0276] Example 20: Synthesis of (S)-cyclopropyl(3-((6-(5,6-dimethoxypyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)methanone (Compound 20)

[0277]

[0278] Step 1 : Synthesis of (S)-l-(3-((6-bromothieno[2,3-d]pyrimidin-4- yl)oxy)pyrrolidin-l-yl)(cyclopropyl)methanone

[0279]

[0280] The title compound is prepared from (S)-6-bromo-4-(pyrrolidin-3-yloxy)thieno[2,3- d]pyrimidine and cyclopropylcarbonyl chloride according to the method of Example 1, Step 3.

[0281] 1 H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 3.5 Hz, 1H), 7.73 (d, J = 3.2 Hz, 1H), 5.89 - 5.76 (m, 1H), 4.16 - 3.39 (m, 4H), 2.42 - 2.19 (m, 2H), 1.90 - 1.65 (m, 1H), 0.84 - 0.62 (m, 4H).

[0282] MS (ESI+) m / z: 368.0, 370.0 [M+H]+.

[0283] Step 2: Synthesis of (S)-cyclopropyl(3-((6-(5,6-dimethoxypyridin-3-yl)thieno[2,3- d]pyrimidin-4-yl)oxy)pyrrolidin-l-yl)methanone (Compound 20)

[0284]

[0285] The title compound is prepared from (S)-l-(3-((6-bromothieno[2,3-d]pyrimidin-4- yl)oxy)pyrrolidin-l-yl)(cyclopropyl)methanone and 2,3-dimethoxy-5-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)pyridine according to the method of Example 1, Step 4.

[0286] 1 H NMR (400 MHz, DMSO-d6) δ 8.69 (d, J = 3.4 Hz, 1H), 8.11 (d, J = 2.0 Hz, 1H), 7.91 (d, J = 3.1 Hz, 1H), 7.79 (d, J = 1.8 Hz, 1H), 5.95 - 5.83 (m, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.87 - 3.43 (m, 4H), 2.46 - 2.16 (m, 2H), 1.90 - 1.69 (m, 1H), 0.81 - 0.67 (m, 4H).

[0287] 13C NMR(101MHz,DMSO-d6)δ171.5,167.7,162.5,154.9,153.5,144.5, 139.0,134.8,123.5,120.5,116.1,114.8,77.1,75.8,56.4,53.9,52.4,52.0,44.7,44.3,31.8,30.2,12.4,7.8,7.6.

[0288] HRMS (ESI) m / z: 427.1428 [M+H] +.

[0289] Example 21: Synthesis of (S)-cyclobutyl(3-((6-(5,6-dimethoxypyridin-3-yl)thien[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)methanone (Compound 21)

[0290]

[0291] Step 1: Synthesis of (S)-1-(3-((6-bromothienyl[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)(cyclobutyl)methanone

[0292]

[0293] The title compound was prepared according to the method of Step 3 in Example 1 from (S)-6-bromo-4-(pyrrolidin-3-yloxy)thiophene[2,3-d]pyrimidine and cyclobutanecarbonyl chloride.

[0294] 1 H NMR (400MHz, DMSO-d6) δ8.69 (d, J = 1.4 Hz, 1H), 7.67 (d, J = 3.3 Hz, 1H), 5.81–5.74 (m, 1H), 3.81–3.41 (m, 4H), 2.34–2.03 (m, 7H), 1.97–1.68(m,2H).

[0295] MS(ESI+)m / z:382.0,384.0[M+H]+.

[0296] Step 2: Synthesis of (S)-cyclobutyl(3-((6-(5,6-dimethoxypyridin-3-yl)thienyl[2,3-d]pyrimidin-4-yl)oxy)pyrrolidin-1-yl)methanone (Compound 21)

[0297]

[0298] The title compound is prepared from (S)-l-(3-((6-bromothieno[2,3-d]pyrimidin-4- yl)oxy)pyrrolidin-l-yl)(cyclobutyl)methanone and 2,3-dimethoxy-5-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)pyridine according to the method of Example 1, Step 4.

[0299] 1 H NMR (400 MHz, DMSO-d6) δ 8.68 (d, J = 1.0 Hz, 1H), 8.10 (d, J = 2.0 Hz, 1H), 7.86 (d, J = 1.5 Hz, 1H), 7.76 - 7.75 (m, 1H), 5.86 - 5.80 (m, 1H), 3.92 (s, 6H), 3.85 - 3.43 (m, 4H), 3.31 - 3.18 (m, 1H), 2.42 - 1.67 (m, 8H).

[0300] 13 C NMR (101 MHz, DMSO-d6) δ 172.6, 167.6, 162.5, 154.9, 153.5, 144.5, 139.0, 134.8, 123.5, 120.5, 116.0, 114.7, 77.1, 75.6, 56.4, 53.9, 51.7, 51.5, 44.1, 44.0, 37.8, 31.9, 30.1, 24.8, 24.7, 24.3, 18.0, 17.9.

[0301] HRMS (ESI) m / z: 427.1428 [M+H]+.

[0302] Pharmacological activity evaluation

[0303] Example 22: Biochemical assay for PI3K a and PI3K5 activity

[0304] The potency of the compounds of the present invention against PI3Kα and PI3Kδ was assessed using an in vitro kinase assay. The kinase activity of PI3Kα and PI3Kδ was determined using a luciferase-based luminescence assay by measuring the level of ADP generated during the kinase reaction. The ADP-Glo ​​Kinase Assay Kit was purchased from Promega. All assays were performed at room temperature using 384-well plates. PI3Kα and PI3Kδ kinases were from Invitrogen. The substrate was PIP2 (Invitrogen). The kinase buffer solution consisted of 50 mM Hepes (pH 7.5), 3 mM MgCl2, 100 mM NaCl, 1 mM EGTA, 0.03% CHAPS, and 2 nM DTT. PI3Kα and PI3Kδ kinase solutions were prepared by diluting PI3Kα and PI3Kδ kinases to 6.6 nM in kinase buffer. The substrate solution consisted of 100 μM PIP2 and 50 μM ATP. Test compounds in 100% DMSO were diluted 25-fold in 1x kinase buffer. 2.5 μL of the diluted compound solution and 2.5 μL of PI3Kα and PI3Kδ kinase solutions were added to each well of a 384-well plate. Reactions were initiated by adding 5 μL of substrate solution per well, for a final reaction volume of 10 μL, containing 25 μM ATP, 50 μM PIP2, and 1.65 nM PI3Kα and PI3Kδ kinases. The plate was covered and the reaction was allowed to proceed at room temperature for 1 hour, after which the reaction was terminated by adding 10 μL of kinase ADP-Glo ​​reagent per well. The plate was incubated for 15 minutes before luminescence reading on an EnVision 2104 Multilabel Microplate Reader.

[0305] The percentage inhibition was calculated based on the following formula:

[0306] Inhibition % = 100 - (max-sample RLU) / (max-min) * 100.

[0307] Where sample RLU is the luminescence reading at a given compound concentration, min refers to the reading of the DMSO control, and max refers to the reading of the no enzyme activity control. The IC value of the compound was calculated using the XLfit program in Excel. 50 , the results are shown in Table 1.

[0308] Table 1: Kinase inhibitory activity against PI3Kα and PI3Kδ and selectivity for PI3Kδ

[0309]

[0310] Example 23: Pfeiffer antiproliferation assay of lymphoma cells

[0311] Pfeiffer cells (diffuse large B-cell lymphoma, DLBCL, ATCC) in the logarithmic growth phase were collected by centrifugation at 850 rpm for 5 minutes. After resuspending and counting, the cells were adjusted to a concentration of 1*10^5 / mL using 1640 medium containing 10% FBS, and 100 μL / well was seeded into a 96-well plate. Different concentrations of drugs were prepared, and medium containing the final concentration of the compound was added to each well. 100 μL of medium without drug was added to the control well. Three replicates were made for each concentration, gently mixed, and the cells were placed in a 37°C, 5% CO2 incubator for further 96 hours. After 96 hours, cell activity was detected using the CellTiter-Glo kit; IC was calculated using Graphpad. 50 , evaluate the ability of the compounds to inhibit tumor cell proliferation in vitro.

[0312] See the results Figure 1

[0313] Summary of pharmacological activities:

[0314] All the compounds in the examples showed strong kinase inhibitory activity against PI3Kδ kinase, with IC 50 The values ​​were all less than 300 nM, and the compound had a high selectivity for PI3Kα (greater than 15 times). Example 6 showed strong anti-proliferative activity against Pfeiffer lymphoma cells, with an IC 50 The value is 1.921 μM.

Claims

1. A compound represented by formula (I), its tautomer or pharmaceutically acceptable salt: in R1 is selected from the group consisting of hydrogen, cyano, trifluoromethyl, halogen, C 1-4 Alkyl, C 1-4 alkoxy; R2 is selected from C 1-4 Alkyl; or phenyl or 3-7 membered cycloalkyl; X is selected from NH and oxygen.

2. The compound according to claim 1, its tautomer or pharmaceutically acceptable salt, characterized in that: R1 is selected from fluorine, methoxy, ethoxy, and propoxy.

3. The compound according to any one of claims 1 or 2, its tautomer or pharmaceutically acceptable salt, characterized in that: R2 is selected from ethyl, propyl, isopropyl, n-butyl, cyclopropyl, cyclobutyl, and cyclopentyl.

4. The compound according to claim 1, its tautomer or pharmaceutically acceptable salt, wherein the compound is selected from:

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises at least one compound according to any one of claims 1 to 4, its tautomer or pharmaceutically acceptable salt and optionally a pharmaceutically acceptable carrier and / or excipient.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition further comprises a pharmaceutically active ingredient other than the compound, its tautomer or pharmaceutically acceptable salt.

7. Use of the compound according to any one of claims 1 to 4, its tautomer or pharmaceutically acceptable salt, or the pharmaceutical composition according to any one of claims 5 or 6 in the preparation of a PI3Kδ kinase inhibitor.

8. Use of the compound according to any one of claims 1 to 4, its tautomer or pharmaceutically acceptable salt in the preparation of a medicament for preventing and / or treating diseases caused by overactivation of the PI3K pathway.

9. Use according to claim 8, characterized in that The diseases in which the PI3K pathway is overactivated include tumors, autoimmune diseases, kidney diseases, cardiovascular diseases, inflammation, metabolic / endocrine dysfunction or neurological diseases.

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