RU2826012C2 - Heterocyclic compounds for tyrosine kinase 2 activity mediation - Google Patents

Abstract

FIELD: chemistry; pharmaceutics.

SUBSTANCE: present invention relates to heterocyclic compounds or pharmaceutically acceptable salts thereof, suitable for controlling or inhibiting Janus kinase (JAK) activity, in particular tyrosine kinase 2 (TYK2), specifically to a compound of formula (I) or a pharmaceutically acceptable salt thereof, a stable isotope, a stereoisomer, in which R¹ is pyrazolyl, where one pyrazolyl hydrogen is optionally substituted -COOR^b, -C(O)R^b, -C(O)NR^bR^c, C_1–6alkyl, CD₃ or piperidinyl; R² is H; R³ is H, halogen, cyano, C_1-6 alkyl, or OC_1–6alkyl; R⁴ and R⁵ are independently selected from H, or C_1-6 alkyl; R⁶ and R⁷ are independently selected from H, or R⁶ and R⁷ are combined in the form of oxo; L is a bond, C_1-6 alkylene, -C(O)-, -C(O)O-, -C(O)N(R^a)- or -S(O)₂-; A is H, C_1-6alkyl, C_3-6 cycloalkyl, 4-membered heterocyclyl, in which one ring atom is N or O, or 5–6-membered heteroaryl, in which from 1 to 3 ring atoms are N or O heteroatoms, where one or more hydrogen atoms of alkyl, cycloalkyl and heteroaryl are optionally substituted with halogen, cyano or -OR^d; R^a represents H; R^b and R^c are independently selected from H or C_1-6alkyl, and R^d is independently selected from H, C_1–6 alkyl or morpholinyl. Invention also relates to specific compounds, as well as to a pharmaceutical composition based on said compounds.

EFFECT: obtaining alternative compounds for treating TYK2-mediated diseases.

11 cl, 101 ex

Description

Description

Field of technology to which the present invention relates

The present invention relates to heterocyclic compounds or pharmaceutically acceptable salts thereof suitable for regulating or inhibiting the activity of Janus kinase (JAK), in particular tyrosine kinase 2 (TYK2). The present invention also relates to methods for preparing said heterocyclic compounds. The present invention further relates to methods for treating and/or preventing diseases mediated by said kinase, in particular autoimmune diseases, inflammatory diseases or malignant tumors.

Prior art of the present invention

JAK is a non-receptor tyrosine protein kinase formed by four members of the family, namely JAK1, JAK2, JAK3, and TYK2. JAK has 7 homology domains in its structure (JAK homology domain, JH), among which JH1 is a kinase domain, JH2 is a pseudokinase domain (which regulates the kinase activity of JH1), and JH6 and JH7 are receptor-binding domains. When the cell surface region of a cytokine receptor binds to a cytokine, its intracellular region, to which JAKs are attached, is phosphorylated, which forms a docking site for signal transducer and activator of transcription (STAT) proteins. STAT proteins are further phosphorylated by activated JAKs to form a dimer that enters the nucleus, regulates the expression and transcription of associated genes, and allows signaling from the cell membrane to the nucleus (Lionard et. al, Ann. Rev. Immunol. 1998, 16, 293-322). Therefore, JAK transmits cytokine-mediated signals through the JAK-STAT pathway and plays an important role in many cellular functions such as cytokine-dependent regulation of cell proliferation, differentiation, apoptosis, and immune response, and is a known target for the treatment of inflammatory diseases, autoimmune diseases, and malignancies (Alicea-Velazquez et. al, Curr. Drug Targets 2011, 12, 546-55). Several pharmaceuticals that regulate JAK have been approved for marketing or submitted for approval, including the JAK1/JAK2 inhibitor ruxolitinib and the JAK2 inhibitor fedratinib for the treatment of myelofibrosis, the pan-JAK inhibitor tofacitinib, the JAK1/JAK2 inhibitor baricitinib, the pan-JAK inhibitor peficitinib and the JAK1 inhibitor upadacitinib for the treatment of rheumatoid arthritis, etc.

Gene knockout studies have demonstrated that JAKs and STATs play highly specific roles in regulating various immune responses. JAK enzyme can participate in signaling processes induced by multiple cytokines, and cytokine signaling pathway can also activate multiple JAK enzymes, but cytokine itself has some selectivity for STAT activation. For example, interleukin-4 (IL-4) activates STAT6, while IL-12 specifically activates STAT4. JAK1, JAK2 and TYK2 are widely expressed in various tissues and cells. JAK1 is closely associated with the activation of inflammatory factors such as IL-6 and interferon (IFN), so selective JAK1 inhibitor is considered to have potential therapeutic effect on autoimmune diseases such as rheumatoid arthritis (RA) and psoriasis. JAK2 independently mediates cytokines. such as erythropoietin (EPO) and thrombopoietin (TPO) (Won et. al, BMC Bioinformatics 2009. 10, S53), and is closely associated with the proliferation and differentiation of blood cells. JAK3 is present only in the bone marrow and lymphatic system, and mediates IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 signaling. These cytokines play an important role in the induction of T cell proliferation and differentiation, the activation of B cells to produce antibodies, the activation of macrophages, the enhancement of natural killer cell (NK cell) activity, and the induction of other cytokines such as IFN. Therefore, a selective JAK3 inhibitor is thought to have an important role in organ transplantation and the treatment of autoimmune diseases and inflammatory pneumonia.

The JAK/STAT pathway can be over-activated by autocrine and paracrine cytokines as well as by certain mutations, and is associated with a variety of malignancies such as breast cancer, liver cancer, prostate cancer, colorectal cancer, lung cancer, pancreatic cancer, bladder cancer, and diffuse large B-cell lymphoma (Tan et. al, Curr. Drug Targets 2014, 15, 1341-53; Lam et. al, Blood 2008, 111, 3701-13). The JAK2 JAK2/V617F mutation occurs in the pseudokinase domain of JH2, causing a conformational change in JAK2, resulting in prolonged activation of the JH1 kinase domain independent of extracellular cytokine signals, which in turn causes cell hyperplasia and hematopoietic malignancy, and is closely associated with polycythemia vera (PV), essential thrombocythemia and myelofibrosis (MF) (O'Shea et. al, Ann. Rev. Med. 2015, 66, 311-28). The JAK2 inhibitor ruxolitinib can be used to treat such hematopoietic disorders, but its efficacy is not associated with the presence of the JAK2/V617F mutation, indicating that the antitumor activity is not solely based on the inhibition of signaling caused by the JAK2/V617F mutation, but may also be associated with the regulation of JAK1-STAT.

TYK2 is involved in signaling of inflammatory cytokines such as interferons (IFN), IL-12 and IL-23, and plays a key role in innate immunity and acquired immunity. TYK2 knockout mice have normal red blood cell counts and are able to survive, but JAK3-deficient mice are characterized by severe immunodeficiency, and JAK1 or JAK2 knockout mice can die during the embryonic period. However, diseases caused by impaired JAK1/2 function have not been detected in humans, indirectly indicating the importance of the physiological functions of JAK1/2. One patient with a TYK2 null mutation had hyperimmunoglobulinemia E syndrome, but seven other cases of TYK2 homositosis null mutation did not have hyperimmunoglobulinemia E syndrome, although susceptibility to mycobacterial or viral infections was increased due to decreased IL-12 and INF-α/β responses. Therefore, TYK2 inhibition does not cause acute toxicity. The absence of TYK2 expression is manifested by impaired signaling of multiple proinflammatory cytokines and severe imbalance of T helper cell differentiation. In addition, genetic studies support the hypothesis that TYK2 is a characteristic susceptibility gene for autoimmune diseases. The importance of TYK2-regulated pathways has been further confirmed by the efficacy of antibody therapies in treating diseases in clinical practice, such as IL-12/IL-23-targeted ustekinumab for psoriasis and type I interferon receptor-targeted anifrolumab for systemic lupus erythematosus (SLE). Therefore, TYK2 has attracted much attention as a drug target for autoimmune diseases. For example, TYK2 inhibitor can be potentially used for the treatment of psoriasis, SLE, and inflammatory bowel disease (IBD), etc.

TYK2 is also associated with some malignancies. For example, abnormal survival of human acute lymphoblastic leukemia (T-ALL) cells is associated with TYK2 activation. Gene knockout experiments demonstrated that 88% of T-ALL cell lines and 63% of patient-derived T-ALL cells are TYK2 dependent, indicating that TYK2 is a T-ALL oncogene (Sanda et. al, Cancer Disc. 2013, 3, 564-77). The selective TYK2 inhibitor NDI-031301 can induce apoptosis to inhibit the growth of human T-ALL cell lines and has the desired safety and efficacy in mouse models of T-ALL tumor KOPT-K1 cells (Akahane et. al, British J. Haematol. 2017, 177, 271-82), demonstrating the promise of a selective TYK2 inhibitor for the treatment of T-ALL.

In addition to many inhibitors primarily targeting JAK1/2/3, the specific TYK2 inhibitor BMS-986165 and the dual JAK1/TYK2 inhibitor PF-06700841 have also entered late-stage clinical trials. Because JAK regulates different immune responses in JAK-STAT, different selective JAK inhibitors exhibit different toxicities and side effects in clinical use and have different clinical applications. Ruxolitinib is used to treat bone marrow fibrosis and exhibits good safety and no toxicities and side effects to non-target organs. Tofacitinib inhibits JAK2 activity in addition to JAK1, affects the production of blood cells and lymphocytes, and exhibits a certain toxicity in the form of anemia, thereby limiting its clinical efficacy in RA. Due to the promise of JAK inhibitors in the treatment of inflammatory diseases, autoimmune diseases and malignancies, the development of selective JAK inhibitors has attracted much attention from the pharmaceutical industry. However, due to the high sequence similarity in the active sites of the JAK family of kinases, it is difficult to develop a selective JAK inhibitor. Although several patent applications have been disclosed for selective TYK2 inhibitors, including WO2010142752, WO2012062704, WO2013180265, WO2015032423, WO2015131080 and WO2017040757, there is an ongoing need to develop novel compounds that have better targeting, greater potency and higher selectivity for TYK2 or TYK2/JAK1.

Detailed description of the present invention

Definitions

Unless otherwise specified, the following terms used in this application have the following meanings.

"C _x-y " refers to a numerical range of carbon atoms where x and y are integers, for example, C _3-8 cycloalkyl means a cycloalkyl containing from 3 to 8 carbon atoms. It should also be understood that "C _3-8 " further includes any subranges such as C _3-7 , C _3-6 , C _4-7 , C _4-6 and C _5-6 .

"Alkyl" refers to a saturated straight-chain or branched hydrocarbyl substituent containing from 1 to 20 carbon atoms, such as from 1 to 8 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl. 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl.

"Alkylene" refers to a saturated straight-chain or branched hydrocarbyl divalent substituent containing from 1 to 20 carbon atoms, such as from 1 to 6 carbon atoms or from 1 to 4 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, _-CH2- , -CH _{( CH3} ) _- , _{-CH2CH2- , -CH2CH2CH2- ,} -( _CH3 )C( _CH3 ) _- , _{-CH2CH2CH2CH2-} , and _-CH2CH ( _{CH3 ) CH2-} .

"Cycloalkyl" refers to a saturated cyclic hydrocarbyl substituent containing from 3 to 14 ring carbon atoms. Cycloalkyl can be a monocarbon ring substituent, typically containing from 3 to 8, from 3 to 7, or from 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Cycloalkyl can also be a substituent with two or three monocarbon rings that are fused together, such as decahydronaphthyl.

"Heterocyclyl or heterocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic group containing from 3 to 20 ring atoms, such as from 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, or 5 to 6 ring atoms, in which one or more ring atoms are selected from N, O, and S(O) _m (where m is an integer from 0 to 2), but do not contain -O-O-, -OS-, or -SS- in the ring structure, and the remainder are carbon atoms. Preferably, it may contain from 3 to 12 ring atoms, from 3 to 10 ring atoms, from 4 to 7 ring atoms and from 4 to 6 ring atoms, wherein from 1 to 4 are heteroatoms, from 1 to 3 are heteroatoms or from 1 to 2 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl include, but are not limited to, pyrrolidinyl, oxetanyl, piperidyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, homopiperazinyl and azetidinyl. The polycyclic heterocyclyl includes fused, bridged or spiro polycyclic heterocycle such as octahydrocyclopenta[c]pyrrole, octahydropyrrole[1,2-a]pyrazine, 3,8-diazabicyclo[3.2.1]octane, 5-azaspiro[2.4]heptane and 2-oxa-7-azaspiro[3.5]nonane.

"Aryl or aryl ring" refers to an aromatic monocyclic or fused polycyclic group containing from 6 to 14 carbon atoms, preferably 6 to 10 membered, such as phenyl or naphthyl, most preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, and non-limiting examples include, but are not limited to:

"Heteroaryl or heteroaryl ring" refers to a heteroaromatic system containing from 5 to 14 ring atoms, of which from 1 to 4 ring atoms are selected from heteroatoms including O, S and N. Heteroaryl is preferably 5-10 membered, and more preferably 5- or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, pyrazolyl, imidazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, isoquinolinyl, indolyl and isoindolyl. The heteroaryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, and non-limiting examples include, but are not limited to:

"Halogen" refers to F, Cl, Br, or I.

"Cyano" refers to -CN.

"Oxo" refers to =O.

"Carbonyl" refers to the -C(=O)- group.

"Sulfonyl" belongs to the -S(O) _2- group.

"Sulfinyl" belongs to the -S(O)- group.

"Optionally substituted or optionally substituted" refers to one or more hydrogen atoms in the group, preferably 5, more preferably 1 to 3 hydrogen atoms, being independently substituted by the appropriate number of substituents. It goes without saying that the substituents are located solely in chemical positions in which they can be located, and those skilled in the art will be able to determine possible or impossible substitutions without much difficulty (by experiment or theory). For example, amino or hydroxyl groups with a free hydrogen atom may be unstable when bonded to carbon atoms of unsaturated bonds (such as olefins). Substituents include, but are not limited to, halogen, cyano, nitro, oxo, -SF ₅ , C _1-4 alkyl, C _3-7 cycloalkyl, etc.

"Isomers" refers to compounds that have the same molecular formula, but the nature or sequence of their atomic bonds or spatial arrangement differ. Isomers with different arrangements of their atoms in space are called "stereoisomers." Stereoisomers include optical isomers, geometric isomers, and conformational isomers.

The compounds of the present invention may exist in the form of optical isomers. Optical isomers include enantiomers and diastereoisomers. An enantiomer is one of two stereoisomers that are mirror images of each other and do not superpose. A racemic mixture or racemate is a mixture that contains equal amounts of the levorotatory and dextrorotatory enantiomers of a chiral molecule. Diastereoisomers are stereoisomers that are not mirror images of each other and do not superpose. Methods for preparing and separating optical isomers are known in the art. If a compound is a single isomer and its absolute configuration is determined, it is called the "R" or "S" isomer depending on the configuration of the substituents around the chiral carbon atom; if its absolute configuration is not determined, it is called the (+) or (-) isomer according to its measured optical rotation value.

The compounds of the present invention may also have geometric isomers resulting from the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyl or heterocyclyl groups. Substituents around a carbon-carbon double bond or carbon-nitrogen bond are designated as being in the Z or E configuration, and substituents around a cycloalkyl or heterocycle are designated as being in the cis or trans configuration.

The compounds of the present invention may also exhibit tautomerism, such as keto-enol tautomerism.

The present invention includes any tautomeric or stereoisomeric forms or mixtures thereof and is not limited to any tautomeric or stereoisomeric forms used in the nomenclature of compounds or in chemical structural formulas.

"Isotopes" include all isotopes of atoms occurring in the compounds of the present invention. Isotopes include atoms with the same atomic number but different masses. Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as, but not limited to, ² H (D), ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl. Isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the embodiments, using appropriate isotopically labeled reagents in place of non-isotopically labeled reagents. Such compounds have various potential uses, such as standards and reagents in the determination of biological activities. In the case of stable isotopes such as deuterium ² H (D), ¹³ C and ¹⁵ N, such compounds are characterized by the potential for a favorable change in biological, pharmacological or pharmacokinetic properties. Deuterium ² H (D) is a preferred isotope according to the present invention. For example, the hydrogen atoms in -CH ₃ can be replaced by D with -CD ₃ . For example, one or more hydrogen atoms of cyclopropyl can be replaced by D.

The compounds of the present invention can be administered in the form of prodrugs. "Prodrugs" refer to derivatives that are converted into biologically active compounds under physiological conditions in vivo, for example, by oxidation, reduction or hydrolysis (each of which may or may not involve enzymes). Examples of a prodrug are a compound of the present invention in which the amino group is acylated, alkylated or phosphorylated, such as eicosanylamino, alanylamino and pivaloyloxymethylamino; the hydroxyl is acylated, alkylated, phosphorylated or borane converted, such as acetoxy, palmitoyloxy, pivaloyloxy, succinyloxy, fumaroyloxy and alanyloxy; the carbonyl is esterified or amidated; and the thiol forms a disulfide bond with a carrier molecule that selectively delivers the drug to the target and/or to the cytosol of cells, such as a peptide. Prodrugs can be prepared from the compounds of the present invention according to well-known methods.

"Pharmaceutically acceptable salts" refer to salts prepared from the compounds of the present invention with pharmaceutically acceptable bases or acids, including inorganic alkalis or acids and organic bases or acids, provided that the compounds contain one or more acidic or basic groups. Therefore, the compounds of the present invention that contain acidic groups can exist in the form of salts, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts. For example, such salts include sodium salts, potassium salts, calcium salts, magnesium or ammonium salts, or organic amine salts such as ethylamine, ethanolamine, triethanolamine or amino acid salts. The compounds of the present invention that contain basic groups can exist in the form of salts of inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propanoic acid, pivalic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acids known to those skilled in the art. If the compounds of the present invention contain both an acidic and a basic group in the molecule, the present invention further includes internal salts in addition to the above-mentioned salt forms. Each salt can be prepared by conventional methods known to those skilled in the art, for example, by mixing a compound of the present invention with an organic or inorganic acid or base in a solvent or dispersing agent, or by anion exchange or cation exchange with another salt.

The term "pharmaceutical composition" refers to a composition comprising one or more compounds described herein, or pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers thereof, and other components such as pharmaceutically acceptable carriers and excipients.

Whenever the term "compounds" is mentioned in this application, it includes all forms of the compound, such as its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof.

"Autoimmune diseases or inflammatory diseases" include, but are not limited to, arthritis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis in pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis, Graves' disease, primary biliary cirrhosis, hepatitis, primary sclerosing cholangitis, chronic invasive hepatitis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, ulcerative colitis, membranous glomerulopathy, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, Sjogren's syndrome, Reiter's syndrome, polymyositis, dermatomyositis, interferon type I disease including Aicardi-Goutieres syndrome and others. systemic sclerosis with increased expression of type I interferon, Mendelian disease, polyarteritis nodosa, multiple sclerosis, relapsing multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, and bullous pemphigoid; additionally, autoimmune diseases associated with O-cells (body fluids) or T-cells, including Cogan's syndrome, ankylosing spondylitis, Wegener's granulomatosis, autoimmune alopecia, diabetes mellitus type I or juvenile diabetes mellitus, and thyroiditis.

As used herein, the term "enteritis" includes, but is not limited to, Crohn's disease, ulcerative colitis, inflammatory bowel disease, celiac disease, proctitis, eosinophilic gastroenteritis, and mastocytosis.

"Malignant neoplasms/tumors" include, but are not limited to, malignant neoplasm of the intestine/gastrointestinal tract, malignant neoplasm of the colorectum, malignant neoplasm of the liver, malignant neoplasm of the skin (including mast cell neoplasm and squamous cell carcinoma), malignant neoplasm of the breast, malignant neoplasm of the ovary, malignant neoplasm of the prostate, lymphoma, leukemia (including acute myeloid leukemia and chronic myelogenous leukemia), malignant neoplasm of the kidney, malignant neoplasm of the lung, malignant neoplasm of the muscle, malignant neoplasm of the bone, malignant neoplasm of the bladder, malignant neoplasm of the brain, melanoma (including intraoral and metastatic melanoma), Kaposi's sarcoma (myeloma, including multiple myeloma), myeloproliferative disorders, proliferative diabetic retinopathy, and vascular hyperplasia-related disorders/tumors.

"Skin disorders" include, but are not limited to, atopic dermatitis, eczema, psoriasis, scleroderma, pruritus and other pruritic symptoms, vitiligo, and hair loss.

"Diabetes" includes, but is not limited to, type 1 diabetes mellitus and complications of diabetes.

"Ocular disorders" include, but are not limited to, keratoconjunctivitis, uveitis (including uveitis associated with Behcet's disease and contact lens-associated uveitis), keratitis, herpetic keratitis, keratoconus, corneal epithelial dystrophy, corneal leukopenia, anterior uveitis, Mooren's ulcer, scleritis, Graves' disease, Vogt-Koyanagi-Harada syndrome, keratoconjunctivitis sicca (dry eye), wheals, iridocyclitis in sarcoidosis, endocrine ophthalmopathy, sympathetic ophthalmia, allergic conjunctivitis, and ocular neovascularization.

"Neurodegenerative diseases" include, but are not limited to, motor neuron disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, cerebral ischemia; neurodegenerative disease caused by trauma, injury, glutamate neurotoxicity, or hypoxia; ischemia/reperfusion injury in stroke, myocardial ischemia, renal ischemia, heart attack, myocardial hypertrophy, atherosclerosis and arteriosclerosis, organ hypoxia, or platelet aggregation.

"Anaphylaxis" includes, but is not limited to, mammalian allergic dermatitis (including equine anaphylactic diseases such as sting hypersensitivity), summer eczema, itchy horseshoes, spasm, inflammatory airway disease, recurrent airway obstruction, airway hyperreactivity, and chronic obstructive pulmonary disease.

"Asthma and other obstructive airways diseases" includes, but is not limited to, chronic or exacerbated asthma, delayed asthma, bronchitis, bronchial asthma, allergic asthma, endogenous asthma, exogenous asthma and dust asthma.

“Graft rejection” includes, but is not limited to, islet cell graft rejection, bone marrow graft rejection, graft-versus-host disease, organ or cell graft rejection (such as bone marrow, cartilage, cornea, heart, intervertebral disc, islet cells, kidney, limb, liver, lung, muscle, myoblast, nerve, pancreas, skin, small intestine, or trachea), and xenotransplantation.

"Therapeutically effective amount" refers to an amount of a compound of the present invention that can effectively inhibit the function of a JAK, in particular TYK2, and/or treat or prevent a disease mediated by this kinase.

"Patients" refers to mammals, preferably humans.

The present invention relates to compounds useful as inhibitors of JAK, in particular TYK2. The compounds are represented by formula (I) or their prodrugs, stable isotopic derivatives, pharmaceutically acceptable salts and isomers.

Where:

R ¹ is aryl or heteroaryl, wherein one or more hydrogen atoms of aryl and heteroaryl are optionally substituted with D, halogen, cyano, -OR ^b , -NR ^b R ^c , -COOR ^b , -C(O)R ^b , -NR ^b C(O)R ^c , -C(O)NR ^b R ^c , -S(O) ₂ R ^b , -S(O) ₂ NR ^b R ^c , -S(O)(NR ^b )R ^c , -P(O)(CH ₃ ) ₂ , C _1-6 alkyl, C _3-6 cycloalkyl, 3-8-membered heterocyclyl or 5-6-membered heteroaryl;

R ² is H, D, or -NHR ^a ;

R ³ is H, D, halogen, cyano, C _1-6 alkyl, C _3-6 cycloalkyl or OC _1-6 alkyl, wherein one or more hydrogen atoms of the alkyl and cycloalkyl are optionally substituted with D or F;

R ⁴ and R ⁵ are independently selected from H, D, halogen, C _1-6 alkyl or OC _1-6 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with D or F;

R ⁶ and R ⁷ are independently selected from H, D, cyano or C _1-6 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with D or F, or R ⁶ and R ⁷ are combined as oxo;

L represents a bond, C _1-6 alkylene, -C(O)-, -C(O)O-, -C(O)N(R ^a )-, -S(O) ₂ -, or -S(O) ₂ N(R ^a )-;

A is H, C _1-6 alkyl, C _3-6 cycloalkyl, 3-8-membered heterocyclyl, aryl or heteroaryl, wherein one or more hydrogen atoms of the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted with D, halogen, cyano, -OR ^d , -NR ^d R ^e , C _1-6 alkyl, C _3-6 cycloalkyl or 3-8-membered heterocyclyl;

R ^a is H, C _1-6 alkyl or C _3-6 cycloalkyl, wherein one or more hydrogen atoms of the alkyl and cycloalkyl are optionally substituted with D or F;

R ^b and R ^c are independently selected from H, C _1-6 alkyl, C _3-6 cycloalkyl or 3-8 membered heterocyclyl containing N and/or O, wherein one or more hydrogen atoms of the alkyl, cycloalkyl and heterocyclyl are optionally further substituted with D, halogen, CN, -OH, -NH ₂ , C _1-6 alkyl and -OC _1-6 alkyl, and

R ^d and R ^e are independently selected from H, C _1-6 alkyl, C _3-6 cycloalkyl or 3-8 membered heterocyclyl, wherein one or more hydrogen atoms of the alkyl, cycloalkyl and heterocyclyl are optionally further substituted with D or F.

According to one embodiment, R ¹ is phenyl.

According to one embodiment, R ¹ is a 5-6 membered heteroaryl, for example, R ¹ is pyridyl, pyrimidyl or pyrazolyl.

According to one embodiment, one or more hydrogen atoms of phenyl and heteroaryl R ¹ are optionally substituted with halogen, -COOR ^b , -C(O)R ^b , -C(O)NR ^b R ^c , C _1-6 alkyl, C _3-6 cycloalkyl or 4-6-membered heterocyclyl containing N and/or O, preferably C _1-6 alkyl, 4-6-membered heterocyclyl, -C(O)R ^b or -C(O)NR ^b R ^c , wherein one or more hydrogen atoms of alkyl, cycloalkyl and heterocyclyl are optionally further substituted with D, halogen, CN, -OH, -NH ₂ , C _1-6 alkyl and -OC _1-6 alkyl. preferably D, F, CN, -OH or C _1-6 alkyl.

According to one embodiment, R ^b and R ^c are independently selected from H, C _1-6 alkyl, C _3-6 cycloalkyl, or 4-6 membered heterocyclyl containing N and/or O, wherein one or more hydrogen atoms of the alkyl, cycloalkyl, and heterocyclyl are optionally further substituted with C _1-6 alkyl.

According to one preferred embodiment, R ² is H.

According to one preferred embodiment, R ³ is H, halogen, cyano, C _1-6 alkyl or OC _1-6 alkyl.

According to one preferred embodiment, R ⁴ and R ⁵ are independently H or C _1-6 alkyl.

According to one preferred embodiment, R ⁶ and R ⁷ are H, or R ⁶ and R ⁷ are combined as oxo.

According to one preferred embodiment, L is a bond, C _1-6 alkylene, -C(O)-, -C(O)O-, -C(O)NH- or -S(O) ₂ -.

According to one embodiment, A is C _1-6 alkyl or C _3-6 cycloalkyl, wherein one or more hydrogen atoms of the alkyl and cycloalkyl are optionally substituted with halogen, cyano, -OH, -OC _1-2 alkyl or C _1-2 alkyl.

According to one embodiment, A is pyridyl, pyrimidyl, 5-membered heteroaryl or 4-6-membered heterocyclyl, wherein one or more hydrogen atoms of the heterocyclyl and heteroaryl are optionally substituted with halogen, cyano, -OH, _-OC1-2alkyl or _C1-2alkyl , preferably _C1-2alkyl .

According to some embodiments, the compounds represented by formula (I) are characterized by the following formula (II):

Where:

R ¹ is phenyl, pyridyl or pyrazolyl, wherein one or more hydrogen atoms of phenyl, pyridyl and pyrazolyl are optionally substituted with halogen, C _1-6 alkyl, 4-6-membered heterocyclyl containing N and/or O (e.g. morpholine, piperazine, piperidine, pyrrolidine and oxetane), -C(O)R ^b or -C(O)NR ^b R ^c , and one or more hydrogen atoms of alkyl and heterocyclyl are optionally further substituted with D, F, CN, -OH or C _1-6 alkyl;

R ^b and R ^c are independently selected from H, C _1-6 alkyl, C _3-6 cycloalkyl, or 4-6 membered heterocyclyl containing N and/or O (e.g. morpholine, piperazine, piperidine, pyrrolidine, and oxetane), wherein one or more hydrogen atoms of the alkyl, cycloalkyl, and heterocyclyl are optionally further substituted with C _1-2 alkyl;

R ³ is H, halogen, cyano, C _1-6 alkyl or OC _1-6 alkyl;

R ⁴ and R ⁵ independently represent H or C _1-6 alkyl;

L is a bond, C _1-6 alkylene, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O) ₂ -, and

A is C _1-6 alkyl, C _3-6 cycloalkyl, 4-6-membered heterocyclyl containing N and/or O (e.g. morpholine, piperazine, piperidine, pyrrolidine and oxetane), pyridyl, pyrimidyl or 5-membered heteroaryl (e.g. isoxazole and pyrazole), wherein one or more hydrogen atoms of the alkyl and cycloalkyl are optionally substituted with halogen, cyano, -OH, -OC _1-2 alkyl or C _1-2 alkyl, and one or more hydrogen atoms of the heterocyclyl and heteroaryl are optionally substituted with C _1-2 alkyl.

According to one preferred embodiment, R ¹ is pyrazolyl.

According to one preferred embodiment, R ³ is H, halogen or C _1-6 alkyl.

According to one preferred embodiment, R ⁴ is H or methyl.

According to one preferred embodiment, R ⁵ is H or methyl.

According to one preferred embodiment, L is -C(O)-.

According to one preferred embodiment, A is C _1-6 alkyl or C _3-6 cycloalkyl, wherein one or more hydrogen atoms of the alkyl and cycloalkyl are optionally substituted with halogen, cyano, -OH or -OC _1-2 alkyl.

According to some embodiments, the compounds represented by formula (II) are characterized by the following formula (III):

Where:

R ³ is H, halogen, cyano, C _1-6 alkyl or OC _1-6 alkyl;

R ⁴ and R ⁵ independently represent H or C _1-6 alkyl;

L represents a bond, C _1-6 alkylene, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O) ₂ -;

A is C _1-6 alkyl, C _3-6 cycloalkyl, 4-6-membered heterocyclyl containing N and/or O (e.g. morpholine, piperazine, piperidine, pyrrolidine and oxetane), pyridyl, pyrimidyl or 5-membered heteroaryl (e.g. isoxazole and pyrazole), wherein one or more hydrogen atoms of the alkyl and cycloalkyl are optionally substituted with D, halogen, cyano, -OH, -OC _1-2 alkyl or C _1-2 alkyl, and one or more hydrogen atoms of the heterocyclyl and heteroaryl are optionally substituted with C _1-2 alkyl, and

R ¹¹ is H, C _1-6 alkyl or 4-6 membered heterocyclyl containing N and/or O (e.g. morpholine, piperazine, piperidine, pyrrolidine and oxetane), wherein one or more hydrogen atoms of the alkyl and heterocyclyl are optionally substituted with D, F, CN, -OH or C _1-6 alkyl.

Preferred A in the compound of formula III is C _1-6 alkyl or C _3-6 cycloalkyl, wherein one or more hydrogen atoms of the alkyl and cycloalkyl are optionally substituted with halogen, cyano, -OH or -OC _1-2 alkyl.

Preferred R ¹¹ is C _1-6 alkyl (eg methyl), wherein one or more hydrogen atoms of the alkyl are optionally substituted with D, F, CN, -OH or C _1-6 alkyl.

The present invention further relates to the following compounds 1-100 or their pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives, isomers and mixtures thereof.

Compounds 61, 62, 63, 64, 65, 68, 69, 70, 71, 72, 73, 74, 92, and 100 are racemates or a pair of diastereoisomers. Compounds 86, 87, 88, 89, 90, 91, 98, and 99 are single optical isomers. Compounds 86/87 and compounds 88/89 are a pair of enantiomers, respectively. Compounds 90 and 91 are a pair of diastereoisomers.

The compounds of the present invention effectively inhibit the activity of JAK, in particular TYK2, preferably having an IC ₅₀ value of 10 to 100 nM, and more preferably having an IC ₅₀ value of less than 10 nM. The compounds of the present invention have a significant effect of inhibiting IL-12-induced IFN-γ secretion in NK92 cells, preferably having an IC ₅₀ value of less than 1000 nM.

The present invention further relates to pharmaceutical compositions comprising compounds of formula (I) or pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives or isomers thereof and pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions are useful for the treatment or prevention of diseases mediated by JAK, in particular TYK2, including but not limited to autoimmune diseases, inflammatory diseases including intestinal diseases, malignant tumors, skin diseases, diabetes, eye diseases, neurodegenerative diseases, anaphylaxis, asthma, obstructive airway diseases and transplant rejection.

The present invention further relates to a method for treating or preventing diseases mediated by JAK, in particular TYK2. Said method comprises administering to a patient in need thereof a therapeutically effective amount of the compounds represented by formula (I) or pharmaceutically acceptable salts, prodrugs, stable isotope derivatives and isomers thereof. The diseases include, but are not limited to, autoimmune diseases, inflammatory diseases including intestinal diseases, malignant tumors, skin diseases, diabetes, eye diseases, neurodegenerative diseases, anaphylaxis, asthma and other obstructive airway diseases such as COPD, and transplant rejection. The compounds of the present invention are particularly useful for treating psoriasis, psoriatic arthritis, ulcerative colitis, Crohn's disease, SLE, lupus nephritis, vitiligo, focal alopecia, dermatitis, asthma, atopic eczema.

According to the present invention, the pharmaceutical agents may be in any dosage form, including, but not limited to, tablets, capsules, solution, lyophilized preparation, and injection preparation.

The dosage form according to the present invention can be administered as a unit dosage form containing a predetermined amount of the active ingredient. Such a unit dosage form can contain from 0.5 mg to 1 g, preferably from 1 mg to 700 mg, more preferably from 5 mg to 300 mg, of the compound according to the present invention, depending on the disease to be treated, the route of administration, and the age, weight and condition of the patients. In addition, the dosage form can be prepared using methods well known in the art of pharmacy, for example, by including the active ingredient in a formulation with one or more auxiliary substances or with one or more adjuvants.

The dosage form according to the present invention is suitable for administration by any suitable route, for example, by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) administration.

The present invention further relates to a method for preparing the compounds. The preparation of the compounds of the present invention represented by formula (I) can be carried out by the following illustrative methods and embodiments, but these methods and embodiments should in no way be considered as limiting the scope of the present invention. Alternatively, the compounds of the present invention can be synthesized by synthesis techniques known to those skilled in the art or by comprehensively using methods known in the art and the methods described in the present invention. The products obtained in each reaction step are isolated by separation techniques known in the art, including but not limited to separation by extraction, filtration, distillation, crystallization and chromatography. The starting materials and chemical reagents used for the synthesis can be conventionally obtained based on the literature (can be found in SciFinder) or purchased.

The heterocyclic compounds represented by formula (I) according to the present invention can be synthesized according to the route shown below: 1) a substitution reaction between starting materials A1 and A2 catalyzed by an organic base to give an intermediate compound A3; 2) an acid-catalyzed substitution reaction or a Buchwald-Hartwig coupling of compound A3 with a primary amine (R ¹ -NH ₂ ) to give a compound A4; 3) deprotection of compound A4 in acid (e.g., PG is Boc) or under hydrogenation conditions (e.g., PG is Bn) to give a compound A5; 4) derivatization of compound A5 to obtain target compounds, for example, by amidation with chlorine anhydride or anhydride, amide coupling with an acid, sulfonation with a sulfonyl chloride, formation of a urea with an amine, Buchwald-Hartwig coupling with a (hetero)aryl halide, etc.

Examples

The structure of the compounds was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). A Bruker ASCEND-400 NMR spectrometer was used for NMR recording, the recording solvent was deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ) or deuterated methanol (CD ₃ OD), the internal standard was tetramethylsilane (TMS), and the chemical shift was reported in parts per million (ppm).

An Agilent SQD (ESI) mass spectrometer (Agilent 6120) was used to register MS.

For HPLC registration, an Agilent 1260 DAD high-pressure liquid chromatograph (column: Poroshell 120 EC-C18, 50×3.0 mm, 2.7 μm) or a Waters Arc high-pressure liquid chromatograph (column: Sunfire C18, 150×4.6 mm, 5 μm) were used.

Silica gel plates GF254 manufactured by Qingdao Haiyang Chemical Co., Ltd. with a thickness of 0.15-0.2 mm were used for thin-layer chromatography (TLC), and silica gel plates with a thickness of 0.4-0.5 mm were used for separation/purification of products by thin-layer chromatography.

For column chromatography, silica gel 200-300 mesh produced by Qingdao Haiyang Chemical Co., Ltd. was generally used.

The known starting materials for the present invention were synthesized according to methods known in the art or purchased from ABCR GmbH&Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Beijing Ouhe Technology Co., Ltd., etc.

Unless otherwise stated in the embodiments, the reactions were carried out under an argon or nitrogen atmosphere using a cylinder with a volume of about 1 L.

Hydrogenation was carried out in a hydrogen atmosphere using a cylinder with a volume of about 1 liter, which was connected to the reaction vessel after three times evacuation and filling with hydrogen in a repeating mode.

A CEM Discover-SP microwave reactor was used to carry out the microwave-assisted reaction.

Unless otherwise stated in the embodiments, reactions were carried out at room temperature.

The reaction was monitored using an Agilent LCMS (1260/6120) or thin layer chromatography. Elution solvent systems for column chromatography and TLC included a) dichloromethane/methanol, b) petroleum ether/ethyl acetate, or other systems as indicated. The solvent ratio was adjusted according to the polarity of the compound and further adjusted by adding a small amount of TEA and an acidic or basic reagent if necessary. Purification of the compound was performed alternatively using a Waters MS-guided automated preparative system (abbreviated prep-HPLC) with an MS detector (SQD2) eluting at a flow rate of 20 ml/min in an appropriate gradient of acetonitrile/water (containing 0.1% TFA or formic acid) or acetonitrile/water (containing 0.05% ammonia) (XBridge-C18, 19×150 mm, 5 μm). After purification by prep-HPLC, some examples were obtained as hydrochloride salts by adding 1 N HCl to the collected fractions followed by drying under reduced pressure.

Example 1. Cyclopropyl((3aR,6aS)-3a,6a-dimethyl-5-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone

Step 1. 3,7-Dimethylene-1,5-ditosyl-1,5-diazocane (1b)

To a mixture of 4-methylbenzenesulfonamide (17.12 g, 100 mmol) and anhydrous potassium carbonate (27.6 g, 200 mmol) in MeCN (200 mL) was slowly added a solution of 3-chloro-2-chloromethyl-1-propylene 1a (12.5 g, 100 mmol) in MeCN (20 mL) over 10 min. Then, the mixture was heated to sublimation and stirred for 18 h. After cooling to room temperature, water (250 mL) was added to the mixture and stirred for 30 min. The resulting mixture was filtered, the precipitate was collected and purified by silica gel column chromatography (petroleum ether/dichloromethane = 100/0 - 0/100) to give the title compound 1b (10.8 g, 48%).

MS m/z (ESI): 447 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.70 - 7.64 (m, 4H), 7.31 (d, J=8.0 Hz, 4H), 5.19 (s, 4H), 3.82 (s, 8H), 2.43 (s, 6H).

Step 2. (3ar,6ar)-3a,6a-dimethyloctahydropyrrolo[3,4-c]pyrrole (1c)

To a solution of compound 1b (10.8 g, 24.2 mmol) in THF (400 mL) at 0 °C was added LAH (9.2 g, 242 mmol) portionwise. After stirring for 4 days, the mixture was cooled to 0 °C and 20% aqueous sodium chloride solution (18 mL) was added dropwise. The resulting mixture was gradually warmed to room temperature and stirred for 1 h. The mixture was filtered through a pad of Celite and the filter cake was washed with THF (3 x 200 mL). The combined filtrate was concentrated to dryness in vacuo to give the title compound 1c (2.25 g, 66%). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 141 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 2.90 (d, J=11.0 Hz, 4H), 2.69 (d, J=11.0 Hz, 4H), 2.39 (br. s, 2H), 0.98 (s, 6H).

Step 3. tert-Butyl (3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (1d)

To a solution of compound 1c (2.25 g, 16 mmol) in _CH2Cl2 ( ₁₀₀ mL) at 0 °C were added TEA (4.8 g, 48 mmol) and a solution of di-tert-butyl dicarbonate (3.5 g, 16 mmol) in _CH2Cl2 ( ₁₀₀ mL). The mixture was gradually warmed to room temperature and stirred for 1 h. The reaction mixture was quenched by adding water (100 mL) and extracted with _CH2Cl2 ( ₂ ×100 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The residue was purified by column chromatography on silica gel (dichloromethane/methanol = 100/1 - 10/1) to give the title compound 1d (1.37 g, 36%).

MS m/z (ESI): 241 [M+l]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 3.47 (s, 2H), 3.20 (d, J=10.2 Hz, 2H), 3.09 (d, J=11.4 Hz, 2H), 2.90 (d, J=11.4 Hz, 2H), 1.45 (s, 9H), 1.06 (s, 6H).

Step 4. tert-Butyl (3aR,6aS)-5-(2-chloropyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (1e)

To a solution of compound 1d (1.37 g, 5.7 mmol) in MeCN (50 mL) were added 2,4-dichloropyrimidine (850 mg, 5.7 mmol) and DIEA (2.21 g, 17.1 mmol). The mixture was heated to sublimation and stirred for 16 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and the residue was diluted with water (50 mL). Then, the mixture was extracted with ethyl acetate (3×100 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 5/1 - 1/1) to give the title compound 1e (1.47 g, 73%).

MS m/z (ESI): 353 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.03 (d, J = 6.0 Hz, 1H), 6.17 (d, J = 5.8 Hz, 1H), 3.77 (d, J = 12.3 Hz, 1H), 3.57 (t, J = 10.9 Hz, 1H), 3.53 - 3.39 (m, 3H), 3.35 (d, J = 11.4 Hz, 1H), 3.32 -3.24 (m, 2H), 1.45 (s, 9H), 1.15 (d, J = 6.5 Hz, 6H).

Step 5. 4-((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine hydrochloride (1f)

To a 30 mL microwave reaction vessel were added compound 1e (353 mg, 1 mmol), 1-methyl-1H-pyrazol-4-amine (102 mg, 1.05 mmol), pTsOH (4 mg, 0.02 mmol) and isopropanol (25 mL). The vessel was sealed and heated in the microwave at 100 °C for 1 h. After cooling to room temperature, HCl solution (33% in ethanol, 2 mL) was added to the mixture and then heated in the microwave at 80 °C for 30 min. The synthesis was repeated for three more batches following the same procedure as described above. The four batches were combined and filtered. The filter cake was collected to give the title compound 1f (1 g, 54%) as the hydrochloride salt.

MS m/z (ESI): 314 [M+1]

Step 6. Cyclopropyl((3aR,6aS)-3a,6a-dimethyl-5-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone (1)

To a solution of compound 1f (46 mg, 0.1 mmol) and TEA (50 mg, 0.5 mmol) in _CH2Cl2 (5 mL) at 0 °C was added _{cyclopropanecarbonyl} chloride (11 mg, 0.1 mmol). The mixture was stirred at 0 °C for 20 min, then quenched with water (20 mL) and extracted with _CH2Cl2 (2×20 mL). The combined organic phase was dried over _anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 1 (23 mg, solid, 61%).

MS m/z (ESI): 382 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.85 (d, J=6.3 Hz, 2H), 7.60 (s, 1H), 5.92 (d, J=6.0 Hz, 1H), 3.90 (d, J=10.0 Hz, 2H), 3.89 (s, 3H), 3.76 (d, J=10.8 Hz, 2H), 3.61 (d, J=12.4 Hz, 2H), 3.48 (d, 1=12.3 Hz, 2H), 1.84 - 1.78 (m, 1H), 1.26 (s, 3H), 1.23 (s, 3H), 0.96 - 0.82 (m, 4H).

Example 2. 4-((4-((3aR,6aS)-5-(cyclopropanecarbonyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)benzoic acid

Step 1. tert-Butyl (3aR,6aS)-5-(2-((4-(tert-butoxycarbonyl)phenyl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (2a)

Into a 10 mL microwave reaction vessel was added compound 1e (100 mg, 0.283 mmol), tert-butyl 4-aminobenzoate (55 mg, 0.283 mmol), pTsOH (5 mg, 0.0283 mmol) and isopropanol (4 mL). The vessel was stirred in the microwave reactor at 100 °C for 1 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2/1 - 1/2) to give the title compound 2a (102 mg, 71%).

MS m/z (ESI): 510 [M+1]

Step 2. 4-((4-((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)benzoic acid (2b)

To a solution of compound 2a (30 mg, 0.06 mmol) in ethanol (2 mL) was added HCl solution (33% in ethanol, 2 mL). The mixture was stirred at room temperature for 2 h and then concentrated to dryness in vacuo to give the title compound 2b (36 mg). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 354 [M+1]

Step 3. 4-((4-((3aR,6aS)-5-(cyclopropanecarbonyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)benzoic acid (2)

To a solution of compound 2b (36 mg, crude) and TEA (30 mg, 0.3 mmol) in _CH2Cl2 (5 mL) _was added cyclopropanecarbonyl chloride (6 mg, 0.06 mmol). The mixture was stirred for 10 min and then quenched by adding water (0.5 mL). The mixture was concentrated to dryness and the residue was dissolved in THF (2 mL). To the resulting mixture was added 20% aqueous sodium chloride solution (2 mL), and stirred at room temperature for 2 h. The mixture was adjusted to pH=3~4 with 1N HCl and extracted with ethyl acetate (3×20 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to dryness in vacuo and purified by prep-HPLC to give the title compound 2 (20 mg, solid, 79%).

MS m/z (ESI): 422 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.96 (dd, J=10.3, 7.5 Hz, 3H), 7.81 (d, J=8.7 Hz, 2H), 6 .07 (d, J=6.1 Hz, 1H), 3.92 (d, J=10.9 Hz, 2H), 3.76 (d, J=10.8 Hz, 2H), 3.62 (d, J=12.4 Hz, 2H), 3.49 (d, J=12.4 Hz, 2H), 1.84 - 1.77 (m, 1H), 1.27 (s, 3H) , 1.24 (s, 3H), 0.95 - 0.84 (m, 4H).

Example 3. 4-((4-((3aR,6aS)-5-(cyclopropanecarbonyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)-N-ethylbenzamide

To a solution of compound 2 (56 mg, 0.133 mmol) in _CH2Cl2 (10 mL) were added DIEA (59 mg, 0.153 mmol) and HATU (58 mg, 0.153 mmol). The mixture was stirred at room temperature for 5 min _, and then 3 drops of aqueous ethylamine solution (65%-70 wt%) were added. The mixture was stirred at room temperature for 30 min, concentrated and purified by prep-HPLC to give the title compound 3 (3.3 mg, solid, 6%).

MS m/z (ESI): 449 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.94 (d, J=6.1 Hz, 1H), 7.84 - 7.77 (m, 4H), 6.04 (d, J=6.1 Hz, 1H), 3.90 (d, J=11.1 Hz, 2H), 3.76 (d, J=10.9 Hz, 2H), 3.62 (d, 1=12.4 Hz, 2H), 3.48 (d, J=12.5 Hz, 2H), 3.43 (q, J=14.5, 7.3 Hz, 2H), 1.85 - 1.77 (m, 1H), 1.28 - 1.19 (m, 9H), 0.95 - 0.83 (m, 4H).

Example 4. 1-((3aR,6aS)-3a,6a-dimethyl-5-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-2,2,2-trifluoroethan-1-one hydrochloride

To a mixture of compound 1f (69 mg, 0.15 mmol) and DIEA (116 mg, 0.9 mmol) in _CH2Cl2 (2 mL) was added a solution of trifluoroacetic anhydride (32 mg, 0.15 mmol) in _CH2Cl2 ( ₂ mL). The mixture was stirred for 20 min, concentrated and purified by prep-HPLC to give _the title compound 4 (36.4 mg, solid, 54%) as a hydrochloride salt.

MS m/z (ESI): 410 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.89 (s, 1H), 7.74 (s, 1H), 7.65 (s, 1H), 6.31 (dd, J=7, 4, 5.0 Hz, 1H), 4.00 - 3.73 (m, 9H), 3.70 - 3.61 (m, 2H), 1.27 (s, 6H).

Example 6 was prepared according to the procedure described for Example 4, except that ethyl chloroformate was used instead of trifluoroacetic anhydride.

Example 5. (2,2-Difluorocyclopropyl)((3aR,6aS)-3a,6a-dimethyl-5-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone hydrochloride

To a mixture of compound 1f (69 mg, _0.15 mmol) in _CH2Cl2 (5 mL) were added DIEA (116 mg, 0.9 mmol), HATU (57 mg, 0.15 mmol) and 2,2-difluorocyclopropane-1-carboxylic acid (19 mg, 0.15 mmol). The mixture was stirred at room temperature for 20 min and then concentrated and purified by prep-HPLC to give the title compound 5 (32.8 mg, solid, 52%) as a hydrochloride salt.

MS m/z (ESI): 418 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.85 (t, J=4.0 Hz, 2H), 7.59 (s, 1H), 5.92 (d, J=6.1 Hz, 1H), 3.90 (d, J=10.9 Hz, 4H), 3.78 (q, J=11.0 Hz, 2H), 3.66 (d, J=11.9 Hz, 3H), 3.52 (d, J=12.5 Hz, 2H), 2.89 - 2.79 (m, 1H), 2.09 - 2.01 (m, 1H), 1.85 - 1.74 (m, 1H), 1.28 -1.19 (m, 6H).

Example 7. 3-((3aR,6aS)-3a,6a-dimethyl-5-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)propanenitrile

To a mixture of 1f (69 mg, 0.15 mmol) in MeCN (5 mL) was added 3-bromopropanenitrile (20 mg, 0.15 mmol) and DIEA (116 mg, 0.9 mmol). The mixture was stirred for 20 h and then heated to 90 °C for 24 h (LCMS showed only trace amounts of the desired product formed). The reaction mixture was transferred to a sealed 15 mL tube and heated to 120 °C for 72 h. After cooling to room temperature, the reaction mixture was concentrated to dryness in vacuo and purified by prep-HPLC to give the title compound 7 (3.2 mg, solid, 4%).

MS m/z (ESI): 367 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.81 (d, J=5.6 Hz, 2H), 7.61 (s, 1H), 5.92 (d, J=6.1 Hz, 1H), 3.88 (s, 3H), 3.77 (dd, J=19.3, 12.2 Hz, 2H), 3.48 - 3.39 (m, 2H), 2.97 (d, J=9.3 Hz, 2H), 2.79 (t, J=6.8 Hz, 2H), 2.61 (dd, J=12.8, 8.0 Hz, 4H), 1.20 (s, 6H).

Example 8. (3aR,6aS)-N-(cyanomethyl)-3a,6a-dimethyl-5-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxamide

To a solution of 2-aminoacetonitrile (14 mg, 0.24 mmol) in DMF (3 mL) was added N,N'-carbonyldiimidazole (49 mg, 0.3 mmol). The resulting mixture was heated to 65 °C and stirred for 2 h, and then compound 1f (69 mg, 0.15 mmol) was added. The mixture was stirred at 65 °C for an additional 2 h and concentrated in vacuo. The residue was dispersed in saturated aqueous sodium hydrogen carbonate solution (20 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by prep-HPLC to give the title compound 8 (19.2 mg, solid, 32%).

MS m/z (ESI): 396 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.81 (d, J=8.0 Hz, 2H), 7.57 (s, 1H), 5.88 (d, J=6.0 Hz, 1H), 4.09 (s, 2H), 3.85 (s, 3H), 3.76 (s, 1H), 3.59 (s, 1H), 3.51 (d, J=9.9 Hz, 3H), 3.37 (d, J=10.3 Hz, 3H), 1.19 (s, 6H).

Example 9. 4-((4-((3aR,6aS)-5-(2,2-difluorocyclopropane-1-carbonyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-fluoropyrimidin-2-yl)amino)-N-ethylbenzamide

Step 1. N-ethyl-4-nitrobenzamide (9b)

To a solution of 4-nitrobenzoic acid compound (5 g, 30 mmol) in _CH2Cl2 ( ₁₀₀ mL) was added dropwise oxalyl chloride (7.6 g, 60 mmol) followed by DME (0.1 mL). The mixture was stirred at 30 min and then concentrated to dryness in vacuo. The residue was dissolved in THF (50 mL) and added dropwise to an aqueous solution of ethylamine (60-70 wt %, 10 mL). The mixture was stirred at room temperature for 30 min, poured into water (200 mL) and extracted with ethyl acetate (2×100 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/1 - 0/100) to give the title compound 9b (5.13 g, 88%).

MS m/z (ESI): 195 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.33 - 8.23 (m, 2H), 7.98 -7.85 (m, 2H), 6.16 (s, 1H), 3.54 (sq.d, J=7.3, 5.7 Hz, 2H), 1.29 (t, J=7.3 Hz, 3H).

Step 2. 4-amino-N-ethylbenzamide (9c)

To a mixture of water (40 mL), acetic acid (4 mL), ammonium chloride (14.13 g, 264.2 mmol), ethanol (100 mL), and compound 9b (5.13 g, 26.42 mmol) was added zinc powder (8.64 g, 132 mmol) portionwise. The mixture was stirred for 1 h, and zinc powder (8.64 g, 132 mmol) was added portionwise again. The resulting mixture was stirred at 60 °C for 1 h. After cooling to room temperature, the mixture was filtered through a pad of Celite. The filtrate was concentrated to dryness in vacuo, the residue was dispersed in saturated aqueous sodium hydrogen carbonate solution (50 mL) and extracted with ethyl acetate (4×50 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/1 - 0/100) to give the title compound 9c (3.25 g, 75%).

MS m/z (ESI): 165 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.94 (t, J = 5.2 Hz, 1H), 7.55 (dd, J = 9.0, 2.2 Hz, 2H), 6.52 (dd, J = 9.0, 2.2 Hz, 2H), 5.54 (s, 2H), 3.26 - 3.16 (m, 2H), 1.08 (t, J = 7.2 Hz, 3H).

Step 3. tert-Butyl (3aR,6aS)-5-(2-chloro-5-fluoropyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (9e)

To a solution of compound 9d (900 mg, 4.24 mmol) in MeCN (50 mL) were added 2,4-dichloro-5-fluoropyrimidine (708 mg, 4.24 mmol) and DIEA (1.64 g, 12.72 mmol). The mixture was stirred for 2 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/1) to give the title compound 9e (1.13 g, 78%).

MS m/z (ESI): 343 [M+1]

Step 4. tert-Butyl (3aR,6aS)-5-(2-((4-(ethylcarbamoyl)phenyl)amino)-5-fluoropyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (9f)

To a 10 mL microwave reactor containing compound 9e (68 mg, 0.2 mmol), 4-amino-N-ethylbenzamide (33 mg, 0.2 mmol), KOtBu (48 mg, 0.5 mmol), and 1,4-dioxane (3 mL) under nitrogen atmosphere was added RuPhos-Pd-G2 (7 mg, 0.01 mmol). The reactor was then heated in a microwave reactor at 120 °C for 1 h. After cooling to room temperature, the reaction mixture was poured into water (20 mL) and extracted with ethyl acetate (3×20 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography (dichloromethane/methanol = 10/1 - 5/1) to give the title product 9f (56 mg, 63%).

MS m/z (ESI): 471 [M+1]

Step 5. N-ethyl-4-((5-fluoro-4-((3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)benzamide hydrochloride (9g)

To a solution of compound 9f (56 mg, 0.112 mmol) in ethanol (2 mL) was added HCl solution (33% in ethanol, 2 mL). The mixture was stirred for 14 h and then concentrated to dryness in vacuo to give the title compound 9g (57 mg). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 371 [M+1]

Step 6. 4-((4-((3aR,6aS)-5-(2,2-difluorocyclopropane-1-carboxyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-fluoropyrimidin-2-yl)amino)-N-ethylbenzamide (9)

To a mixture of compound 9g (57 mg, _0.112 mmol) in _CH2Cl2 (5 mL) were added DIEA (91 mg, 0.696 mmol), 2,2-difluorocyclopropane-1-carboxylic acid (15 mg, 0.12 mmol) and HATU (46 mg, 0.12 mmol). The mixture was stirred for 1 h and concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 9 (21 mg, solid, 40%).

MS m/z (ESI): 475 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.84 (d, J=5.8 Hz, 1H), 7.77 - 7.70 (m, 4H), 4.09 - 3.90 ( m, 3H), 3.82 - 3.56 (m, 4H), 3.52 - 3.43 (m, 1H), 3.40 (kV, J=7.2 Hz, 2H), 3.22 - 3.05 (m, 2H), 2.88 -2.77 (m, 1H), 2.08 - 1.97 (m, 1H), 1.83 - 1.70 (m, 1H), 1 .22 (t, J=7.2 Hz, 3H).

Example 10 was prepared according to the procedure described for Example 9, except that compound 1d was used instead of compound 9d.

Example 11. Cyclopropyl((3aR,6aS)-5-(5-fluoro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone

Step 1. tert-Butyl (3aR,6aS)-5-(2-chloro-5-fluoropyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (11a)

To a solution of 2,4-dichloro-5-fluoropyrimidine (680 mg, 4 mmol) and compound 1d (1.96 g, crude product, purity ca. 50%) in MeCN (40 mL) was added TEA (1.55 g, 12 mmol). The mixture was heated to 90 °C and stirred for 18 h. After cooling to room temperature, the mixture was added saturated aqueous ammonium chloride solution (100 mL) and extracted with ethyl acetate (3×50 mL). The combined organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 7/3) to give the title compound 11a (245 mg, 17%).

MS m/z (ESI): 371 [M+l]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.91 (d, J=4.9 Hz, 1H), 3.83 (s, 2H), 3.67 (s, 2H), 3.46 ( s, 2H), 3.33 (s, 2H), 1.46 (s, 9H), 1.15 (s, 6H).

Step 2. tert-Butyl (3aR,6aS)-5-(5-fluoro-2-((1-methyl-1H-pyrazol-4-yl)amino)-pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (11b)

In a 30 mL microwave reactor, compound 11a (245 mg, 0.66 mmol), 1-methyl-1H-pyrazol-4-amine (64 mg, 0.66 mmol), NaOtBu (159 mg, 1.65 mmol), 1,4-dioxane (8 mL) and RuPhos-Pd-G2 (24 mg, 0.033 mmol) were added, and the reaction mixture was heated to 120 °C in the microwave reactor for 1 h under nitrogen atmosphere. After cooling to room temperature, the reaction mixture was filtered through a pad of Celite. The filtrate was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (dichloromethane/ethyl acetate = 100/0 - 0/100) to give the title compound 11b (147 mg, 52%).

MS m/z (ESI): 432 [M+1]

Step 3. 4-((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-fluoro-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine hydrochloride (11c)

To a solution of compound 11b (147 mg, _0.34 mmol) in _CH2Cl2 (10 mL) was added TFA (2 mL). The mixture was stirred for 18 h and then concentrated to dryness in vacuo. The residue was dissolved in _CH2Cl2 ( ₁₀ mL) and a solution of HCl in ethanol (9 M, 1 mL) was added. The resulting mixture was concentrated to dryness to give the title compound 11c as a hydrochloride salt (160 mg, 98%).

MS m/z (ESI): 332 [M+1]

Step 4. Cyclopropyl((3aR,6aS)-5-(5-fluoro-2-((1-methyl-1H-pyrazol-4-yl)amino)-pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone (11)

To a mixture of compound 11c (160 mg, 0.34 mmol) and TEA (243 mg, 2.4 mol) in _CH2Cl2 (10 mL) at 0 °C was added _{cyclopropanecarbonyl} chloride (42 mg, 0.4 mmol). The mixture was stirred at 0 °C for 30 min, diluted _{with CH2Cl2} (20 mL) and washed with water (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The residue was purified by prep-HPLC to give the title compound 11 (12.5 mg, solid, 9%).

MS m/z (ESI): 400 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.76 (d, J=5.7 Hz, 1H), 7.61 (s, 1H), 7.53 (s, 1H), 6.80 ( s, 1H), 3.87 (s, 3H), 3.81 (dd, J=20.0, 10.7 Hz, 3H), 3.64 (t, J=11.8 Hz, 4H), 3.48 (d, J=12.4 Hz, 1H), 1.60 - 1.52 (m, 1H), 1.20 (s, 3H), 1.16 (s, 3H), 1.05 - 0.96 (m, 2H), 0.83 - 0.73 (m, 2H).

Example 13 was synthesized according to the procedure described for Example 11, except that 2-(4-amino-1H-pyrazol-1-yl)ethan-1-ol was used in place of 1-methyl-1H-pyrazol-4-amine in step 3.

Example 15 and Example 24 were synthesized according to the procedure described for Example 11, except that in Step 1, different compounds were used instead of 2,4-dichloro-5-fluoropyrimidine.

Example 12. ((3aR,6aS)-5-(5-chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone (12)

Step 1. tert-Butyl (3aR,6aS)-5-(2,5-dicloropyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (12a)

To a solution of 2,4,5-trichloropyrimidine (183 mg, 1 mmol) and compound 1d (770 mg, crude product, ca. 1 mmol) in MeCN (40 mL) was added DIEA (388 mg, 3 mmol). The mixture was heated to 90 °C and stirred for 24 h. After cooling to room temperature, the reaction mixture was added saturated aqueous ammonium chloride solution (50 mL), and extracted with ethyl acetate (3×20 mL). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 7/3) to give the title compound 12a (90 mg, 23%).

MS m/z (ESI): 387 [M+1]

Step 2. tert-Butyl (3aR,6aS)-5-(5-chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)-pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (12b)

Into a 30 mL microwave reactor were added compound 12a (90 mg, 0.23 mmol), 1-methyl-1H-pyrazol-4-amine (23 mg, 0.23 mmol), 1,4-dioxane (5 mL), NaOtBu (49 mg, 0.506 mmol) and RuPhos-Pd-G2 (9 mg, 0.0115 mmol). The reaction mixture was heated to 110 °C in the microwave reactor for 1 h under nitrogen atmosphere. After cooling to room temperature, the mixture was filtered through a pad of Celite. The filtrate was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (dichloromethane/ethyl acetate = 100/0 - 0/100) to give the title compound 12b (61 mg, 59%).

MS m/z (ESI): 448 [M+1]

Step 3. 5-Chloro-4-((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine hydrochloride (12c)

To a solution of compound 12b (61 mg, _0.136 mmol) in _CH2Cl2 (5 mL) was added TFA (2 mL). The mixture was stirred for 20 h and then concentrated to dryness in vacuo. The residue was dissolved in a mixture of methanol (2 mL) and water (10 mL) and a solution of HCl (9 M in ethanol, 0.2 mL) was added. The mixture was again concentrated to dryness to give the title compound 12c (67 mg, 100%) as a hydrochloride salt.

MS m/z (ESI): 348 [M+1]

Step 4. ((3aR,6aS)-5-(5-chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone (12)

To a mixture of compound 12c (67 mg, 0.136 mmol) and TEA (55 mg, 0.544 mol) in _CH2Cl2 (20 mL) at 0 °C was added _{cyclopropanecarbonyl} chloride (14 mg, 0.136 mmol). The mixture was stirred at 0 °C for 30 min and then washed with saturated ammonium chloride solution (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 12 (1.9 mg, solid, 3%).

MS m/z (ESI): 416 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.83 (s, 1H), 7.75 (s, 1H), 7.53 (s, 1H), 3.97 (dd, J=15, 1, 11.8 Hz, 2H), 3.90 - 3.78 (m, 6H), 3.71 (d, J=10.8 Hz, 1H), 3.59 (d, J=12.4 Hz, 1H), 3.42 (d, J=12.4 Hz, 1H), 1.78 (dd, J=8.7, 3.9 Hz, 1H), 1.21 (s, 3H), 1.18 (s, 3H), 0.92 - 0.87 (m, 2H), 0.86 - 0.82 (m, 2H).

Examples 19, 34, 37, 41 and 96 were synthesized according to the procedure described for Example 12, except that in Step 4, different compounds were used in place of cyclopropanecarbonyl chloride.

Examples 23, 30 and 32 were synthesized according to the procedure described for Example 12, except that in Step 2, different compounds were used instead of 1-methyl-1H-pyrazol-4-amine.

Example 14. 4-((3aR,6aS)-5-(2,2-difluoroethyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-fluoro-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine

To a solution of compound 11c (135 mg, 0.255 mmol) in DMF (2 mL) were added DIEA (132 mg, 1.02 mmol) and 1,1-difluoro-2-iodoethane (49 mg, 0.255 mmol), and the mixture was stirred at 70 °C for 24 h. After cooling to room temperature, the mixture was transferred to a 15 mL sealed tube, and then DIEA (132 mg, 1.02 mmol) and 1,1-difluoro-2-iodoethane (98 mg, 0.51 mmol) were added. The mixture was stirred in a sealed tube at 80 °C for 72 h. After cooling to room temperature, the mixture was purified by prep-HPLC to give the title compound 14 (16.9 mg, solid, 17%).

MS m/z (ESI): 396 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.77 (s, 1H), 7.74 (d, J=6.2 Hz, 1H), 7.55 (s, 1H), 5.89 (tt, J=56.1, 4.3 Hz, 1H), 3.96 (dd, J=11.7, 1.9 Hz, 2H), 3.87 (s, 3H), 3.58 (dd, J=11.6, 1.2 Hz, 2H), 2.99 (d, J=9.5 Hz, 2H), 2.89 (td, J=15.3, 4.3 Hz, 2H), 2.66 (d, J=9.5 Hz, 2H), 1.17 (s, 6H).

Example 16. ((3aR,6aS)-5-(5-Chloro-2-((1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone

Step 1. 1-Methyl-4-(4-nitro-1H-pyrazol-1-yl)piperidine (16b)

To a mixture of 4-nitro-1H-pyrazole 16a (1.13 g, 10 mmol), 1-methylpiperidin-4-ol (1.15 g, 10 mmol), and triphenylphosphine (3.15 g, 12 mmol) in THF (25 mL) at 0 °C under nitrogen atmosphere was added dropwise diisopropyl azodicarboxylate (2.63 g, 13 mmol). After stirring for 4 h, the mixture was diluted with water (100 mL) and adjusted to pH 1 with 6 N HCl. The resulting mixture was washed with ethyl acetate (2×50 mL). Solid LiOH was added to the aqueous layer to adjust pH to 10, and then extracted with ethyl acetate (3×50 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/0 - 94/6) to give the title compound 16b (800 mg, 38%).

MS m/z (ESI): 211 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.17 (s, 1H), 8.08 (s, 1H), 4.19 - 4.11 (m, 1H), 3.04 - 2.96 (m, 2H), 2.35 (s, 3H), 2.24 - 2.12 (m, 4H), 2.10 - 2.00 (m, 2H).

Step 2. 1-(1-Methylpiperidin-4-yl)-1H-pyrazol-4-amine (16c)

To a solution of compound 16b (800 mg, 3.8 mmol) in MeOH (10 mL) was added 10% Pd/C (400 mg). The mixture was stirred under hydrogen atmosphere for 2 h and then filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 16c (688 mg, 100%). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 181 [M+1]

Step 3. 5-Chloro-4-((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)pyrimidin-2-amine (16d)

To a 10 mL microwave reactor were added compound 12a (39 mg, 0.1 mmol), compound 16c (18 mg, 0.1 mmol), pTsOH (38 mg, 0.2 mmol), and isopropanol (2 mL). The mixture was stirred in the microwave reactor at 100 °C for 1 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo to give the title compound 16d (60 mg). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 431 [M+1]

Step 4. ((3aR,6aS)-5-(5-chloro-2-((1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone (16)

To a solution of compound 16d (60 mg) and TEA (51 _mg , 0.5 mmol) in _CH2Cl2 (5 mL) was added a solution of cyclopropanecarbonyl chloride (11 mg, 0.1 mmol) in _CH2Cl2 ( ₁ mL). The mixture was stirred for 30 min and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 16 (22.3 mg, solid, 45% over two steps).

MS m/z (ESI): 499 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.92 (s, 1H),7.85 (s, 1H),7.56 (s, 1H), 4.19-4.11 (m, 1H), 4.19-4.11 (dd, J=18.7, 11.6 Hz, 2H), 3.89 - 3.83 (m, 3H), 3.73 (d, J=10.8 Hz, 1H), 3.62 (d, J=12.4 Hz, 1H), 3.44 (d, J=12.4 Hz, 1H), 3.03 (d, J=12.5 Hz, 2H), 2.37 (s, 3H), 2.29 (t, J=11.7 Hz, 2H), 2.16 (d, J=11.6 Hz, 2H), 2.07 (dd, J=16.7, 8.0 Hz, 2H), 1.84 - 1.76 (m, 1H), 1.24 (s, 3H), 1.21 (s, 3H), 0.94 - 0.89 (m, 2H), 0.88 - 0.82 (m, 2H).

Example 17. ((3aR,6aS)-5-(5-Chloro-2-((1-((R)-2-hydroxypropyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone

Step 1. (R)-1-(4-nitro-1H-pyrazol-1-yl)propan-2-ol (17a)

To a solution of 4-nitro-1H-pyrazole 16a (2.26 g, 20 mmol) and (R)-2-methyloxirane (3.48 g, 60 mmol) in DMF (15 mL) was added cesium carbonate (13 g, 40 mmol). The mixture was stirred in a sealed tube at 100 °C for 4 h. After cooling to room temperature, the mixture was poured into water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic phase was washed with water (3 x 100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 7/3) and then purified by prep-HPLC to give the title compound 17a (1.2 g, 35%).

MS m/z (ESI): 172 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.77 (s, 1H), 8.26 (s, 1H), 5.03 (d, J=4.8 Hz, 1H), 4.17 - 4.09 (m, 1H), 4.06 - 3.97 (m, 2H), 1.08 (d, J=6.0 Hz, 3H).

Step 2. (R)-1-(4-amino-1H-pyrazol-1-yl)propan-2-ol (17b)

To a solution of compound 17a (1.2 g, 7 mmol) in MeOH (20 mL) was added 10% Pd/C (120 mg). The mixture was stirred under hydrogen atmosphere for 16 h and then filtered. The filtrate was concentrated to dryness, and the residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/0 - 19/1) to give the title compound 17b (848 mg, 86%).

MS m/z (ESI): 142 [M+1]

Step 3. (R)-1-(4-((5-chloro-4-((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propan-2-ol (17c)

To a 10 mL microwave reactor were added compound 12a (39 mg, 0.1 mmol), compound 17b (15 mg, 0.1 mmol), pTsOH (38 mg, 0.2 mmol), and isopropanol (2 mL). The reactor was stirred in the microwave at 100 °C for 1 h. After cooling to room temperature, the mixture was concentrated to dryness to give the title compound 17c (60 mg). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 392 [M+1]

Step 4. ((3aR,6aS)-5-(5-chloro-2-((1-((R)-2-hydroxypropyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone (17)

To a solution of compound 17c (60 mg, crude) and TEA (51 _mg , 0.5 mmol) in _CH2Cl2 (5 mL) was added a solution of cyclopropanecarbonyl chloride (11 mg, 0.1 mmol) in _CH2Cl2 (1 mL). The mixture was stirred for 30 min and then concentrated _to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 17 (20.1 mg, solid, 44% over two steps).

MS m/z (ESI): 460 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.92 (s, 1H), 7.84 (s, 1H), 7.54 (s, 1H), 4.13 - 3.97 (m, 5H), 3.91 - 3.83 (m, 3H), 3.72 (d, J=10.8 Hz, 1H), 3.61 (dd, J=12.4, 3.7 Hz, 1H), 3.44 (dd, J=12.4, 1.7 Hz, 1H), 1.83 - 1.77 (m, 1H), 1.23 (s, 3H), 1.20 (s, 3H), 1.17 (d, J=6.1 Hz, 3H), 0.94 - 0.89 (m, 2H), 0.88 - 0.82 (m, 2H).

Example 18 was synthesized according to the procedure described for Example 12, except that (S)-2-methyloxirane was used in place of (R)-2-methyloxirane in step 1.

Example 20. 3-((3aR,6aS)-5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

A mixture of compound 12c (35 mg, 0.1 mmol), 2-cyanoacetic acid (9 mg, 0.1 mmol), DIEA (39 mg, 0.3 mmol) and HATU (46 mg, 0.12 mmol) in _CH2Cl2 (5 mL) was stirred for 30 min. The mixture was quenched with water (20 mL) and extracted _{with CH2Cl2} (2×100 mL). The combined organic phase was dried over _anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 20 (10.5 mg, solid, 25%).

MS m/z (ESI): 415 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.84 (s, 1H), 7.77 (s, 1H), 7.55 (s, 1H), 3.99 (t, J=10.8 Hz, 2H), 3.86 (s, 3H), 3.84 (d, J=11.8 Hz, 2H), 3.66 (dd, J=18.8, 11.6 Hz, 2H), 3.50 (dd, J=19.0, 11.6 Hz, 2H), 1.21 (s, 3H), 1.20 (s, 3H).

Examples 21, 22, 35, 36, 39, 42, 43, 44, 45, 46, 47, 48 and 52 were synthesized according to the procedure described for Example 20, except that in Step 1, different compounds were used instead of 2-cyanoacetic acid.

Example 25. 5-Chloro-4-((3aR,6aS)-3a,6a-dimethyl-5-(pyrimidin-4-yl)-hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine

A mixture of compound 12c (100 mg, 0.26 mmol), 4-chloropyrimidine hydrochloride (59 mg, 0.39 mmol) and DIEA (336 mg, 2.6 mmol) in MeCN (4 mL) was heated to 80 °C and stirred for 8 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo and the residue was purified by prep-HPLC to give the title compound 25 (65.1 mg, solid, 58%).

MS m/z (ESI): 426 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.06 (s, 1H), 8.46 (s, 1H), 8.14 (d, J=6.1, 1H), 7.89 (s, 1H), 7.69 (s, 1H), 7.43 (s, 1H), 6.46 (dd, J=6.1, 1.0, 1H), 3.88 (s, 2H), 3.76 (s, 3H), 3.74 (s, 2H), 3.67 (s, 2H), 3.52 (s, 2H), 1.15 (s, 6H).

Example 26. 3-((3aR,6aS)-5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

Step 1. tert-Butyl (3aR,6aS)-5-(2,5-dichloropyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (26b)

To a solution of 2,4,5-trichloropyrimidine (182 mg, 1 mmol) in MeCN (10 ml) were added compound 26a (212 mg, 1 mmol) and potassium carbonate (207 mg, 1.5 mmol). The mixture was stirred at 80°C for 3 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 3/1) to give the title compound 26b (300 mg, 84%).

MS m/z (ESI): 359 [M+1]

Step 2. 5-Chloro-4-((3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine(26c)

To a mixture of compound 26b (300 mg, 0.84 mmol) and 1-methyl-1H-pyrazol-4-amine hydrochloride (81.3 mg, 0.84 mmol) in isopropanol (50 mL) was added pTsOH (318 mg, 1.68 mmol). The mixture was heated to 100 °C in a microwave reactor for 2 h. After cooling to room temperature, the precipitate was collected by filtration to give the title compound 26c (200 mg, 75%).

MS m/z (ESI): 320 [M+1]

Step 3. 3-((3aR,6aS)-5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile (26)

To a mixture of compound 26c (100 mg, 0.313 mmol), 2-cyanoacetic acid (26.6 mg, 0.313 mmol) and TEA (47.5 mg, 0.47 mmol) in DMF (5 mL) was added HATU (178.6 mg, 0.47 mmol). The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 26 (61.9 mg, solid, 50%).

MS m/z (ESI): 387 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.85 (s, 1H), 7.78 (s, 1H), 7.54 (s, 1H), 4.19 - 4.11 (m, 2H), 3.93 - 3.72 (m, 9H), 3.52 - 3.45 (m, 2H), 3.18 - 3.12 (m, 1H), 3.09 - 3.03 ( m, 1H).

Example 27 was synthesized according to the procedure described for Example 26, except that (S)-2,2-difluorocyclopropane-1-carboxylic acid was used in place of 2-cyanoacetic acid in step 3.

Example 28. 3-((3aR,6aS)-5-(2-((1H-pyrazol-4-yl)amino)-5-chloropyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

Step 1. 5-chloro-4-((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1H-pyrazol-4-yl)pyrimidine -2-amine (28a)

To a 30 mL microwave reactor were added compound 12a (161 mg, 0.42 mmol), N-pyrazol-4-amine (35 mg, 0.42 mmol), pTsOH (160 mg, 0.84 mmol), and isopropanol (10 mL). The mixture was heated to 100 °C in the microwave reactor for 1 h. The mixture was cooled to room temperature and concentrated to dryness in vacuo to give the title compound 28a (260 mg). The crude product was used directly in the next step without purification.

MS m/z (ESI): 334 [M+1]

Step 2. 3-((3aR,6aS)-5-(2-((1H-Pyrazol-4-yl)amino)-5-chloropyrimidine-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile (28)

To a mixture of compound 28a (130 mg, crude material, 0.21 mmol), 2-cyanoacetic acid (18 mg, 0.21 mmol) and DIEA (82 mg, 0.63 mmol) in DMF (5 mL) was added HATU (80 mg, 0.21 mmol). The mixture was stirred for 1 h and then purified by prep-HPLC to give the title compound 28 (7.6 mg, solid, 9%).

MS m/z (ESI): 401 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.85 (s, 2H), 7.65 (s, 1H), 4.02 (d, J=11.8 Hz, 1H), 3.98 (d, J=11.8 Hz, 1H), 3.85 (dd, J=11.6, 2.8 Hz, 2H), 3.66 (t, J=12.1 Hz, 2H), 3.53 (d, J=10.7 Hz, 1H), 3.47 (d, J=12.5 Hz, 1H), 1.21 (s, 3H), 1.20 (s, 3H).

Example 29 was synthesized according to the procedure described for Example 28, except that (S)-2,2-difluorocyclopropane-1-carboxylic acid was used in place of 2-cyanoacetic acid in step 2.

Example 31. 3-((3aR,6aS)-5-(5-Chloro-2-((1-(2-hydroxyethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

Step 1. 2-(4-((5-Chloro-4-((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl) amino)-1H-pyrazol-1-yl)ethan-1-ol (31a)

In a 30 mL microwave reactor were added compound 12a (330 mg, 0.852 mmol), 2-(4-amino-1H-pyrazol-1-yl)ethan-1-ol (108 mg, 0.852 mmol), pTsOH (324 mg, 1.704 mmol) and isopropanol (10 mL). The mixture was heated to 100 °C in the microwave reactor for 1 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo and the residue was purified by prep-HPLC to give the title compound 31a (210 mg, 65%).

MS m/z (ESI): 378 [M+1]

Step 2. 3-((3aR,6aS)-5-(5-Chloro-2-((1-(2-hydroxyethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile (31)

To a mixture of compound 31a (100 mg, 0.26 mmol), 2-cyanoacetic acid (23 mg, 0.26 mmol) and _DIEA (101 mg, 0.87 mmol) in _CH2Cl2 (10 mL) was added HATU (101 mg, 0.26 mmol). The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 31 (18.6 mg, solid, 16%).

MS m/z (ESI): 445 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.96 (s, 1H), 7.84 (s, 1H), 7.53 (s, 1H), 4.20 (t, J=5.2 Hz, 2H), 4.02 (d, J=5.8 Hz, 1H), 3.99 (d, J=5.7 Hz, 1H), 3.92 - 3.81 (m, 4H), 3.68 (d, J=10.7 Hz, 1H), 3.63 (d, J=12.5 Hz, 1H), 3.52 (d, J=10.7 Hz, 1H), 3.48 (d, J=12.5 Hz, 1H), 1.21 (s, 3H), 1.20 (s, 3H).

Example 33. 2-(4-((5-Chloro-4-((3aR,6aS)-5-(cyclopropanecarbonyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)acetonitrile

Step 1. 2-(4-Nitro-1H-pyrazol-1-yl)acetonitrile (33a)

To a solution of 4-nitro-1H-pyrazole 16a (1.13 g, 10 mmol) in DMF (12 mL) was added cesium carbonate (9.75 g, 30 mmol). After cooling to 0 °C, 2-bromoacetonitrile (2.4 g, 20 mmol) was added dropwise to the mixture. The mixture was stirred for 1 h, diluted with water (150 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic phase was washed with water (3 x 100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 1/1) to give the title compound 33a (960 mg, 63%).

MS m/z (ESI): 153 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.36 (s, 1H), 8.16 (s, 1H), 5.15 (s, 2H).

Step 2. 2-(4-Amino-1H-pyrazol-1-yl)acetonitrile (33b)

A mixture of compound 33a (940 mg, 6.18 mmol), ammonium chloride (6.6 g, 123.6 mmol), ethanol (40 mL), and water (10 mL) was heated to 60 °C, and zinc powder (4 g, 61.8 mmol) was added portionwise. The mixture was stirred at 60 °C for 10 min, cooled to room temperature, and filtered. The filtrate was diluted with water (80 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/0 - 19/1) to give the title compound 33b (130 mg, 17%).

MS m/z (ESI): 123 [M+1]

Step 3. 2-(4-((5-Chloro-4-((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)acetonitrile (33c)

To a 10 mL microwave flask were added compound 12a (155 mg, 0.4 mmol), compound 33b (49 mg, 0.4 mmol), pTsOH (16 mg, 0.08 mmol), and isopropanol (4 mL). The mixture was stirred in the microwave at 100 °C for 1 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo to give the title compound 33c. The crude product was used directly in the next step without purification.

MS m/z (ESI): 373 [M+1]

Step 4. 2-(4-((5-Chloro-4-((3aR,6aS)-5-(cyclopropanecarbonyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)acetonitrile (33)

To a mixture of compound 33c (crude material, 0.4 mmol) in _CH2Cl2 ( ₂₀ mL) were added TEA (121 mg, 1.2 mmol) and a solution of cyclopropanecarbonyl chloride (42 mg, 0.4 mmol) in _CH2Cl2 (1 mL). _The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 233 (81 mg, solid, 46% over two steps).

MS m/z (ESI): 441 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 8.04 (s, 1H), 7.87 (s, 1H), 7.63 (s, 1H), 5.29 (s, 2H), 4 .06 - 3.96 (m, 2H), 3.95 - 3.82 (m, 3H), 3.72 (d, J=10.8 Hz, 1H), 3.62 (d, J=12 .4 Hz, 1H), 3.45 (d, J=12.4 Hz, 1H), 1.83 - 1.77 (m, 1H), 1.24 (s, 3H), 1.20 (s , 3H), 0.94 - 0.88 (m, 2H), 0.88 - 0.82 (m, 2H).

Example 38. Azetidin-3-yl((3aR,6aS)-5-(5-chloro-2-((1-methyl-1H-pyrazol-4-yl)-amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone

Step 1. tert-Butyl 3-((3aR,6aS)-5-(5-chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)-pyrimidin-4-yl)-3a,6a-dimethyloctahydropyrrolo[3,4-c]pyrrole-2-carb6onyl)azetidine-1-carboxylate (38a)

To a mixture of compound 12c (35 mg, 0.1 mmol), 1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid (21 mg, 0.1 mmol) and DIEA (39 mg, 0.3 mmol) in _CH2Cl2 (5 mL) _was added HATU (38 mg, 0.1 mmol). After stirring at room temperature for 30 min, the mixture was diluted with saturated ammonium chloride solution (20 mL) and extracted with _CH2Cl2 (3×20 mL). The combined organic phase was dried over _anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo to give the title compound 38a. This crude product was used directly in the next step without purification.

MS m/z (ESI): 531 [M+1]

Step 2. Azetidin-3-yl((3aR,6aS)-5-(5-chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)-pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone (38)

To a solution of compound 38a (crude material, ca. _0.1 mmol) in _CH2Cl2 (2 mL) was added TEA (2 mL). The mixture was stirred for 30 min and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 38 (19.9 mg, solid, 46% over two steps).

MS m/z (ESI): 431 [M+1]

¹ H-NMR(400 MHz, CD ₃ OD) δ 7.85 (s, 1H), 7.76 (s, 1H), 7.55 (s, 1H), 4.05 - 3.93 (m, 4H), 3.90 - 3.78 (m, 8H), 3.63 (d, J=12.5 Hz, 1H), 3.57 (d, J=10.8 Hz, 1H), 3, 46 (d, J=12.5 Hz, 1H), 3.41 [d, J=10.8 Hz, 1H], 1.19 (s, 6H).

Example 40. 5-Chloro-4-((3aR,6aS)-3a,6a-dimethyl-5-(2,2,2-trifluoroethyl)-hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine

To a mixture of compound 12c (50 mg, 0.14 mmol) in 1,4-dioxane (4 mL) were added DIEA (54 mg, 0.42 mmol) and 2,2,2-trifluoroethyl trifluoromethanesulfonate (65 mg, 0.28 mmol). The mixture was stirred in a sealed tube at 100 °C for 2 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and the residue was purified by prep-HPLC to give the title compound 40 (7.3 mg, solid, 12%).

MS m/z (ESI): 430 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.84 (s, 1H), 7.77 (s, 1H), 7.56 (s, 1H), 4.06 (d, J=11.5 Hz, 2H), 3.86 (s, 3H), 3.69 (d, J=11.5 Hz, 2H), 3.22 (q, J=9.8 Hz, 2H), 3.04 (d, J=9.3 Hz, 2H), 2.79 (d, J=9.3 Hz, 2H), 1.16 (s, 6H).

Example 49. 2-((3aR,6aS)-5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)ethan-1-ol

A mixture of compound 12c (35 mg, 0.1 mmol), 2-bromoethanol (15 mg, 0.12 mmol), and potassium carbonate (42 mg, 0.3 mmol) in MeCN (5 mL) was heated to reflux for 20 h with stirring. After cooling to room temperature, the mixture was concentrated to dryness in vacuo. The residue was dissolved in DMSO (5 mL), followed by the addition of 2-bromoethanol (0.2 mL) and cesium carbonate (98 mg, 0.3 mmol). The mixture was heated to 100 °C for 2 h. After cooling to room temperature, the mixture was filtered, and the filtrate was purified by prep-HPLC to give the title compound 49 (16.7 mg, solid, 43%).

MS m/z (ESI): 392 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.84 (s, 1H), 7.77 (s, 1H), 7.56 (s, 1H), 4.12 (d, J=11, 5 Hz, 2H), 3.87 (s, 3H), 3.65 (dd, J=8.7, 5.1 Hz, 4H), 2.93 (d, J=9.6 Hz, 2H) , 2.70-2.61 (m, 4H), 1.17 (s, 6H).

Example 50. 5-Chloro-4-((3aR,6aS)-5-(isoxazol-5-ylmethyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine

Step 1: From oxazol-5-yl-methyl methanesulfonate (50b)

To a solution of oxazol-5-ylmethanol 50a (76 mg, 0.77 mmol) in _CH2Cl2 ( ₁₀ mL) at 0 °C were added TEA (390 mg, 3.85 mmol) and methylsulfonyl chloride (93 mg, 0.81 mmol). After stirring at room temperature for 1 h, the resulting solution was used directly in the next step.

MS m/z (ESI): 178 [M+1]

Step 2. 5-Chloro-4-((3aR,6aS)-5-(isoxazol-5-ylmethyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (50)

To a mixture of compound 12c (70 mg, 0.2 mmol) in _CH2Cl2 (10 mL) was added _dropwise the solution obtained in step 1. The mixture was stirred for 2 h and concentrated to dryness in vacuo. The residue was dissolved in DMSO (2 mL) and cesium carbonate (195 mg, 0.6 mmol) was added. The mixture was heated to 50 °C and then immediately cooled to room temperature. Then, the mixture was filtered and the filtrate was purified by prep-HPLC to give the title compound 50 (7.7 mg, solid, 9%).

MS m/z (ESI): 429 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 8.31 (d, J=1.4 Hz, 1H), 7.84 (s, 1H), 7.76 (s, 1H), 7.56 (s, 1H), 6.32 (d, J=1.1 Hz, 1H), 4.08 (d, J=11.5 Hz, 2H), 3.87 (s, 2H), 3.87 (s, 3H), 3.62 (d, J=11.4 Hz, 2H), 2.94 (d, J=9.4 Hz, 2H), 2.66 (d, J=9.4 Hz, 2H), 1.15 (s, 6H).

Example 51 -yl)amino)-N-methylpicolinamide

Step 1. (3aR,6aS)-5-Benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (51b)

To a solution of 3,4-dimethylfuran-2,5-dione 51a (6 g, 47.6 mmol) in _CH2Cl2 ( ₁₂₀ mL) was added N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (14.8 g, 61.9 mmol). After cooling to 0 °C, a solution of TFA (543 mg, 4.76 mmol) in _CH2Cl2 ( ₃₀ mL) was added dropwise to the mixture under nitrogen atmosphere. The resulting mixture was stirred for 3 h and then concentrated to dryness in vacuo to give the title compound 51b (17 g). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 260 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.45 - 7.18 (m, 5H), 3.53 (s, 2H), 3.46 (d, J=10.2 Hz, 2H), 2.18 (d, J=10.1 Hz, 2H), 1.28 (s, 6H).

Step 2. (3aR,6aS)-5-Benzyl-3a,6a-dimethyltetrahydropyrrolo[3,4-c]pyrrole-1,3(2H,3aH)-dione (51c)

To a solution of compound 51b (17 g, crude product, 47.6 mmol) in THF (60 mL) was added ammonium hydroxide (60 mL). The mixture was heated in a sealed tube at 100 °C for 4 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo. The residue was diluted with water (150 mL) and extracted with CH ₂ Cl ₂ (3 x 150 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 100/0 - 1/4) to give the title compound 51c (7 g, 57% over two steps).

MS m/z (ESI): 259 [M+1]

^1H -NMR (400 MHz, DMSO- _d6 ) δ 11.24 (s, 1H), 7.35 - 7.12 (m, 5H), 3.48 (s, 2H), 3.12 (d , J=9.7 Hz, 2H), 2.03 (d, J=9.6 Hz, 2H), 1.09 (s, 6H).

Step 3. (3aR,6aS)-2-Benzyl-3a,6a-dimethyloctahydropyrrolo[3,4-c]pyrrole (51d)

To a solution of compound 51c (2.67 g, 10.34 mmol) in THF (30 mL) was added LAH (1.18 g, 31 mmol) portionwise. The mixture was heated to reflux for 2 h with stirring. After cooling to 0 °C, water (8 mL), 20% aqueous sodium hydroxide solution (16 mL), and water (8 mL) were added. The mixture was stirred for 10 min and filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 51d (2.44 g, 100%).

MS m/z(ESI): 231 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.34 - 7.17 (m, 5H), 3.45 (s, 2H), 2.61 (d, J=8.8 Hz, 2H), 2.46 (d, J=8.7 Hz, 2H), 2.24 (dd, J=10.6, 8.9 Hz, 4H), 0.97 (s, 6H).

Step 4. ((3aR,6aS)-5-Benzyl-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone (51e)

To a solution of compound 51d (2.44 g, 10.34 mmol) in _CH2Cl2 ( ₄₀ mL) were added TEA (3.14 g, 31 mmol) and cyclopropanecarbonyl chloride (1.08 g, 10.34 mmol). The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 1/4) to give the title compound 51e (1.59 g, 52%).

MS m/z (ESI): 299 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.31 - 7.27 (m, 4H), 7.25 - 7.20 (m, 1H), 3.81 (d, J=10.4 Hz , 1H), 3.75 (d, J=12.1 Hz, 1H), 3.59 (kv, J=13.3 Hz, 2H), 3.46 (d, J=10.4 Hz, 1H ), 3.34 (d, J=12.1 Hz, 1H), 2.81 (d, J=9.3 Hz, 1H), 2.77 (d, J=9.2 Hz, 1H), 2.37 (d, J=9.1 Hz, 2H), 1.59 (tt, J=8.0, 4.7 Hz, 1H), 1.09 (s, 3H), 1.05 (s, 3H), 1.03 - 0.95 (m, 2H), 0.79 - 0.68 (m, 2H).

Step 5. Cyclopropyl((3aR,6aS)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone (51f)

To a solution of compound 51e (1.59 g, 5.38 mmol) in ethanol (30 mL) was added 10% Pd/C (320 mg). The mixture was stirred under hydrogen atmosphere for 18 h and then filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 51f (1.11 g, 100%).

MS m/z (ESI): 209 [M+1]

Step 6. Cyclopropyl((3aR,6aS)-5-(2,5-dichloropyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone (51g)

To a solution of compound 51f (1.11 g, 5.33 mmol) in MeCN (20 mL) were added DIEA (2.07 g, 16 mmol) and 2,4,5-trichloropyrimidine (977 mg, 5.33 mmol). The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 3/7) to give the title compound 51g (1.59 g, 86%).

MS m/z (ESI): 355 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 3.96 (s, 2H), 3.87 - 3.72 (m, 3H), 3.62 (d, J =7.0 Hz, 2H), 3.48 (d, J=12.3 Hz, 1H), 1.60 - 1.51 (m, 1H), 1.19 (s, 3H), 1.16 (s, 3H), 1.03 - 1.00 (m, 2H), 0.82 - 0.75 (m, 2H).

Step 7. Methyl 5-((5-chloro-4-((3aR,6aS)-5-(cyclopropanecarbonyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)picolinate (51h)

A mixture of compound 51g (355 mg, 4.74 mmol), methyl 5-aminopicolinate (167 mg, 1.1 mmol), cesium carbonate (975 mg, 3 mmol), tris(dibenzylideneacetone)dipalladium (92 mg, 0.1 mmol) and 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (116 mg, 0.2 mmol) in 1,4-dioxane (15 mL) was heated in a microwave reactor at 100 °C for 1 h under nitrogen atmosphere. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/0 - 9/1) to give the title compound 51h (88 mg, 19%).

MS m/z (ESI): 471 [M+1]

Step 8. 5-((5-Chloro-4-((3aR,6aS)-5-(cyclopropanecarbonyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)picolinic acid (51i)

To a mixture of compound 51h (88 mg, 0.19 mmol) in THF (2 mL) and MeOH (2 mL) was added 1N NaOH (4 mL). The mixture was stirred for 2 h and then concentrated to dryness in vacuo. The residue was added water (10 mL) and washed with ethyl acetate (20 mL). The aqueous layer was adjusted to pH = 3 with 1N HCl and extracted with ethyl acetate (3 x 20 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo to give the title compound 51i (30 mg, 35%).

MS m/z (ESI): 457 [M+1]

Step 9. ,5-((5-Chloro-4-((3aR,6aS)-5-(cyclopropanecarbonyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-yl)amino)-N-methylpicolinamide (51)

To a solution of compound 51i (30 mg, _0.066 mmol) in _CH2Cl2 (10 mL) were added sequentially DIEA (74 mg, 0.57 mmol), HATU (72 mg, 0.19 mmol) and methylamine solution (2 M in THF, 0.05 mL, 1 mmol). The resulting mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 51 (8.2 mg, solid, 27%).

MS m/z (ESI): 470 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 8.77 (d, J=2.3 Hz, 1H), 8.17 (dd, J=8.6, 2.6 Hz, 1H), 7.88 (d, J=10.1 Hz, 2H), 3.91 (t, J=12.2 Hz, 2H), 3.82 - 3.73 (m, 3H), 3.62 (d, J=10.8 Hz, 1H), 3.50 (d, J=12.5 Hz, 1H), 3.34 (d, J=12.4 Hz, 1H), 2.85 (s, 3H), 1.72 - 1.66 (m, 1H), 1.13 (s, 3H), 1.09 (s, 3H), 0.82 - 0.77 (m, 2H), 0.77 - 0.71 (m, 2H).

Example 53. ((3aR,6aS)-5-(5-Chloro-2-((6-(morpholine-4-carbonyl)pyridin-3-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone

Step 1. Morpholino(5-nitropyridin-2-yl)methanone (53b)

To a solution of 5-nitropicolinic acid 53a (505 mg, 3 mmol) in _CH2Cl2 ( ₃₀ mL) were added morpholine (262 mg, 3 mmol), DIEA (1.16 g, 9 mmol) and HATU (1.14 g, 3 mmol). The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/ethyl acetate = 100/0 - 1/4) to give the title compound 53b (880 mg, contaminated with DIEA and tetramethylurea).

MS m/z (ESI): 238 [M+1]

Step 2. (5-Aminopyridin-2-yl)(morpholino)methanone (53c)

To a solution of compound 53b (880 mg, ca. 3 mmol) in MeOH (20 mL) was added 10% Pd/C (200 mg). The mixture was stirred under hydrogen atmosphere for 2 h and then filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 53c (740 mg, contaminated with DIEA and tetramethylurea).

MS m/z (ESI): 208 [M+1]

Step 3. ((3aR,6aS)-5-(5-Chloro-2-((6-(morpholine-4-carbonyl)pyridin-3-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone (53)

To a mixture of compound 51g (71 mg, 0.2 mmol) and compound 53c (42 mg, 0.2 mmol) in 1,4-dioxane (2 mL) were added NaOEt (30 mg, 0.44 mmol) and RuPhos-Pd-G2 (8 mg, 0.01 mmol). The mixture was heated to 100 °C in a microwave reactor under nitrogen atmosphere for 1 h. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/0 - 9/1) and then prep-HPLC to give the title compound 53 (11.4 mg, solid, 11%).

MS m/z (ESI): 526 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 8.92 (d, J=2.4 Hz, 1H), 8.29 (dd, J=8.6, 2.6 Hz, 1H), 7.99 (s, 1H), 7.62 (d, J=8.6 Hz, 1H), 4.02 (t, J=11.0 Hz, 2H), 3.95 - 3.58 (m, 13H), 3.46 (d, J=12.4 Hz, 1H), 1.81 (ddd, J=12.8, 7.9,4.7 Hz, 1H), 1.24 (s, 3H), 1.21 (s, 3H), 0.92 (dt, J=5.4, 3.8 Hz, 2H), 0.89 - 0.81 (m, 2N).

Example 59 was synthesized according to the procedure described for Example 53, except that 1-methyl piperazine was used instead of morpholine in Step 1.

Example 54. 5-Chloro-4-((3aR,6aS)-3a,6a-dimethyl-5-(pyridin-2-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine

A mixture of compound 12c (100 mg, 0.26 mmol), 2-bromopyridine (62 mg, 0.39 mmol), DBU (4 mL) was heated to 140 °C and stirred for 16 h. After cooling to room temperature, water (4 mL) was added to the mixture and extracted with ethyl acetate (3 x 5 mL). The combined organic phase was washed with water (10 mL) and concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 54 (36.4 mg, solid, 33%).

MS m/z (ESI): 425 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.05 (s, 1H), 8.04 (d, J=3.7 Hz, 1H), 7.89 (s, 1H), 7. 69 (s, 1H), 7.47 (s, 1H), 7.43 (s, 1H), 6.54 (d, J=1.6 Hz, 1H), 6.40 (d, J=8 .4 Hz, 1H), 3.89 (d, J=11.4 Hz, 2H), 3.75 (s, 3H), 3.75 - 3.71 (m, 2H), 3.56 (d , J=10.9 Hz, 2H), 3.38 (d, J=10.8 Hz, 2H), 1.15 (s, 6H).

Example 55. 4-((3aR,6aS)-3a,6a-Dimethyl-5-(methylsulfonyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-methyl-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine

Step 1. (3aR,6aS)-2-Benzyl-5-(2-chloro-5-methylpyrimidin-4-yl)-3a,6a-dimethyloctahydropyrrolo[3,4-c]pyrrole (55a)

To a solution of compound 51d (2.02 g, 8.8 mmol) in MeCN (20 mL) were added 2,4-dichloro-5-methylpyrimidine (1.63 g, 10 mmol) and DIEA (3.88 g, 30 mmol). The mixture was stirred for 2 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 45/55) to give the title compound 55a (1.45 g, 46%).

MS m/z (ESI): 357 [M+1]

Step 2. 4-((3aR,6aS)-5-Benzyl-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-methyl-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (55b)

To a mixture of compound 55a (1.45 g, 4.1 mmol) and 1-methyl-1H-pyrazol-4-amine hydrochloride (4.1 g, 1 mmol) in isopropanol (40 mL) was added pTsOH (78 mg, 0.41 mmol). The mixture was stirred at 100 °C for 18 h. After cooling to room temperature, MeOH (20 mL) and then TEA (0.5 mL) were added. The mixture was stirred for additional 5 min and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol = 19/1) to give the title compound 55b (540 mg, 32%).

MS m/z (ESI): 418 [M+1]

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.68 (s, 2H), 7.51 (s, 1H), 7.30 (dd, J=6.6, 3.5 Hz, 4H), 7.25 - 7.20 (m, 1H), 6.86 (s, 1H), 3.85 (d, J=10.8 Hz, 2H), 3.85 (s, 3H), 3.60 (s, 2H), 3.48 (d, J=10.8 Hz, 2H), 2.80 (d, J=9.3 Hz, 2H), 2.41 (d, J=9.3 Hz , 2H), 2.20 (s, 3H), 1.10 (s, 6H).

Step 3. 4-((3aR,6aS)-3a,6a-Dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-methyl-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (55c)

To a solution of compound 55b (540 mg, 1.29 mmol) in MeOH (30 mL) was added 10% Pd/C (300 mg). The mixture was stirred under hydrogen atmosphere at 36 °C for 16 h and then filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 55c (340 mg, 81%).

MS m/z (ESI): 328 [M+1]

Step 4. 4-((3aR,6aS)-3a,6a-Dimethyl-5-(methylsulfonyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-methyl-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (55)

To a solution of compound 55c (33 mg, 0.1 mmol) in _CH2Cl2 (10 mL) _was added TEA (30 mg, 0.3 mmol) followed by a solution of MsCl (12 mg, 0.1 mmol) in _CH2Cl2 (1 mL). _The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 55 (18.7 mg, solid, 46%).

MS m/z (ESI): 406 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.64 (s, 1H), 7.50 (s, 1H), 7.44 (s, 1H), 3.82 (d, J=11.2 Hz, 2H), 3.74 (s, 3H), 3.60 (d, J=11.2 Hz, 2H), 3.40 (d, J=10.1 Hz, 2H), 3.23 (d, J=10.1 Hz, 2H), 2.83 (s, 3H), 2.15 (s, 3H), 1.09 (s, 6H).

Example 56. ((S)-2,2-difluorocyclopropyl)((3aR,6aS)-3a,6a-dimethyl-5-(5-methyl-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone

To a solution of compound 54c (33 mg, 0.1 mmol) in _CH2Cl2 ( ₁₀ mL) were added (S)-2,2-difluorocyclopropane-1-carboxylic acid (13 mg, 0.1 mmol) and DIEA (39 mg, 0.3 mmol), followed by HATU (38 mg, 0.1 mmol). The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 56 (21.3 mg, solid, 49%).

MS m/z (ESI): 432 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.76 (d, J=3.1 Hz, 1H), 7.62 (s, 1H), 7.54 (d, J=11.0 Hz , 1H), 3.98 - 3.88 (m, 2H), 3.86 (d, J=2.7 Hz, 3H), 3.82 - 3.71 (m, 3H), 3.68 - 3.56 (m, 2H), 3.48 (dd, J=12.6, 2.7 Hz, 1H), 2.89 - 2.79 (m, 1H), 2.28 (d, J= 5.7 Hz, 3H), 2.08 - 2.00 (m, 1H), 1.85 - 1.73 (m, 1H), 1.24 (d, J=3.4 Hz, 2H), 1.21 (d, J=3.9 Hz, 3H).

Examples 57, 58 and 60 were synthesized according to the procedure described for Example 56, except that in Step 1, different compounds were used in place of (S)-2,2-difluorocyclopropane-1-carboxylic acid.

Example 61. cis-5-chloro-N-(1-methyl-1H-pyrazol-4-yl)-4-(3a-methyl-5-(methylsulfonyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-amine

Step 1. cis-5-Benzyl-3a-methyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (61b)

To a solution of 3-methylfuran-2,5-dione (5.33 g, 47.6 mmol) and N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (14.8 g, 61.9 mmol) in _CH2Cl2 ( ₁₇₀ mL) was added trifluoroacetic acid (543 mg, 4.76 mmol). The mixture was stirred for 3 h and then concentrated to dryness in vacuo to give the title compound 61b (11.62 g). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 246 [M+1]

Step 2. cis-5-Benzyl-3a-methyltetrahydropyrrolo[3,4-c]pyrrole-1,3(2H,3aH)-dione (61c)

To a solution of compound 61b (11.66 g, ca. 47.6 mmol) in THF (60 mL) was added ammonium hydroxide (60 mL). The mixture was heated in a sealed tube at 100 °C for 5 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and the residue was dissolved in _CH2Cl2 ( ₂₀₀ mL). The resulting mixture was washed with water (20 mL) and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/1 - 1/1) to give the title compound 61c (8 g, 68%).

MS m/z (ESI): 245 [M+1]

Step 3. cis-2-Benzyl-3a-methyloctahydropyrrolo[3,4-c]pyrrole (61d)

To a solution of compound 61c (8 g, 32.8 mmol) in THF (100 mL) was added LAH (3.73 g, 98.36 mmol). The mixture was heated to 70 °C and stirred for 3 h. After cooling to 0 °C, water (10 mL), 20% NaOH solution (20 mL), and water (10 mL) were added successively. The mixture was stirred for 10 min and then filtered. The filtrate was concentrated to dryness in vacuo to give the title compound (8 g). The crude product was used directly in the next step without further purification.

MS m/z(ESI): 217 [M+1]

Step 4. tert-Butyl-cis-5-benzyl-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (61e)

To a solution of compound 61d (8 g, crude) and TEA (7.48 g, 74.07 mmol) in _CH2Cl2 ( ₁₀₀ mL) was added di-tert-butyl dicarbonate (8.07 g, 37.03 mmol). The mixture was stirred for 3 h and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/1 - 7/3) to give the title compound 61e (10 g, 96% over two steps).

MS m/z (ESI): 317 [M+1]

Step 5. tert-Butyl cis-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (61f)

To a solution of compound 61e (10 g, 31.64 mmol) in MeOH (100 mL) was added 10% Pd/C (1 g). The mixture was stirred under hydrogen atmosphere for 18 h and then filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 61f (7 g, 97%).

MS m/z (ESI): 227 [M+1]

Step 6. tert-Butyl cis-5-(2,5-dichloropyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (61g)

To a solution of 2,4,5-trichloropyrimidine (546 mg, 3 mmol) in MeCN (10 ml) were added compound 61f (678 mg, 3 mmol) and potassium carbonate (828 mg, 6 mmol). The mixture was heated to 80 °C and stirred for 3 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 9/1) to give the title compound 61g (850 mg, 76%).

MS m/z (ESI): 373 [M+1]

Step 7. cis-5-Chloro-N-(1-methyl-1H-pyrazol-4-yl)-4-(3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-amine hydrochloride (61h)

To a mixture of compound 61 g (850 mg, 2.28 mmol) and 1-methyl-1H-pyrazol-4-amine hydrochloride (221 mg, 2.28 mmol) in isopropanol (10 mL) was added pTsOH (868 mg, 4.57 mmol). The mixture was heated in a microwave reactor at 100 °C for 2 h. After cooling to room temperature, the precipitate was collected by filtration to give the title compound 61h (200 mg, 75%) as a hydrochloride salt.

MS m/z (ESI): 334 [M+1]

Step 8. cis-5-Chloro-N-(1-methyl-1H-pyrazol-4-yl)-4-(3a-methyl-5-(methylsulfonyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-amine (61)

To a mixture of compound 61h (33.3 mg, 0.1 mmol) and TEA (20.2 mg, 0.2 mmol) in _CH2Cl2 (5 mL) _was added MsCl (11.4 mg, 0.1 mmol). The mixture was stirred for 1 h and concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 61 (15.4 mg, solid, 37%).

MS m/z (ESI): 412 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.74 (s, 1H),7.66 (s, 1H),7.43 (s, 1H), 4.06 - 4.01 (m, 1H ), 3.88 (d, J=11.5 Hz, 1H), 3.76 - 3.64 (m, 6H), 3.61 - 3.56 (m, 1H), 3.35 (d, J=9.9 Hz, 2H), 2.83 (s, 3H), 2.56 - 2.51 (m, 1H), 1.22 (s, 3H).

Example 62 was synthesized according to the procedure described for Example 61, except that cyclopropanecarbonyl chloride was used instead of MsCl in step 8.

Example 63. cis-3-(5-(5-chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

To a solution of compound 61h (66.6 mg, 0.2 mmol), HATU (152 mg, 0.4 mmol) and TEA (40.4 mg, 0.4 mmol) in _CH2Cl2 (5 mL) _was added 2-cyanoacetic acid (17 mg, 0.2 mmol). The mixture was stirred for 2 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 63 (12.4 mg, solid, 16%).

MS m/z (ESI): 401 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.85 (s, 1H), 7.78 (s, 1H), 7.54 (s, 1H), 4.20 - 4.15 (m, 1H), 4.00 - 3.92 (m, 1H), 3.88 - 3.79 (m, 6H), 3.66 - 3.61 (m, 1H), 3.52 - 3.45 ( m, 2H), 2.71 - 2.62 (m, 1H), 1.32 (d, J=4.0 Hz, 3H).

Examples 64, 65 and 68 were synthesized according to the procedure described for Example 63, except that different compounds were used in place of 2-cyanoacetic acid in Step 1.

Example 66. 5-Chloro-4-((3aR,6aS)-5-(isoxazol-5-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1 H-pyrazol-4-yl)pyrimidin-2-amine

Step 1. 01-((3aR,6aS)-5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)prop-2-yn-1-one (66a)

To a mixture of compound 12c (139 mg, 0.4 mmol) in _CH2Cl2 (20 mL) _were added propiolic acid (28 mg, 0.4 mmol), DIEA (155 mg, 1.2 mmol) and HATU (152 mg, 0.4 mmol). The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/0 - 19/1) to give the title compound 66a (90 mg, 56%).

MS m/z (ESI): 400 [M+1]

Step 2. 5-Chloro-4-((3aR,6aS)-5-(isoxazol-5-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (66)

To a solution of compound 66a (90 mg, 0.225 mmol) in THF (2 mL) were added water (2 mL), ammonium chloride (60 mg, 1.125 mmol), and sodium azide (73 mg, 1.125 mmol). The mixture was stirred for 18 h and then heated to 70 °C and stirred for an additional 2 h. After cooling to room temperature, the mixture was diluted with water (20 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 66 (12.1 mg, solid, 13%).

MS m/z (ESI): 415 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 8.06 (d, J=2.1 Hz, 1H), 7.85 (s, 1H), 7.76 (s, 1H), 7.54 (s, 1H), 5.00 (d, J=2.1 Hz, 1H), 4.01 (d, J=11.6 Hz, 2H), 3.86 (d, J=11.5 Hz, 2H), 3.85 (s, 3H), 3.62 (d, J=10.3 Hz, 2H), 3.47 (d, J=10.3 Hz, 2H), 1.24 (s, 6H).

Example 69. cis-4-(5-(Isoxazol-5-ylmethyl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine

Step 1. tert-Butyl-cis-5-(2-chloropyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (69a)

To a solution of 2,4-dichloropyrimidine (444 mg, 3 mmol) in MeCN (10 mL) was added compound 61f (678 mg, 3 mmol) and potassium carbonate (621 mg, 4.5 mmol). The mixture was stirred at 80 °C for 3 h and then cooled to room temperature. The mixture was filtered and the filtrate was concentrated to dryness in vacuo to give the title compound 69a (700 mg, 69%).

MS m/z (ESI): 339 [M+1]

Step 2. cis-N-(1-methyl-1H-pyrazol-4-yl)-4-(3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-amine (69b )

To a mixture of compound 69a (700 mg, 2.07 mmol) and 1-methyl-1H-pyrazol-4-amine hydrochloride (200 mg, 2.07 mmol) in isopropanol (15 mL) was added pTsOH (787 mg, 4.14 mmol). The mixture was stirred at 100 °C for 18 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (CH ₂ Cl ₂ /MeOH = 100/1 - 4/1) to give the title compound 69b (300 mg, 48%).

MS m/z (ESI): 300 [M+1]

Step 3. cis-4-(5-(Isoxazol-5-ylmethyl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-H-(1-methyl-1H-pyrazol-4 -yl)pyrimidin-2-amine (69)

Compound 69 was synthesized according to Step 2 of Example 50 except that compound 69b was used instead of compound 12c.

MS m/z (ESI): 381 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 8.33 (d, J=1.8 Hz, 1H), 7.83 - 7.80 (m, 2H), 7.58 (s, 1H) , 6.35 (d, J=1.8 Hz, 1H), 5.92 (d, J=6.1 Hz, 1H), 3.87 (s, 5H), 3.82 (s, 2H) , 3.63 (s, 1H), 3.52 (s, 1H), 2.98 (dd, J=9.4, 7.8 Hz, 1H), 2.84 (d, J=9.2 Hz, 1H), 2.69 (dd, J=9.5, 4.6 Hz, 1H), 2.58 (d, J=9.3 Hz, 1H), 2.55 - 2.49 (m, 1H), 1.32 (s, 3H).

Examples 73 and 74 were synthesized according to Step 2 of Example 50 except that different chemical compounds were used in place of compound 12c.

Example 70. cis-Cyclopropyl(3a-methyl-5-(5-methyl-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone

Step 1. tert-Butyl-cis-5-(2-chloro-5-methylpyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (70a)

To a solution of 2,4-dichloro-5-methyl pyrimidine (324 mg, 2 mmol) in MeCN (10 ml) were added compound 61f (452 mg, 2 mmol) and potassium carbonate (552 mg, 4 mmol). The mixture was stirred at 80°C for 3 h and then cooled to room temperature. The mixture was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 7/3) to give the title compound 70a (600 mg, 85%).

MS m/z (ESI): 353 [M+1]

Step 2. cis-5-Methyl-N-(1-methyl-1H-pyrazol-4-yl)-4-(3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-amine hydrochloride (70b)

To a mixture of compound 70a (600 mg, 1.7 mmol) and 1-methyl-1H-pyrazol-4-amine hydrochloride (165 mg, 1.7 mmol) in isopropanol (15 mL) was added pTsOH (646 mg, 3.4 mmol). The mixture was heated at 100 °C in a microwave reactor for 1 h. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel (CH ₂ Cl ₂ /MeOH = 100/1 - 67/33) to give the title compound 70b as a hydrochloride salt (400 mg, 75%).

MS m/z (ESI): 314 [M+1]

Step 3. cis-Cyclopropyl(3a-methyl-5-(5-methyl-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methanone (70)

To a mixture of compound 70b (62.6 mg, 0.2 mmol) in _CH2Cl2 (5 mL) were added TEA (30.3 mg, 0.3 mmol) and cyclopropanecarbonyl chloride (20.8 mg, 0.2 _mmol ). The mixture was stirred at room temperature for 1 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 70 (7.3 mg, solid, 10%).

MS m/z (ESI): 382 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.65 (s, 1H),7.49 (s, 1H),7.43 (s, 1H), 4.05 - 3.92 (m, 2H ), 3.81 - 3.71 (m, 4H), 3.69 - 3.48 (m, 4H), 3.36 - 3.29 (m, 1H), 2.60 - 2.47 (m , 1H), 2.17 (s, 3H), 1.74 - 1.66 (m, 1H), 1.22 (d, J=12.1 Hz, 3H), 0.84 - 0.66 ( m, 4H).

Example 71. cis-3-(3a-methyl-5-(5-methyl-2-((1-methyl-1H-pyrazol-4-yl)amino)-pyrimidin-4-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

To a mixture of compound 70b (62.6 mg, _0.2 mmol) in _CH2Cl2 (5 mL) were added 2-cyanoacetic acid (17 mg, 0.2 mmol), TEA (40.4 mg, 0.4 mmol) and HATU (114 mg, 0.3 mmol). The mixture was stirred for 2 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 71 (9.4 mg, solid, 10%).

MS m/z (ESI): 382 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.64 (s, 1H),7.50 (s, 1H),7.43 (s, 1H), 4.01 - 3.93 (m, 1H ), 3.83 -3.56 (m, 8H), 3.54 - 3.49 (m, 1H), 3.43 -3.27 (m, 3H), 2.59 - 2.49 (m , 1H), 2.15 (d, J=12.8 Hz, 3H), 1.22 (dd, J=10.7, 3.4 Hz, 3H).

Example 72 was synthesized according to the procedure described for Example 7, except that (S)-2,2-difluorocyclopropane-1-carboxylic acid was used in place of 2-cyanoacetic acid in step 1.

Example 75. 5-Chloro-4-((3aR,6aS)-3a,6a-dimethyl-5-((1-methyl-1H-pyrazol-3-yl)methyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine hydrochloride

To a mixture of compound 12c (35 mg, 0.1 mmol) in DMF (4 mL) were added cesium carbonate (98 mg, 0.3 mmol) and 33-(chloromethyl)-1-methyl-1H-pyrazole (14 mg, 0.11 mmol). The mixture was stirred for 20 h and then filtered. The filtrate was purified directly by prep-HPLC to afford the title compound 75 as the hydrochloride salt (3.4 mg, solid, 8%).

MS m/z (ESI): 442 [M+1]

¹ H-NMR (400 MHz, CD ₃ OD) δ 7.94 (t, J=31.0 Hz, 2H), 7.74 - 7.56 (m, 2H), 6.56 (d, J=5.3 Hz, 1H), 4.45 (d, J=6.5 Hz, 2H), 4.15 - 3.85 (m, 6H), 3.81 (d, J=12.8 Hz, 2H), 3.71 (d, J=11.8 Hz, 2H), 3.60 (d, J=11.9 Hz, 2H), 3.54 - 3.48 (m, 2H), 1.28 (s, 3H), 1.22 (s, 3H).

Examples 76, 77, 78, 79, 80, 81 and 82 were synthesized according to the procedure described for Example 75, except that in Step 1, different compounds were used in place of 1,3-(chloromethyl)-1-methyl-1H-pyrazole.

Example 83. 5-Chloro-4-((3aR,6aS)-3a,6a-dimethyl-5-(5-methyl-1,3,4-oxadiazol-2-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine

To a mixture of compound 12c (80 mg, 0.208 mmol) in DMF (3 mL) were added 5-methyl-1,3,4-oxadiazol-2(3H)-one (21 mg, 0.208 mmol) and DIEA (161 mg, 1.25 mmol). The mixture was stirred at for 5 min and then (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (101 mg, 0.229 mmol) was added. The resulting mixture was heated to 50 °C and stirred for 12 h. After cooling to room temperature, the mixture was added water (5 mL) and extracted with ethyl acetate (3×5 mL). The combined organic phase was concentrated to dryness in vacuo and the residue was purified by prep-HPLC to give the title compound 83 (8.5 mg, solid, 10%).

MS m/z (ESI): 430 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.06 (s, 1H), 7.89 (s, 1H), 7.70 (s, 1H), 7.44 (s, 1H), 3.92 (d, J=11.6 Hz, 2H), 3.77 (s, 3H), 3.73 (d, J=11.3 Hz, 2H), 3.57 (d, J=10 .4 Hz, 2H), 3.40 (d, 1=10.4 Hz, 2H), 2.31 (s, 3H), 1.14 (s, 6H).

Example 84. 5-Chloro-4-((3aR,6aS)-5-(cyclopropylmethyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine

Step 1. (3aR,6aS)-2-benzyl-5-(2,5-dichloropyrimidin-4-yl)-3a,6a-dimethyloctahydropyrrolo[3,4-c]pyrrole (84a)

To a solution of compound 51d (400 mg, 1.74 mmol) in MeCN (10 ml) were added potassium carbonate (720 mg, 5.22 mmol) and 2,4,5-trichloropyrimidine (410 mg, 2.26 mmol). The mixture was stirred for 2 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 3/2) to give the title compound 84a (200 mg, 31%).

MS m/z (ESI): 377 [M+1]

Step 2. (3aR,6aS)-2-(2,5-dichloropyrimidin-4-yl)-3a,6a-dimethyloctahydropyrrolo[3,4-c]pyrrole (84b)

To a solution of compound 84a (0.2 g, 0.53 mmol) in _CH2Cl2 (10 mL) was added 1-chloroethyl chloroformate (226 mg, 1.59 mmol). The mixture was heated to reflux for 2 h. _After cooling to room temperature, the mixture was concentrated to dryness in vacuo and then MeOH (10 mL) was added. The mixture was heated to reflux for additional 2 _h and then cooled to room temperature. The mixture was concentrated to dryness in vacuo and the residue was purified by silica gel column chromatography ( _CH2Cl2 /MeOH=9/1) to give the title compound 84b (140 mg, 92%).

MS m/z (ESI): 287 [M+1]

Step 3. (3aR,6aS)-2-(cyclopropylmethyl)-5-(2,5-dichloropyrimidin-4-yl)-3a,6a-dimethyloctahydropyrrolo[3,4-c]pyrrole (84c)

A mixture of (bromomethyl)cyclopropane (56.3 mg, 0.42 mmol), compound 84b (60 mg, 0.21 mmol), potassium carbonate (87 mg, 0.63 mmol) in MeCN (4 ml) was heated to 50 °C and stirred for 16 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 2/3) to give the title compound 84c (50 mg, 70%).

MS m/z (ESI): 341 [M+1]

Step 4. 5-Chloro-4-((3aR,6aS)-5-(cyclopropylmethyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (84)

A mixture of compound 84c (50 mg, 0.147 mmol), 1-methyl-1H-pyrazol-4-amine (21 mg, 0.122 mmol) and pTsOH (55 mg, 0.284 mmol) in isopropanol (2 mL) was heated to 100 °C and stirred for 4 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo and the residue was purified by prep-HPLC to give the title compound 84 (16.4 mg, solid, 28%).

MS m/z (ESI): 402 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.06 (s, 1H), 7.89 (s, 1H), 7.70 (s, 1H), 7.45 (s, 1H), 3.98 (d, J=10.9 Hz, 2H), 3.77 (s, 3H), 3.56 (d, J=11.2 Hz, 2H), 2.82 (s, 2H), 2.32 (d, J=63.4 Hz, 4H), 1.07 (s, 6H), 0.79 (s, 1H), 0.41 (d, J=6.1 Hz, 2H), 0.05 (s, 2H).

Example 85. 2-((3aR,6aS)-5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)acetonitrile

A mixture of compound 12c (42 mg, 0.115 mmol), 2-bromoacetonitrile (30 mg, 0.23 mmol), TEA (50 mg, 0.46 mmol) in THF (1 mL) was stirred for 2 h and then concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 85 (16 mg, solid, 34%).

MS m/z (ESI): 402 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.07 (s, 1H), 7.89 (s, 1H), 7.71 (s, 1H), 7.44 (s, 1H), 4.00 (d, J=11.3 Hz, 2H), 3.80 (s, 2H), 3.77 (s, 3H), 3.56 (d, J=11.3 Hz, 2H), 2.85 (d, J=9.2 Hz, 2H), 2.55 (d, J=9.3 Hz, 2H), 1.09 (s, 6H).

Example 86. ((3aS,6aR)-5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone

Step 1. (S)-1-Phenyl-N-((trimethylsilyl)methyl)ethan-1-amine (86b)

A mixture of (chloromethyl)trimethylsilane (20.24 g, 165 mmol), compound 86a (20 g, 165 mmol) and TEA (20 g, 165 mmol) was heated to sublimation for 16 h. After cooling to room temperature, n-heptane (500 ml) was added to the mixture, stirred and filtered. The filtrate was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/20) to give the title compound 86b (26 g, 76%).

MS m/z (ESI): 208 [M+1]

Step 2. (S)-N-(methoxymethyl)-1-phenyl-N-((trimethylsilyl)methyl)ethan-1-amine (86c)

To 37% aqueous formaldehyde solution (6.6 g, 81.49 mmol) at 0 °C was added compound 86b (13.0 g, 62.68 mmol). The mixture was stirred at 0 °C for 0.5 h, and then MeOH (3.4 g, 106.56 mmol) and potassium carbonate (8.7 g, 62.68 mmol) were added. The mixture was stirred at 0 °C for additional 2 h, water (50 mL) was added and extracted with ethyl acetate (3 x 50 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by distillation to give the title compound 86c (6.816 g, 43%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.35 - 7.30 (m, 4H), 7.23 (s, 1H), 4.18 (d, J=9.3 Hz, 1H ), 3.99 (d, J=9.3 Hz, 1H), 3.90 (d, J=6.8 Hz, 1H), 3.14 (s, 3H), 2.06 (kv, J =14.4 Hz, 2H), 1.33 (d, J=6.8 Hz, 3H), 0.00 (s.9H).

Step 3. cis-3a-methyl-5-((R)-1-phenylethyl)tetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (86d)

To a solution of compound 86c (6.62 g, 26.34 mmol) in _CH2Cl2 ( ₂₅ mL) at 0 °C was added a solution of 3-methylfuran-2,5-dione (2.95 g, 26.34 mmol) in _CH2Cl2 ( ₁₀ mL), followed by dropwise addition of a solution of TFA (0.3 g, 2.63 mmol) in _CH2Cl2 ( ₅ mL). The resulting mixture was stirred for 6 h and then concentrated to dryness in vacuo to give the title compound 86d (6.83 g). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 260 [M+1]

Step 4. cis-3a-Methyl-5-((R)-1-phenylethyl)tetrahydropyrrolo[3,4-c]pyrrole-1,3(2H,3aH)-dione (86e)

To a solution of compound 86d (6.83 g, 26.34 mmol) in THF (30 mL) was added ammonium hydroxide (30 mL). The resulting mixture was heated to 100 °C in a sealed tube and stirred for 3 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/2) to give the title compound 86e (4.30 g, 63%).

MS m/z (ESI): 259 [M+1]

Step 5. cis-3a-Methyl-2-((R)-1-phenylethyl)octahydropyrrolo[3,4-c]pyrrole (86f)

To a solution of compound 86e (4.3 g, 16.65 mmol) in THF (80 mL) at 0 °C was added LAH (1.90 g, 49.95 mmol) portionwise. The mixture was heated to 70 °C and stirred for 2 h. After cooling to 0 °C, the mixture was quenched by adding saturated sodium sulfate solution (20 mL) and filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 86f (3.62 g, 94%).

MS m/z(ESI): 231 [M+1]

Step 6. tert-Butyl (3aS,6aR)-3a-methyl-5-((R)-1-phenylethyl)-hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (86g) and tert-butyl (3aR,6aS)-3a-methyl-5-((R)-1-phenylethyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (86h)

To a solution of compound 86f (3.62 g, _15.73 mmol) in _CH2Cl2 (40 mL) at 0 °C were added TEA (3.18 g, 31.46 mmol) and a solution of di-tert-butyl pyrocarbonate (4.12 g, 18.88 mmol) in _CH2Cl2 (10 mL). The mixture was stirred for 1 h, added water (20 mL) and extracted _{with CH2Cl2} (3×50 mL). The combined organic phase _was dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 20/1) to give the title compound 86g (1.89 g) and compound 86h (1.65 g) and a mixture of compound 86g and compound 86h (1.04 g). The combined yield of compound 86g and compound 86h was 88%.

tert-Butyl (3aS,6aR)-3a-methyl-5-((R)-1-phenylethyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate 86g:

MS m/z (ESI): 331 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.29 (d, J = 4.5 Hz, 4H), 7.21 (d, J = 4.2 Hz, 1H), 3.57 -3.48 (m, 1H), 3.25 (d, J = 10.8 Hz, 1H), 3.22 - 3.14 (m, 2H), 3.06 (d, J = 10.8 Hz, 1H), 2.54 (dd, J = 5.5, 3.8 Hz, 2H), 2.35 (d, J = 9.1 Hz, 1H), 2.18 (s, 1H), 2.12 (d, J = 9.1 Hz, 1H), 1.40 (s, 9H), 1.24 (d, J = 6.5 Hz, 3H), 1.08 (s, 3H).

tert-Butyl-(3aR,6aS)-3a-methyl-5-((R)-1-phenylethyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate 86h:

MS m/z (ESI): 331 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.29 (d, J=4.4 Hz, 4H), 7.21 (d, J=4.3 Hz, 1H), 3.49 - 3.41 (m, 1H), 3.34 (d, J=10.9 Hz, 1H), 3.20 (d, J=6.6 Hz, 1H), 3.10 (dd, J=11 ,3, 3.5 Hz, 1H), 3.06 (d, J=11.0 Hz, 1H), 2.65 (s, 1H), 2.57 (d, J=8.9 Hz, 1H ), 2.18 (d, J=3.5 Hz, 1H), 2.16 (s, 2H), 1.39 (s, 9H), 1.24 (d, J=6.5 Hz, 3H), 1.11 (s, 3H).

Step 7. tert-Butyl (3aS,6aR)-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (86i)

To a solution of compound 86g (1.89 g, 5.72 mmol) in MeOH (50 mL) was added 10% Pd/C (0.19 g). The mixture was stirred under hydrogen atmosphere for 12 h and then concentrated to dryness in vacuo to give the title compound 86i (1.30 g, 100%).

MS m/z (ESI): 227 [M+1]

Step 8. tert-Butyl (3aS,6aR)-5-(2,5-dichloropyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (86j)

To a solution of compound 86i (1.3 g, 5.73 mmol) in MeCN (40 mL) were added DIEA (1.48 g, 11.46 mmol) and 2,4,5-trichloropyrimidine (1.05 g, 5.73 mmol). The mixture was stirred for 2 h and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 7/3) to give the title compound 86j (1.83 g, 86%).

MS m/z (ESI): 373 [M+1]

Step 9. 5-Chloro-N-(1-methyl-1H-pyrazol-4-yl)-4-((3aR,6aS)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-amine (86k)

To a solution of compound 86j (1.83 g, 4.91 mmol) in isopropanol (30 mL) were added 1-methyl-1H-pyrazol-4-amine hydrochloride (656 mg, 4.91 mmol) and pTsOH (93 mg, 0.49 mmol). The mixture was heated to 100 °C and stirred for 12 h. After cooling to room temperature, the reaction mixture was filtered and the filter cake was collected to give the title compound 86k (1.28 g, 82%) as a hydrochloride salt.

MS m/z (ESI): 334 [M+1]

Step 10. ((3aS,6aR)-5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone (86)

To a solution of compound 86k (100 mg, 0.27 mmol) in _CH2Cl2 (5 mL) _were added cyclopropanecarboxylic acid (23 mg, 0.27 mmol) and DIEA (175 mg, 1.35 mmol). The mixture was stirred for 10 min and then HATU (205 mg, 0.54 mmol) was added. The mixture was stirred for an additional 2 h, water (5 mL) was added and extracted with ethyl acetate (3×5 mL). The combined organic phase was concentrated to dryness in vacuo and the residue was purified by prep-HPLC to give the title compound 86 (64.3 mg, solid, 59%).

MS m/z (ESI): 402 [M+1]

Specific rotation =-9.2°× ^dm2 /kg

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.06 (s, 1H), 7.89 (s, 1H), 7.71 (s, 1H), 7.44 (s, 1H), 4.04 (s, 1H), 3.95 (dd, J=10.7, 7.7 Hz, 1H), 3.88 - 3.80 (m, 1H), 3.78 (s, 3H), 3.75 - 3.69 (m, 1H), 3.65 (dd, J=13.6, 6.6 Hz, 1H), 3.58 (dd, J=12.7, 8.0 Hz, 1H), 3.44 (d, J=12.2 Hz, 1H), 3.28 (s, 1H), 2.69 - 2.60 (m, 1H), 1.73 (dd, J=13.0, 6.2 Hz, 1H), 1.23 (d, J=12.9 Hz, 3H), 0.77 - 0.66 (m, 4H).

Examples 87, 88, 89, 90 and 91 were synthesized according to the procedure described for Example 86, except that in Step 7 different compounds were used in place of 86g, and in Step 10 different compounds were used in place of cyclopropanecarboxylic acid.

The characteristic data of Examples 87, 88, 89, 90 and 91 are presented below.

Example 92. cis-2-(5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethyl-1-oxohexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)acetonitrile

Step 1. 2-((3aR,6aS)-5-Benzyl-3a,6a-dimethyl-1,3-dioxohexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)acetonitrile (92a)

To a mixture of compound 51b (1.5 g, 5.4 mmol) and 2-aminoacetonitrile (500 mg, 5.4 mmol) in 1,2-dichloroethane (6 mL) was added TEA (1.6 g, 16.2 mmol). The resulting solution was heated to 60 °C and stirred for 12 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and the residue was purified by prep-HPLC to give the title compound 92a (1 g, 62%).

MS m/z (ESI): 298 [M+1]

Step 2. cis-2-(5-Benzyl-1-hydroxy-3a,6a-dimethyl-3-oxohexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)acetamide (92b)

To a solution of compound 92a (800 mg, 2.7 mmol) in EtOH (10 mL) at 0 °C was added sodium borohydride (260 mg, 6.7 mmol). The mixture was heated to reflux for 12 h. After cooling to room temperature, the residue was concentrated to dryness in vacuo. The residue was diluted _{with CH2Cl2} (10 mL) and filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 92b (900 mg). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 318 [M+1]

Step 3. cis-2-(5-Benzyl-3a,6a-dimethyl-1-oxohexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)acetamide (92c)

To a solution of compound 92b (900 mg, crude product) in TFA (8 mL) was added triethylsilane (4 mL). The solution was heated to 120 °C in a sealed tube and stirred for 48 h. After cooling to room temperature, the solution was concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 92c (130 mg, 16% over two steps).

MS m/z (ESI): 302 [M+1]

Step 4. cis-2-(3a,6a-Dimethyl-1-oxohexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)acetamide (92d)

To a solution of compound 92c (130 mg, 0.42 mmol) in MeOH (5 mL) was added 10% Pd/C (50 mg). The mixture was stirred under hydrogen atmosphere for 3 h and then filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 92d (90 mg, 100%).

MS m/z(ESI): 212 [M+1]

Step 5. cis-2-(5-(2,5-Dichloropyrimidin-4-yl)-3a,6a-dimethyl-1-oxohexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)acetamide (92e )

A mixture of compound 92d (90 mg, 0.42 mmol), 2,4,5-trichloropyrimidine (91 mg, 0.5 mmol) and DIEA (0.3 mL) in MeCN (2 mL) was heated to 50 °C and stirred for 12 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo and the residue was purified by prep-HPLC to give the title compound 92e (150 mg, 99%).

MS m/z (ESI): 358 [M+1]

Step 6. cis-2-(5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethyl-1-oxohexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)acetamide (92f)

A mixture of compound 92e (100 mg, 0.26 mmol), 1-methyl-1H-pyrazol-4-amine hydrochloride (40 mg, 0.29 mmol) and pTsOH (6 mg, 0.026 mmol) in isopropanol (4 mL) was heated to 120 °C and stirred for 1 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo to give the title compound 92f (150 mg). The crude product was used directly in the next step without further purification.

MS m/z (ESI): 419 [M+1]

Step 7. cis-2-(5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethyl-1-oxohexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)acetonitrile (92)

A mixture of 92f (150 mg, crude product) in phosphorus trichloride (2 mL) was heated to 100 °C for 12 h with stirring. After cooling to room temperature, the mixture was concentrated to dryness in vacuo and dissolved in MeCN (5 mL), to which TEA was slowly added until pH = 7. The mixture was concentrated to dryness in vacuo, and the residue was purified by prep-HPLC to give the title compound 92 (15 mg, solid, 14% over two steps).

MS m/z (ESI): 401 [M+1]

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.11 (br s, 1H), 7.91 (s, 1H), 7.70 (s, 1H), 7.44 (s, 1H), 4.44 (s, 2H), 4.15 (d, J=11.4 Hz, 1H), 3.81 (d, 1=11.8 Hz, 1H), 3.78 (s, 3H), 3.70 (d, 1=11.8 Hz, 1H), 3.65 (d, J=12.1 Hz, 1H), 3.43 (d, J=10.0 Hz, 1H), 3.34 (d, J=4.8 Hz, 1H), 1.18 (s, 3H), 1.09 (s, 3H).

Example 93. ((3aR,6aS)-5-(5-chloro-2-((5-methylisoxazol-3-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)(cyclopropyl)methanone

To a solution of compound 51g (0.25 g, 0.71 mmol) and 5-methylisoxazol-3-amine (83 mg, 0.85 mmol) in isopropanol (6 mL) was added pTsOH (270 mg, 1.42 mmol). The mixture was stirred at 100 °C for 18 h and then cooled to room temperature. The mixture was concentrated to dryness in vacuo, and the residue was purified by prep-HPLC to give the title compound 93 (51.3 mg, solid, 17%).

MS m/z (ESI): 417 [M+1]

^1H -NMR (400 MHz, DMSO- _d6 ) δ 9.91 (s, 1H), 7.97 (s, 1H), 6.65 (s, 1H), 3.92 - 3.66 (m , 5H), 3.59 (d, J=10.7 Hz, 1H), 3.47 (d, J=12.3 Hz, 1H), 3.29 (d, J=12.5 Hz, 1H ), 2.36 (s, 3H), 1.76 - 1.64 (m, 1H), 1.10 (d, J=12.9 Hz, 6H), 0.77 - 0.65 (m, 4H).

Examples 94 and 95 were synthesized according to the procedure described for Example 93, except that different compounds were used in place of 5-methylisoxazol-3-amine in Step 1.

Example 97. 3-((3aR,6aS)-5-(5-Methoxy-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

Step 1. (3aR,6aS)-2-Benzyl-5-(2-chloro-5-methoxypyrimidin-4-yl)-3a,6a-dimethyloctahydropyrrolo[3,4-c]pyrrole (97a)

To a solution of compound 51d (0.26 g, 1.13 mmol) in MeCN (10 mL) were added potassium carbonate (0.47 g, 3.39 mmol) and 2,4-dichloro-5-methoxypyrimidine (0.263 mg, 1.47 mmol). The mixture was stirred for 2 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 100/0 - 0/100) to give the title compound 97a (70 mg, 20%).

MS m/z (ESI): 373 [M+1]

Step 2. 4-((3aR,6aS)-5-Benzyl-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-methoxy-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (97b)

A mixture of compound 97a (70 mg, 0.188 mmol), 1-methyl-1H-pyrazol-4-amine (27.3 mg, 0.182 mmol) and pTsOH (71.4 mg, 0.376 mmol) in n-butanol (5 ml) was stirred at 120 °C for 8 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (CH ₂ Cl ₂ /MeOH = 9/1) to give the title compound 97b (70 mg, 86%).

MS m/z (ESI): 434 [M+1]

Step 3. 4-((3aR,6aS)-3a,6a-Dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-methoxy-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (97c)

To a solution of compound 97b (70 mg, 0.16 mmol) in MeOH (5 mL) was added 10% Pd/C (34 mg, 0.32 mmol). The mixture was stirred under hydrogen atmosphere for 3 h and then filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 97c (50 mg, 91%).

MS m/z (ESI): 344 [M+1]

Step 4. 3-((3aR,6aS)-5-(5-Methoxy-2-((1-methyl-1H-pyrazol-4-yl)amino)-pyrimidin-4-yl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile (97)

To a mixture of compound 97c (50 mg, 0.146 mmol), 2-cyanoacetic acid (25 mg, 0.292 mmol), DIEA (38 mg, 0.292 mmol) in DMF (1 mL) was added HATU (67 mg, 0.175 mmol). The mixture was stirred for 1 h and then purified by prep-HPLC to give the title compound 97 (31.9 mg, solid, 53%).

MS m/z (ESI): 411[M+1]

^1H -NMR (400 MHz, DMSO- _d6 ) δ 8.56 (s, 1H), 7.70 (s, 1H), 7.67 (s, 1H), 7.41 (s, 1H), 3.91 (d, J=1.1 Hz, 2H), 3.82 - 3.71 (m, 5H), 3.66 (s, 3H), 3.63 - 3.52 (m, 3H) , 3.47 (d, J=12.2 Hz, 1H), 3.39 (d, J=10.5 Hz, 1H), 3.31 (d, J=5.3 Hz, 1H), 1 .07 (t, J=7.5 Hz, 6H).

Example 98. 3-((3aS,6aR)-5-(2-((1H-pyrazol-4-yl)amino)-5-chloropyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

Step 1. 5-Chloro-4-((3aR,6aS)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-N-(1H-pyrazol-4-yl)pyrimidin-2-amine dihydrochloride (98a)

To a solution of compound 86j (4.00 g, 10.72 mmol) in EtOH (50 mL) were added 1H-pyrazol-4-amine (1.16 g, 13.94 mmol) and pTsOH monohydrate (0.21 g, 1.07 mmol). The mixture was heated to 70 °C and stirred for 16 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and a solution of HCl (10 N in ethanol, 50 mL) was added. The mixture was heated to 50 °C and stirred for an additional 1 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo, and a mixture of methanol and 2-methoxy-2-methylpropane (60 mL, 1/1 v/v) was added. The resulting mixture was stirred for 30 min and then filtered. The filter cake was collected and dried to give the title compound 98a (3.4 g, 81%) as a hydrochloride salt.

MS m/z (ESI): 320 [M+1]

Step 2. 3-((3aS,6aR)-5-(2-((1H-pyrazol-4-yl)amino)-5-chloropyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile hemiformate (98)

To a solution of compound 98a (3.30 g, 8.40 mmol) in methanol (40 mL) were added ethyl cyanoacetate (1.90 g, 16.80 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (5.10 g, 33.60 mmol). The mixture was stirred for 5 h and concentrated to dryness in vacuo. The residue was purified by prep-HPLC to give the title compound 98 (1.04 g, solid, 30%) as a formate.

MS m/z (ESI): 387 [M+1]

Specific rotation =-4.6°× ^dm2 /kg

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.50 (s, 1.5H), 9.07 (s, 1H), 8.13 (s, 0.5H), 7.89 (s , 1H), 7.65 (s, 2H), 4.09 - 3.99 (m, 1H), 3.92 (d, J=2.7 Hz, 2H), 3.84 (dd, J= 26.5, 11.3 Hz, 1H), 3.76 - 3.72 (m, 1H), 3.69 (d, J=11.1 Hz, 1H), 3.64 (dd, J=12 ,5, 7.3 Hz, 1H), 3.49 (dd, J=22.9, 11.3 Hz, 1H), 3.38 (dd, J=15.7, 7.9 Hz, 1H), 3.29 (d, J=12.3 Hz, 1H), 2.57 (d, J=33.9 Hz, 1H), 1.21 (d, J=2.7 Hz, 3H).

Example 99. 3-((3aS,6aR)-5-(5-Chloro-2-((1-(methyl-d3)-1H-pyrazol-4-yl)amino)-pyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

Step 1. 1-(Methyl-d3)-4-nitro-1H-pyrazole (99b)

To a solution of 4-nitro-1H-pyrazole 99a (2.5 g, 22.11 mmol) in MeCN (20 mL) were added iodomethane-d ₃ (3.85 g, 26.53 mmol) and potassium carbonate (4.58 g, 33.17 mmol). The mixture was heated to 70 °C and stirred for 12 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo. The residue was added water (50 mL) and extracted with CH ₂ Cl ₂ (3×50 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 4/1) to give the title compound 99b (2.50 g, 87%).

MS m/z (ESI): 131 [M+1]

Step 2. 1-(Methyl-d3)-1H-pyrazol-4-amine (99c)

To a solution of compound 99b (2.50 g, 19.2 mmol) in MeOH (30 mL) was added 10% Pd/C (0.5 g). The mixture was stirred under hydrogen atmosphere for 16 h and then filtered. The filtrate was concentrated to dryness in vacuo to give the title compound 99c (1.92 g, 100%).

MS m/z (ESI): 101 [M+1]

Step 3. 5-Chloro-N-(1-(methyl-d3)-1H-pyrazol-4-yl)-4-((3aR,6aS)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-amine dihydrochloride (99d)

To a solution of compound 86j (3.00 g, 8.04 mmol) in EtOH (50 mL) were added compound 99c (1.05 g, 10.45 mmol) and pTsOH monohydrate (0.15 g, 0.80 mmol). The mixture was heated to 70 °C and stirred for 8 h. After cooling to room temperature, ethanolic HCl solution (10 N, 50 mL) was added to the mixture. The mixture was again heated to 50 °C and stirred for 1 h. After cooling to room temperature, the mixture was concentrated to dryness in vacuo. To the residue was added a mixture of methanol and 2-methoxy-2-methylpropane (60 mL, 1/1 v/v). After stirring for 30 min, the mixture was filtered. The filter cake was collected and dried to give the title compound 99d (3.3 g, 100%) as a hydrochloride salt.

MS m/z (ESI): 337 [M+1]

Step 4. 3-((3aS,6aR)-5-(5-Chloro-2-((1-(methyl-d3)-1H-pyrazol-4-yl)amino)-pyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile (99)

To a mixture of compound 99d (3.30 g, 8.05 mmol) in MeCN (35 mL) was added ethyl cyanoacetate (1.82 g, 16.10 mmol) and DBU (4.90 g, 32.2 mmol). The mixture was heated to 30 °C and stirred for 4 h. The mixture was concentrated to dryness in vacuo and then EtOH (20 mL) was added. After stirring for 1 h, the mixture was filtered and the filter cake was collected to give the title compound 99 (2.60 g, solid, 80%).

MS m/z (ESI): 404 [M+1]

Specific rotation =-7.2°× ^dm2 /kg

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.07 (s, 1H), 7.89 (s, 1H), 7.71 (s, 1H), 7.44 (s, 1H), 4.03 (dd, J=11.2, 5.9 Hz, 1H), 3.93 (d, J=3.0 Hz, 2H), 3.84 (dd, J=18.6, 11, 5 Hz, 1H), 3.73 (d, J=10.2 Hz, 1H), 3.69 (s, 1H), 3.64 (dd, J=12,1,7,1 Hz, 1H) , 3.49 (dd, J=27.8, 11.2 Hz, 1H), 3.42 - 3.33 (m, 1H), 3.33 - 3.24 (m, 1H), 2.57 (d, J=31.9 Hz, 1H), 1.20 (t, J=8.1 Hz, 3H).

Example 100. trans-3-(5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile

Step 1. Dimethyl trans-1-benzyl-3-methylpyrrolidine-3,4-dicarboxylate (100b)

To a mixture of dimethyl 2-methyl fumarate 100a (9.63 g, 60.89 mmol) and TFA (0.69 g, 6.09 mmol) in CH ₂ Cl ₂ (10 mL) at 0 °C was slowly added a solution of N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (17.35 g, 73.07 mmol) in CH ₂ Cl ₂ (200 mL). After the addition, the resulting yellow solution was gradually warmed to room temperature over 16 h, and then saturated aqueous sodium bicarbonate solution (200 mL) and CH ₂ Cl ₂ (200 mL) were added. The mixture was stirred for 15 min. The separated organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness in vacuo to give a residue, which was purified by column chromatography on silica gel (CH ₂ Cl ₂ /MeOH = 100/3) to give the title compound 100b (17.74 g, 100%).

MS m/z (ESI): 292 [M+1]

Step 2. 1-(tert-Butyl)-3,4-dimethyl-trans-3-methylpyrrolidine-1,3,4-tricarboxylate (100c)

To a mixture of compound 100b (17.74 g, 60.79 mmol) and di-tert-butyl pyrocarbonate (14.62 g, 66.67 mmol) in MeOH (200 mL) was added 10% Pd/C (1.8 g). The resulting mixture was heated to 50 °C under hydrogen atmosphere and stirred for 20 h. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/1) to give the title compound 100c (18.00 g, 98%).

MS m/z (ESI): 202 [M+1-100]

Step 3. tert-Butyl trans-3,4-bis(hydroxymethyl)-3-methylpyrrolidine-1-carboxylate (100d)

To a solution of compound 100c (18.00 g, 22.75 mmol) in THF (200 mL) at 0 °C was added lithium borohydride solution (2 M in THF, 65 mL, 131.41 mmol). After stirring for 30 min, the mixture was gradually warmed to room temperature and stirred for an additional 16 h. The mixture was again cooled to 0 °C, and 1 N HCl was slowly added until pH = 1. The resulting solution was extracted with CH ₂ Cl ₂ (400 mL). The organic phase was washed with water and brine in that order, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2/1) to give the title compound 100d (14.00 g, 96%).

MS m/z (ESI): 190 [M+1-56]

Step 4. tert-Butyl trans-3-methyl-3,4-bis(((methylsulfonyl)oxy)methyl)-pyrrolidine-1-carboxylate (100e)

To a solution of compound 100d (14.00 g, 57.07 mmol) and DIEA (44.00 g, 342.41 mmol) in _CH2Cl2 ( ₂₀₀ mL) at 0 °C was added MsCl (20.00 g, 171.21 mmol) dropwise. The mixture was stirred for 1 h, and then saturated aqueous ammonium chloride solution (100 mL) and _CH2Cl2 ( ₂₀₀ mL) were added. The mixture was stirred for 10 min and left to stand. The separated organic layer was washed with saturated brine and saturated sodium bicarbonate solution in that order, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10/1) to give the title compound 100e (8.00 g, 35%).

MS m/z (ESI): 346 [M+1-56]

Step 5. tert-Butyl trans-5-benzyl-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (100f)

A mixture of compound 100e (8.00 g, 19.93 mmol), TEA (12.10 g, 119.55 mmol), and benzylamine (8.54 g, 79.72 mmol) in toluene (100 mL) was heated to reflux for 16 h. After cooling to room temperature, 1N NaOH (100 mL) and saturated brine were added to the reaction mixture. The mixture was stirred for 10 min, and the separated organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 5/1) to give the title compound 100f (3.00 g, 48%).

MS m/z(ESI): 317 [M+1]

Step 6. tert-Butyl-trans-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (100g)

To a solution of compound 100f (3.00 g, 9.48 mmol) in MeOH (100 mL) was added 20% Pd/C (0.6 g). The mixture was heated to 50 °C under hydrogen atmosphere and stirred for 12 h. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated to dryness in vacuo to give the title compound 100g (2.15 g, 100%).

MS m/z (ESI): 227 [M+1]

Step 7. tert-Butyl trans-5-(2,5-dichloropyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (100h)

To a solution of compound 100g (2.15 g, 9.50 mmol) in ethyl acetate (40 ml) at 0°C were added DIEA (1.47 g, 11.40 mmol) and 2,4,5-trichloropyrimidine (1.74 g, 9.50 mmol). The mixture was stirred for 1 h and then concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10/3) to give the title compound 100h (3.30 g, 93%).

MS m/z (ESI): 373 [M+1]

Step 8. trans-5-Chloro-N-(1-methyl-1H-pyrazol-4-yl)-4-(3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidin-2-amine dihydrochloride (100i)

To a solution of compound 100h (3.30 g, 8.84 mmol) in ethanol (40 mL) were added 1-methyl-1H-pyrazol-4-amine hydrochloride (1.54 g, 11.49 mmol) and pTsOH monohydrate (0.17 g, 0.88 mmol). The mixture was heated to 70 °C and stirred for 16 h. After cooling to room temperature, the mixture was concentrated to dryness, and a mixture of 2-methoxy-2-methylpropane and methanol (40 mL, 1/1 v/v) was added thereto. After stirring for 30 min, the mixture was filtered and the filter cake was collected to give the title compound 100i (3.15 g, 88%) as a hydrochloride salt.

MS m/z (ESI): 334 [M+1]

Step 9. trans-3-(5-(5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3a-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanenitrile (100)

To a mixture of compound 100i (2.00 g, 4.92 mmol) in acetonitrile (40 mL) was added ethyl 2-cyanoacetate (1.11 g, 9.84 mmol) and DBU (3.74 g, 24.60 mmol). The mixture was heated to 30 °C and stirred for 2 h. The mixture was concentrated to dryness, ethanol (30 mL) was added, and stirred at room temperature for 1 h. The mixture was filtered and the filter cake was collected to give the title compound 100 (1.15 g, solid, 58%).

MS m/z (ESI): 401 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.10 (s, 1H), 7.90 (s, 1H), 7.70 (s, 1H), 7.46 (s, 1H), 4.00 - 3.90 (m, 3H), 3.78 (s, 3H), 3.69 - 3.47 (m, 4H), 3.42 - 3.33 (m, 1H), 3.28 (dd, J=11.9, 9.3 Hz, 1H), 3.09 (dd, J=20.0, 10.8 Hz, 1H), 2.40 (dd, J=24.7, 17.6 Hz, 1H), 0.94 (d, J=8.8 Hz, 3H).

Example 101. Biological experiments

JAK2 inhibition assay

The effect of the compounds of the present invention on the enzymatic activity of JAK2 was assessed by determining the phosphorylation level of the substrate in the kinase reaction using the HTRF Kinase Activity Detection Assay Kit (Cisbio, Cat. No. 62TK0PEC).

The experimental method is described below in general terms:

The reaction buffer contained the following components: enzyme reaction buffer (1×), 5 mM _MgCl2 , 1 mM DTT, and 0.01% Brij35 from the kit. JAK2 kinase solution contained recombinant human JAK2 protein (Carna Biosciences, 08-045) diluted with reaction buffer to 0.15 ng/μl. Substrate reaction solution contained 2.5 μM ATP and 0.25 μM biotinylated tyrosine kinase substrate from the kit in reaction buffer. Detection solution contained 0.1 ng/μl ^Eu3+ -labeled cage antibodies (Cisbio, 61T66KLB) and 12.5 nM streptavidin-labeled XL665 (Cisbio, 610SAXLB) in reaction buffer.

The test compound was dissolved in DMSO to 10 μM, followed by serial 4-fold dilutions with DMSO to a minimum concentration of 0.061 nM. Each concentration was further diluted 40-fold with reaction buffer.

In a 384-well assay plate (Corning, 3674), 4 μl of the compound solution and 2 μl of the JAK2 kinase solution were added. The mixture was mixed and incubated at room temperature for 15 min, and then 4 μl of the reaction substrate solution were added. After an additional incubation at room temperature for 30 min, an equal volume of 10 μl of the detection solution was added to the reaction mixture and left to stand at room temperature for 30 min. Then, an Envision plate reader (Perkin Elmer) was used to measure the reaction progress at 620 nm and 665 nm. The ratio of the absorbance values at 665 nm and 620 nm positively correlated with the degree of substrate phosphorylation, thereby revealing the activity of JAK2 kinase. In the experiment, the group without JAK2 kinase protein was considered as 100% inhibition group, and the group with JAK2 kinase protein but without test compound was considered as 0% inhibition group. The percentage inhibition of JAK2 kinase activity by the test compound was calculated using the following formula:

Percent inhibition = 100 - 100 × (degree _{of compound} - degree _{of 100% inhibition} ) / (degree of _{0% inhibition} - degree of _{100% inhibition} )

The IC ₅₀ value of the test compound was calculated from 8 concentration points using XLfit software (ID Business Solutions Ltd., UK) using the following formula:

Y = Minimum value + (Maximum value - Minimum value) / (1+10^((log IC ₅₀ - X) × Slope coefficient)),

where Y was the percentage inhibition, X was the logarithm of the concentration of the test compound, “Minimum Value” was the value of the minimum plateau of the S-shaped curve, “Maximum Value” was the value of the maximum plateau of the S-shaped curve, and “Slope Factor” was the slope factor of the curve.

TYK2 inhibition assay method

The effect of the compounds of the present invention on the enzymatic activity of TYK2 was assessed by determining the phosphorylation level of the substrate in the kinase reaction using the HTRF Kinase Activity Detection Assay Kit (Cisbio, Cat. No. 62TK0PEC).

The experimental method is described below in general terms:

The reaction buffer contained the following components: enzyme reaction buffer (1×), 5 mM _MgCl2 , 1 mM DTT, and 0.01% Brij35 from the kit. TYK2 kinase solution contained recombinant human TYK2 protein (Carna Biosciences, 08-147) diluted with reaction buffer to 0.25 ng/μl. Substrate reaction solution contained 11.25 μM ATP and 0.5 μM biotinylated tyrosine kinase substrate from the kit in reaction buffer. Detection solution contained 0.1 ng/μl ^Eu3+ -labeled cage antibodies (Cisbio, 61T66KLB) and 12.5 nM streptavidin-labeled XL665 (Cisbio, 610SAXLB) in reaction buffer.

The test compound was dissolved in DMSO to 10 μM, followed by serial 4-fold dilutions with DMSO to a minimum concentration of 0.061 nM. Each concentration was further diluted 40-fold with reaction buffer.

In a 384-well assay plate (Corning, 3674), 4 μl of the compound solution and 2 μl of the TYK2 kinase solution were added. The mixture was mixed and incubated at room temperature for 15 minutes, and then 4 μl of the reaction substrate solution were added. After an additional incubation at room temperature for 40 minutes, an equal volume of 10 μl of the detection solution was added to the reaction mixture and left to stand at room temperature for 30 minutes. Then, an Envision plate reader (Perkin Elmer) was used to measure the reaction progress at 620 nm and 665 nm. The ratio of the absorbance values at 665 nm and 620 nm positively correlated with the degree of substrate phosphorylation, thereby revealing the activity of TYK2 kinase. In the experiment, the group without TYK2 kinase protein was considered as 100% inhibition group, and the group with TYK2 kinase protein but without test compound was considered as 0% inhibition group. The percentage inhibition of TYK2 kinase activity by the test compound was calculated using the following formula:

Percent inhibition = 100 - 100 × (extent _{of compound} - extent of _{100% inhibition} ) / (extent of _{0% inhibition} - extent of _{100% inhibition} )

The IC ₅₀ value of the test compound was calculated from 8 concentration points using XLfit software (ID Business Solutions Ltd., UK) using the following formula:

Y = Minimum value + (Maximum value - Minimum value) / (1+10^((log IC ₅₀ - X) × Slope coefficient)),

where Y was the percentage inhibition, X was the logarithm of the concentration of the test compound, “Minimum Value” was the value of the minimum plateau of the S-shaped curve, “Maximum Value” was the value of the maximum plateau of the S-shaped curve, and “Slope Factor” was the slope factor of the curve.

The compounds of the present invention have a significant TYK2 activity inhibitory effect, preferably with an IC ₅₀ value of 10 to 100 nM, and more preferably with an IC ₅₀ value of less than 10 nM.

Inhibition of IL-12-induced IFN-γ secretion in NK92 cells

The effect of the compounds of the present invention on IL-12-induced IFN-γ secretion in NK92 cells was assessed by enzyme-linked immunosorbent assay (ELISA).

The IL-12 receptor is expressed primarily in activated T cells, NK cells (NK92 is an NK cell line), DC cells, and B cells. When bound to IL-12, it activates the JAK2/TYK2 signaling pathway in NK cells and T lymphocytes, thereby inducing IFN-γ secretion.

The experimental method is described below in general terms:

The test compound was dissolved in DMSO to 2.5 mM, followed by serial 4-fold dilutions with DMSO to a minimum concentration of 0.31 nM. Each concentration was further diluted 50-fold with FBS-free MEMα medium (Gibco, 12561-056).

NK92 cells (Nanjing Cobioer, CBP60980) were cultured in complete MEMα medium containing 12.5% FBS (Ausbian, VS500T), 12.5% horse serum (Gibco, 16050-122), 0.02 mM folic acid (Sigma, F8758), 0.2 mM inositol (Sigma, 17850), 0.55 mM β-mercaptoethanol (Gibco, 21985-023), 200 units/ml IL-2 (R&D Systems, 202-1L) and 100 units/ml penicillin (Thermofisher, 15140122). When 80-90% of the culture container surface was covered, the cells were dispersed and plated into a 96-well plate (Thermofisher, 167425) at 100,000 cells per well (80 µl complete MEM medium without IL-2). The 96-well plate was then incubated overnight in an incubator at 37°C/5% CO ₂ .

After overnight incubation, 10 μl of the test compound and 10 μl of 50 ng/ml IL-12 (R&D Systems, 219-1L) were added to each well, mixed gently, and the 96-well plate was incubated in an incubator at 37°C/5% _CO2 for an additional 24 h. The plate was centrifuged at 800 rpm for 10 min at room temperature, and 50 μl of the supernatant from each well was transferred to another 96-well plate (Sigma, CLS3695) coated with anti-IFN-γ antibodies. The amount of IFN-γ secretion was determined following the instruction of the human IFN-γ DuoSet ELISA kit (R&D Systems, DY285B). In the experiment, the group in which IL-12 and the test compound were replaced by MEM medium was the control group without stimulation (100% inhibition), and the group treated with IL-12 and 0.2% DMSO was the stimulation group (0% inhibition). The percentage inhibition of IL-12-induced IFN-γ secretion in NK-92 cells by the test compound was calculated using the following formula:

Percent inhibition = 100 - 100 × (signal _connection - signal _{without stimulation} ) / (signal _stimulation - signal _{without stimulation} )

The IC ₅₀ value of the test compound was calculated from 8 concentration points using XLfit software (ID Business Solutions Ltd., UK) using the following formula:

Y = Minimum value + (Maximum value - Minimum value) / (1+10^((log IC ₅₀ - X) × Slope coefficient)),

where Y was the percentage inhibition, X was the logarithm of the concentration of the test compound, “Minimum Value” was the value of the minimum plateau of the S-shaped curve, “Maximum Value” was the value of the maximum plateau of the S-shaped curve, and “Slope Factor” was the slope factor of the curve.

The compounds of the present invention have a significant effect of inhibiting IFN-γ secretion in NK92 cells induced by IL-12, and the IC ₅₀ value is preferably less than 1000 nM.

Claims (37)

1. A compound of formula (I) or a pharmaceutically acceptable salt, stable isotope, stereoisomer thereof: Where: R ¹ is pyrazolyl, wherein one hydrogen of the pyrazolyl is optionally substituted with -COOR ^b , -C(O)R ^b , -C(O)NR ^b R ^c , C _1-6 alkyl, CD ₃ or piperidinyl; R ² is H; R ³ is H, halogen, cyano, C _1-6 alkyl, or OC _1-6 alkyl; R ⁴ and R ⁵ are independently selected from H or C _1-6 alkyl; R ⁶ and R ⁷ are independently selected from H, or R ⁶ and R ⁷ are combined as oxo; L represents a bond, C _1-6 alkylene, -C(O)-, -C(O)O-, -C(O)N(R ^a )-, or -S(O) ₂ -; A is H, C _1-6 alkyl, C _3-6 cycloalkyl, 4-membered heterocyclyl in which one ring atom is N or O, or 5-6-membered heteroaryl in which from 1 to 3 ring atoms are N or O heteroatoms, wherein one or more hydrogen atoms of alkyl, cycloalkyl and heteroaryl are optionally substituted with halogen, cyano or -OR ^d ; R ^a represents H; R ^b and R ^c are independently selected from H or C _1-6 alkyl, and R ^d is independently selected from H, C _1-6 alkyl or morpholinyl. 2. The compound according to claim 1, which is a compound of formula (II) or a pharmaceutically acceptable acid salt, stable isotope, stereoisomer thereof: Where: L represents a bond, C _1-6 alkylene, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O) ₂ -. 3. The compound according to claim 1 or 2, wherein L is -C(O)-. 4. A compound according to claim 1 or 2, wherein A is C _1-6 alkyl or C _3-6 cycloalkyl, wherein one or more hydrogen atoms of the alkyl and cycloalkyl are optionally substituted with halogen, cyano or -OC _1-2 alkyl. 5. The compound according to claim 2, which is a compound of formula (III) or a pharmaceutically acceptable acid salt, stable isotope, stereoisomer thereof: Where: L represents a bond, C _1-6 alkylene, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O) ₂ -; R ¹¹ is H, COOR ^b , -C(O)R ^b , -C(O)NR ^b R ^c , C _1-6 alkyl or piperidinyl. 6. A compound according to claim 5, wherein A is C _1-6 alkyl or C _3-6 cycloalkyl, wherein one or more hydrogen atoms of the alkyl and cycloalkyl are optionally substituted with halogen, cyano or -OC _1-2 alkyl. 7. A compound according to claim 5, wherein R ¹¹ is C _1-6 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with D. 8. A compound having the following structure, or a pharmaceutically acceptable salt, stable isotope, stereoisomer thereof: or (the stereochemistry of carbon atoms marked * is not determined). 9. The compound according to item 8, which is: 10. The compound according to claim 8, which is: 11. A pharmaceutical composition for the prevention or treatment of a disease mediated by tyrosine kinase 2 (TYK2), comprising a therapeutically effective amount of a compound according to any one of claims 1-10 and a pharmaceutically acceptable carrier or excipient thereof.