Patent Application
20240336594
The present invention belongs to the field of medicine, and specifically relates to a six-membered aryl or heteroaryl amide, and composition and use thereof.
MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), as a key mediator of the classic NF-κB signaling pathway, is currently the only paracaspase known in humans that transduces signals from B cell receptors (BCR) and T cell receptors (TCR). The engagement of TCR/BCR results in the assembly of CARD11-BCL10-MALT1 (CBM) complex, which is composed of three proteins: CARD11 (caspase recruitment domain family member 11), BCL10 (B-cell lymphoma factor 10), and MALT1, to trigger the downstream activation. MALT1 affects NF-κB signaling through two mechanisms: first, MALT1 functions as a scaffolding protein, recruiting NF-κB signaling proteins such as TRAF6, TAB-TAK1 or NEMO-IKKα/μ; second, MALT1 use its protease function to cleave and inactivate negative regulators of NF-κB signaling, such as RelB, A20 or CYLD. The caspade signaling ultimately leads to the nuclear translocation of the NF-κB transcription factor complex and the activation of genes involved in proliferation, apoptosis inhibition, and inflammation (Jaworski et al., Cell Mol Life Sci, 2016, Vol. 73, 459-473).
Sustained activation of the NF-κB signaling pathway is a hallmark of ABC-DLBCL (activated B-cell-like subtype of diffuse large B-cell lymphoma). The ABC subtype is a more aggressive form of DLBCL. DLBCL is the most common form of non-Hodgkin lymphoma (NHL), accounting for approximately 25% of lymphoma cases, while ABC-DLBCL accounts for approximately 40% of DLBCL cases. In ABC-DLBCL patients, NF-κB pathway activation is driven by mutations in CD79A/B, CARD11, MYD88 or A20 (Staudt, ColdSpring Harb Perspect Biol, 2010, Vol. 2; Lim et al., Immunol Rev, 2012 Vol. 246, 359-378). Small molecule tool compound inhibitor of MALT1 protease has proven effective in preclinical models of ABC-DLBCL (Fontan et al., Cancer Cell, 2012, Vol. 22, 812-824; Nagel et al., Cancer ell, 2012, Vol. 22, 825-837).
In addition to lymphoma, MALT1 has also been shown to play a crucial role in both innate and adaptive immunity (Jaworski M et al., Cell Mol Life Sci., 2016). MALT1 protease inhibitors showed the anti-inflammatory activity by slowing down the disease onset and progression of experimental allergic encephalomyelitis (mouse model of multiple sclerosis) in mice (Mc Guire et al., J. Neuroinflammation, 2014, Vol. 11, 124). Furthermore, MALT1 knockout mice have fewer Tregs with Teff cell function severely impaired, and the immunity is generally compromised. In contrast, only knockout of the protease domain of MALT1 significantly diminishes Tregs but preservers the functions of Teff cell. Therefore, MALT1 protease inhibitors can benefit patients with solid tumors by regulating Treg function and promoting the body's immune response (Arianna Bertossi et al., EMBO J, 2014, Vol. 33, 2740-2742).
Therefore, the MALT1 inhibitors of the present invention may provide beneficial therapeutic effects to patients suffering from cancer and/or immune diseases.
The present invention relates to a six-membered aryl or heteroaryl amide and its composition and use, which can be used as an inhibitor of MALT1 for the treatment of cancer and/or immune-related diseases.
A first object of the present invention is to provide a compound of Formula I, or a stereoisomer, tautomer, deuterated compound, pharmaceutically acceptable salt, prodrug, chelate, non-covalent complex or solvate thereof,
wherein,
is not phenyl.
In some embodiments of Formula I,
is selected from
In some embodiments of Formula I,
is selected from
In some embodiments of Formula I, R1, R3 are the same or different, and are each independently selected from the group consisting of halogen, —C1-6 alkyl, —OR5, —NR5R6, —C0-6 alkyl —C3-8 carbocyclyl and —C0-6 alkyl-3-8 membered heterocyclyl; wherein the —C1-6 alkyl, —C0-6 alkyl —C3-8 carbocyclyl, —C0-6 alkyl-3-8 membered heterocyclyl, R5 and R6 are optionally substituted with one or more substituents selected from hydrogen, halogen, CN, OH, NH2, —C1-6 alkyl, —C2-6 alkenyl, —C2-6 alkynyl, —C1-6 alkoxy, —C0-6 alkyl —C3-8 carbocyclyl or —C0-6 alkyl-3-8 membered heterocyclyl.
In some embodiments of Formula I, R2, R4 are the same or different, and are each independently selected from the group consisting of halogen, —C1-6 alkyl, —OR5, —NR5R6, —C0-6 alkyl —C3-8 carbocyclyl and —C0-6 alkyl-3-8 membered heterocyclyl; wherein the —C1-6 alkyl, —C0-6 alkyl —C3-8 carbocyclyl, —C0-6 alkyl-3-8 membered heterocyclyl, R5 and R6 are optionally substituted with one or more substituents selected from hydrogen, halogen, CN, OH, NH2, —C1-6 alkyl, —C2-6 alkenyl, —C2-6 alkynyl, —C1-6 alkoxy, —C0-6 alkyl —C3-8 carbocyclyl or —C0-6 alkyl-3-8 membered heterocyclyl.
In some embodiments of Formula I, R1, R2, R3, R4 are the same or different, and are each independently selected from the group consisting of H, —F, —Cl, —Br, —I, —OH, —NH2, —CN, —CH3, —CF3, —CH2CH3, —CH(CH3)2, —CH2CH(CH3)2, —C(CH3)3, —NHCH(CH3)2, —NHC(O)CH3, —NHC(O)OCH2CH3,
OCH3, —OCH(CH3)2,
In some embodiments of Formula I,
is selected from
In some embodiments of Formula I, ring A is selected from —C6 carbocyclyl, membered heterocyclyl, —C6 aryl or 6 membered heteroaryl.
In some embodiments of Formula I, ring A is selected from Phenyl, pyridyl, furyl, thienyl, pyrrolyl, pyrazolyl, pyrimidinyl, pyridazinyl, diazinyl, cyclopentenyl, pyridin-2(1H)-one or pyrimidine-2(1H)-keto group, etc.
In some embodiments of Formula I, ring A is
wherein Y1 is selected from CRA or N; Y2 is selected from CRA or N; Y3 is selected from CRA or N; Y4 is selected from CRA or N; RA is defined as described for the compound represented by formula I above.
In some embodiments of Formula I, each RA is the same or different, and is independently selected from the group consisting of hydrogen, oxo, —F, —Cl, —Br, —I, —CN, —OH, —NH2, —NO2, —CH3, —CF3, —OCF3, —NHCH3, —N(CH3)2, —OCH3, —SCH3, —CHF2, —OCHF2, —C(O)NH2, —C(O)CH3, —OC(O)NH2, —NHC(O)CH3, —CH2CH3, —CH(CH3)2,
—SCH3, —SOCH3, —CH2SO2CH3, —SO2CH3, —NHC(O)NH2, —CH2OH, —CH2NH2, —NHC(O)OCH2CH3, —NHOH, —CH2OCH3,
In some embodiments of Formula I,
is selected from
In some embodiments of Formula I, ring E is selected from 6-10 membered heterocyclyl or 6-10 membered heteroaryl.
In some embodiments of Formula I, ring E is selected frompyridyl, benzo[d]oxazolyl, 3,4-dihydro-2H-benzo[b][1,4]oxazinyl, 1,6-naphthyridinyl, 2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazinyl, 1H-pyrazolo[4,3-b]pyridyl, 2H-pyrazolo[4,3-b]pyridyl, 1H-pyrazolo[3,4-b]pyridyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, thiazolo[5,4-b] pyridyl or 1,5-naphthyridinyl.
In some embodiments of Formula I, each RE is the same or different, and is independently selected from the group consisting of hydrogen, halogen, oxo, CN, —C1-6 alkyl, —C0-6 alkyl-5-10 membered heteroaryl, —C0-6 alkyl —C3-8 carbocyclyl, —C0-6 alkyl-3-8 membered heterocyclyl, —OR5, —NR5R6, —SR5, —C(O)R5, —S(O)R5, —S(O)2R5 and —C(O)OR5; wherein the —C1-6 alkyl, —C0-6 alkyl-5-10 membered heteroaryl, —C0-6 alkyl —C3-8 carbocyclyl and —C0-6 alkyl-3-8 membered heterocyclyl are optionally substituted with one or more substituents selected from hydrogen, halogen, CN, oxo, —C1-6 alkyl, —C2-6 alkenyl, —C2-6 alkynyl, —OR8, —NR8R9, —C0-6 alkyl —C3-8 carbocyclyl and —C0-6 alkyl-3-8 membered heterocyclyl, —C(O)OR8 or —C(O)NR8R9.
In some embodiments of Formula I, each RE is the same or different, is independently selected from the group consisting of hydrogen, oxo, —F, —Cl, —CN, —CH3, —OCH3, —CH2OH, —CH2CN, —CF3, —C(O)NH2, —C(O)NHCH3, —C(O)NHCH2CN, —C(O)NHCH2CH3, —C(O)NHCH2CF3, —C(O)NHCH2CH2OCF3, —C(O)NHCH2CH2CH3, —OCHF2, —OCF3, —SO2CH3, —SCH3, —SCF3, —NHC(O)NH2, —NHC(O)CH3, —NHC(O)OCH3, —NHS(O)2CH3,
In some embodiments of Formula I,
is selected from
preferably,
is selected from
more preferably,
is selected from
Wherein RE and e are defined as described in any embodiment of the present invention.
In some embodiments of Formula I
is selected from
In some embodiments of Formula I, the aforementioned —C3-8 carbocyclyl, 3-8 membered heterocyclyl can be respectively preferably —C3-6 carbocyclyl, 3-6 membered heterocyclyl.
In some embodiments, the compound of Formula I is selected from Formula II:
wherein, ring E, X1, X3, Y1, Y3, Y5, RE, e are defined as described in any embodiment of the invention.
In some embodiments, the compound of Formula I is selected from III:
wherein, ring E, X2, X4, Y1, Y4, Y5, RE, e are defined as described in any embodiment of the invention.
In some embodiments of Formula I, the compound is selected from:
A second object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of at least a compound of Formula I, or a stereoisomer, tautomer, deuterated compound, pharmaceutically acceptable salt, prodrug, chelate, non-covalent complex, or solvate thereof as an active ingredient, and at least one pharmaceutically acceptable ingredient, such as carrier or excipient.
A third object of the present invention is to provide use of a compound of Formula I or a stereoisomer, tautomer, deuterated compound, pharmaceutically acceptable salt, prodrug, chelate, non-covalent complex, or solvate thereof for the manufacture of a medicament for the treatment of diseases, syndromes, disorders or obstacles.
In some embodiments, the diseases, syndromes, disorders and obstacles are affected by MALT1 inhibition.
In some embodiments, the diseases, syndromes, disorders and obstacles are selected from diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), mucosa-associated lymphoid tissue (MALT) lymphoma, rheumatoid arthritis (RA), psoriatic arthritis (PsA), psoriasis (Pso), ulcerative colitis (UC), Crohn's disease, systemic lupus erythematosus (SLE), asthma and chronic obstructive pulmonary disease (COPD).
A forth object of the present invention is to provide a method of treating diseases, syndromes, disorders or obstacles, wherein the diseases, syndromes, disorders and obstacles are affected by MALT1 inhibition, comprising administering to a patient in need the compound of Formula I, or a stereoisomer, tautomer, deuterated compound, pharmaceutically acceptable salt, prodrug, chelate, non-covalent complex, or solvate thereof.
In some embodiments, the diseases, syndromes, disorders and obstacles are selected from diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), mucosa-associated lymphoid tissue (MALT) lymphoma, rheumatoid arthritis (RA), psoriatic arthritis (PsA), psoriasis (Pso), ulcerative colitis (UC), Crohn's disease, systemic lupus erythematosus (SLE), asthma and chronic obstructive pulmonary disease (COPD).
The general chemical terms used in the formula above have their usual meanings. For example, the term “halogen”, as used herein, unless otherwise indicated, means fluoro, chloro, bromo or iodo.
As used herein, unless otherwise indicated, alkyl includes saturated monovalent hydrocarbon radicals having straight, or branched moieties. For example, alkyl radicals include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, 3-(2-methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-hexyl, and 2-methylpentyl. The alkyl group is preferably a C1-8 alkyl, the C1-8 alkyl is further preferably a C1-6 alkyl, the C1-6 alkyl is further preferably a C1-3 alkyl. Among them, C1-8, as in C1-8 alkyl is defined to identify the group as having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms in a linear or branched arrangement.
Alkoxy radicals are oxygen ethers formed from the previously described straight, branched chain or cyclic alkyl groups.
Alkenyl and alkynyl groups include straight, branched chain or cyclic alkenes and alkynes. Likewise, “C2-8 alkenyl” and “C2-8 alkynyl” means an alkenyl or alkynyl radicals having 2, 3, 4, 5, 6, 7 or 8 carbon atoms in a linear or branched arrangement. For example, alkenyl radicals include ethenyl, propenyl, etc. For example, alkynyl radicals include ethynyl, propynyl, etc.
Unless otherwise stated, the term “heteroaryl” as used herein refers to a monocyclic or polycyclic (e.g., fused bicyclic) aromatic heterocycle having at least one heteroatom ring member selected from the group consisting of N, O, and/or S. and wherein the nitrogen or sulfur heteroatom may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroaryl group is preferably a 5-10-membered heteroaryl group, and the 5-10-membered heteroaryl group is further preferably a 5-6-membered heteroaryl group. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, imidazolyl, isoxazolyl, oxazolyl, pyrazolyl, pyrrolyl, thiazolyl, thiadiazolyl, triazolyl, pyridyl, pyridyl Azinyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, benzofuranyl, benzothienyl, benzisoxazolyl, benzoxazolyl, benzopyrazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyladenyl, quinolyl or isoquinolyl.
Unless otherwise stated, the term “carbocyclyl” refers to a stable monocyclic, bicyclic or tricyclic ring with carbon atoms; they can be cyclic saturated alkyl groups or structures with partially unsaturated bonds. The carbocyclic group is preferably a C3-14 carbocyclyl, the C3-14 carbocyclic group is further preferably a C3-8 carbocyclyl, the C3-8 carbocyclic group is further preferably a C3-6 carbocyclic, the C3-6 carbocyclic group is further preferably a C5-6 carbocyclyl. Examples of carbocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutyl, cyclopentyl, and cyclohexenyl.
The term “heterocyclyl”, as used herein, unless otherwise indicated, represents stable heteroatom-containing monocyclic, bicyclic or tricyclic rings, which can be saturated or partially unsaturated. They contain carbon atoms and 1-4 heteroatoms selected from N, O or S, and wherein nitrogen or Sulfur heteroatoms can optionally be oxidized, and nitrogen heteroatoms can optionally be quaternized. Heterocyclyl groups can be attached to any heteroatom or carbon atom, resulting in a stable structure. The heterocyclyl group is preferably a 3-14-membered heterocyclyl group, the 3-14-membered heterocyclyl group is further preferably a 3-8-membered heterocyclyl group or a 5-10-membered heterocyclyl group, and the 3-8-membered heterocyclic group The ring group is further preferably a 3-6-membered heterocyclyl group, and the 3-6-membered heterocyclyl group is further preferably a 5-6-membered heterocyclyl group. Examples of heterocyclic groups include, but are not limited to, oxiranyl, piperazinyl, morpholinyl, piperidinyl, tetrahydropyrrolyl, tetrahydrofuranyl, 4,5,6,7-tetrahydro-2H-[1,2,3]triazolyl[4,5-c]pyridyl, 4,5,6,7-tetrahydro-2H-indazolyl, 2,4,5,6-tetrahydrocyclopentyl[c] ]pyrazolyl, 2,4,5,6-tetrahydrocyclopentyl[d][1,2,3]triazolyl, 5,6,7,8-tetrahydro-[1,2,4]triazole[1,5-a]pyrazinyl, 5,6,7,8-tetrahydro[1,2,4]triazole[4,3-a]pyrazinyl, 4,5,6,7-tetrahydro-1H-phenyl[d]imidazolyl, 2-azaspiro[3.3]heptyl, 2-oxo-7-azaspiro[4.4]nonanyl, 7-aza Bicyclo[2.2.1]heptyl, octahydrocyclopenta[c]pyrrolyl, 3-azabicyclo[3.1.0]hexyl, 3,4-dihydro-2H-benzo[b] [1,4]oxazine, 2H-benzo[b][1,4]oxazine-3(4H)-one, etc.
The term “substituted” refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s). Typical substituents include, but are not limited to, halogen (F, Cl, Br or I), C1-8 alkyl, C2-12 cycloalkyl, —OR11, SR11, ═O, ═S, —C(O)R11, —C(S)R11, ═NR11, —C(O)OR11, —C(S)OR11, —NR11R12, —C(O)NR11R12, cyano, nitro, —S(O)2R11, —OS(O2)OR11, —OS(O)2R11, —OP(O)(OR11)(OR12); wherein Ru and R12 is independently selected from —H, C1-8 alkyl, C1-8 haloalkyl. In some embodiments, the substituent(s) is independently selected from the group consisting of —F, —Cl, —Br, —I, —OH, trifluromethoxy, ethoxy, propyloxy, iso-propyloxy, n-butyloxy, isobutyloxy, t-butyloxy, —SCH3, —SC2H5, formaldehyde group, —C(OCH3), cyano, nitro, CF3, —OCF3, amino, dimethylamino, methyl thio, sulfonyl and acetyl.
Examples of substituted alkyl group include, but not limited to, 2-aminoethyl, 2-hydroxyethyl, pentachloroethyl, trifluoromethyl, methoxymethyl, pentafluoroethyl and piperazinylmethyl.
Examples of substituted alkoxy groups include, but not limited to, aminomethoxy, tetrafluoromethoxy, 2-diethylaminoethoxy, 2-ethoxycarbonylethoxy, 3-hydroxypropoxy.
The term “one or more” or “at least one” means one, two, three, four, five, six, seven, eight, nine or more.
The present invention includes within its scope the prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds that are readily converted in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term “administering” shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the subject. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.
It is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definitions elsewhere in that molecule. It is understood that substituents and substitution patterns on the compounds of this invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques know in the art as well as those methods set forth herein.
The term “stereoisomer” refers to a compound that contains one or more asymmetric centers (or axes) in its structure and is capable of stereoisomerism, including geometric isomers, optical isomers (including atropisomers)) and conformational isomers. The present invention includes all possible diastereoisomers and their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers and their pharmaceutical acceptable salt.
The above Formula I are shown without a definitive stereochemistry at certain positions. The present invention includes all stereoisomers of Formula I and pharmaceutically acceptable salts thereof. Further, mixtures of stereoisomers as well as isolated specific stereoisomers are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.
When a tautomer of the compound of Formula I exists, the present invention includes any possible tautomers and pharmaceutically acceptable salts thereof, and mixtures thereof, except where specifically stated otherwise.
When the compound of Formula I and pharmaceutically acceptable salts thereof exist in the form of solvates or polymorphic forms, the present invention includes any possible solvates and polymorphic forms. A type of a solvent that forms the solvate is not particularly limited so long as the solvent is pharmacologically acceptable. For example, water, ethanol, propanol, acetone or the like can be used.
The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (ic and ous), ferric, ferrous, lithium, magnesium, manganese (ic and ous), potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N′,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
When the compound of the present invention is basic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic, formic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids, particularly preferred are formic and hydrochloric acid. Since the compounds of Formula I are intended for pharmaceutical use they are preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure, especially at least 98% pure (% are on a weight for weight basis).
The pharmaceutical compositions of the present invention comprise a compound represented by Formula I (or a pharmaceutically acceptable salt thereof) as an active ingredient, a pharmaceutically acceptable carrier and optionally other therapeutic ingredients or adjuvants. The compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
In practice, the compounds represented by Formula I, or a prodrug, or a metabolite, or pharmaceutically acceptable salts thereof, of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion, or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compound represented by Formula I, or a pharmaceutically acceptable salt thereof, may also be administered by controlled release means and/or delivery devices. The compositions may be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.
Thus, the pharmaceutical compositions of this invention may include a pharmaceutically acceptable carrier and a compound, or a stereoisomer, tautomer, deuterated compound, pharmaceutically acceptable salt, prodrug, chelate, non-covalent complex, or solvate thereof. The compounds of Formula I, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds.
The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers include such as sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include such as carbon dioxide and nitrogen. In preparing the compositions for oral dosage form, any convenient pharmaceutical media may be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like may be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets may be coated by standard aqueous or nonaqueous techniques.
A tablet containing the composition of this invention may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Each tablet preferably contains from about 0.05 mg to about 5 g of the active ingredient and each cachet or capsule preferably containing from about 0.05 mg to about 5 g of the active ingredient. For example, a formulation intended for the oral administration to humans may contain from about 0.5 mg to about 5 g of active agent, compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Unit dosage forms will generally contain between from about 1 mg to about 2 g of the active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.
Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.
Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, or the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations may be prepared, utilizing a compound represented by Formula I of this invention, or a pharmaceutically acceptable salt thereof, via conventional processing methods. As an example, a cream or ointment is prepared by admixing hydrophilic material and water, together with about 5 wt % to about 10 wt % of the compound, to produce a cream or ointment having a desired consistency.
Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.
In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above may include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including antioxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a compound described by Formula I, or pharmaceutically acceptable salts thereof, may also be prepared in powder or liquid concentrate form.
Generally, dosage levels on the order of from about 0.01 mg/kg to about 150 mg/kg of body weight per day are useful in the treatment of the above-indicated conditions, or alternatively about 0.5 mg to about 7 g per patient per day. For example, colon cancer, rectal cancer, mantle cell lymphoma, multiple myeloma, breast cancer, prostate cancer, glioblastoma, squamous cell esophageal cancer, liposarcoma, T-cell lymphoma melanoma, pancreatic cancer, or lung cancer, may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day, or alternatively about 0.5 mg to about 3.5 g per patient per day.
It is understood, however, that lower or higher doses than those recited above may be required. Specific dose level and treatment regimens for any particular subject will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, the severity and course of the particular disease undergoing therapy, the subject disposition to the disease, and the judgment of the treating physician.
These and other aspects will become apparent from the following written description of the invention.
The following Examples are provided to better illustrate the present invention. All parts and percentages are by weight and all temperatures are degrees Celsius, unless explicitly stated otherwise.
The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any subject matter claimed. All parts and percentages are by weight unless expressly stated otherwise. All temperatures are in degrees Celsius. The raw materials for the synthesis method are not given in this invention, and the reagents can be obtained from commercial sources or synthesized by conventional methods as shown below using commercially available raw materials and reagents.
The following abbreviations have been used in the examples:
To a solution of A05-1 (4-bromo-2-fluorobenzoic acid) (2.19 g, 10.0 mmol) and 6-(2H-1,2,3-triazol-2-yl)-5-(trifluoromethyl)pyridin-3-amine (2.52 g, 11.0 mmol) in dichloromethane (25 mL) was added pyridine (1.19 g, 15.0 mmol) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (4.59 g, 30.0 mmol). The reaction was stirred for 1 hour and quenched by water, extracted with dichloromethane (250 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A05-2 (N-(6-(2H-1,2,3-triazol-2-yl)-5-(trifluoromethyl)pyridin-3-ly)-4-bromo-2-fluorobenzamide) (2.5 g). LC-MS (ES+): m/z 430 (M+H)+
Into a 100-mL round-bottom flask, was placed A05-2 (215 mg, 0.5 mmol), (3-methylpyridin-2-yl)boronic acid (82 mg, 0.6 mmol), Pd(dppf)Cl2 (36.6 mg, 0.05 mmol), K2CO3 (414 mg, 3.0 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A05 (47 mg). LC-MS (ES+): m/z 443 [M+H]+
To a solution of A12-1 (2.17 g, 10.0 mmol) and 6-(2H-1,2,3-triazol-2-yl)-5-(trifluoromethyl)pyridin-3-amine (2.52 g, 11.0 mmol) in dichloromethane (25 mL), was added pyridine (1.19 g, 15.0 mmol) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride (4.59 g, 30.0 mmol) was added dropwise. The reaction was stirred at room temperature for 1 hour and quenched by water, extracted with dichloromethane (250 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A12-2 (2.1 g). LC-MS (ES+): m/z 428,430 [M+H]+
Into a 50-mL round-bottom flask, was placed A12-2 (215 mg, 0.5 mmol), 2-(2-chloro-4-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (154 mg, 0.6 mmol), Pd(dppf)Cl2, (36.6 mg, 0.05 mmol), K2CO3 (414 mg, 3.0 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A12 (41 mg), LC-MS (ES+): m/z 478 [M+H]+
To a solution of A35-1 (2.15 g, 10.0 mmol) and 6-(2H-1,2,3-triazol-2-yl)-5-(trifluoromethyl)pyridin-3-amine (2.52 g, 11.0 mmol) in dichloromethane (25 mL), was added pyridine (1.19 g, 15.0 mmol) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (4.59 g, 30.0 mmol). The reaction was stirred at room temperature for 1 hour and quenched by water, extracted with dichloromethane (250 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A35-2 (1.8 g), LC-MS (ES+): m/z 426 [M+H]+
Into a 50-mL round-bottom flask, was placed A35-2 (213 mg, 0.5 mmol), 2-(4,4,5,5-Tetramethyl-1,3,2-dioxaboran-2-yl) aniline (154 mg, 0.6 mmol), Pd(dppf)Cl2, (36.6 mg, 0.05 mmol), K2CO3 (414 mg, 3.0 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography to give compound A35 (60 mg), LC-MS (ES+): m/z 439 [M+H]+
To a solution of A41-1 (2.35 g, 10.0 mmol) and 5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-amine (2.15 g, 11.0 mmol) in dichloromethane (25 mL), was added pyridine (1.19 g, 15.0 mmol) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (4.59 g, 30.0 mmol). The reaction was stirred at room temperature for 1 hour and quenched by water, extracted with dichloromethane (250 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A41-2 4-bromo-2-chloro-N-(5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-yl)benzamide (3.1 g, yellow solid). LC-MS (ES+): m/z 412,414 [M+H]+
Into a 50-mL round-bottom flask, was placed A41-2 (314 mg, 0.76 mmol), 3-methylpyridine-4-boronic acid (158 mg, 1.15 mmol), Pd(dppf)Cl2 (58.5 mg, 0.08 mmol), K2CO3 (476 mg, 3.45 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 75° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A41 (50 mg, off-white solid). LC-MS (ES+): m/z 425 [M+H]+
Into a 100-mL round-bottom flask, was placed A41-2 (314 mg, 0.76 mmol), 2-chlorophenylboronic acid (179.8 mg, 1.15 mmol), Pd(dppf)Cl2 (58.5 mg, 0.08 mmol), K2CO3 (476 mg, 3.45 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 75° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A42 (40 mg off-white solid). LC-MS (ES+): m/z 444 [M+H]+
Into a 100-mL round-bottom flask, was placed A35-2 (213 mg, 0.5 mmol), 2-(2-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (140 mg, 0.6 mmol), Pd(dppf)Cl2, (36.6 mg, 0.05 mmol), K2CO3 (414 mg, 3.0 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A64 (50 mg), LC-MS (ES+): m/z 454 [M+H]+
Into a 100-mL round-bottom flask, was placed A41-2 (314 mg, 0.76 mmol), 4-fluoro-2-hydroxyphenylboronic acid (179 mg, 1.15 mmol), Pd(dppf)Cl2 (58.5 mg, 0.08 mmol), K2CO3 (476 mg, 3.45 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 75° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A98 (49 mg off-white solid). LC-MS (ES+): m/z 444 [M+H]+
Into a 100-mL round-bottom flask, was placed A41-2 (314 mg, 0.76 mmol), 3-chloropyridine-4-boronic acid (180 mg, 1.15 mmol), Pd(dppf)Cl2 (58.5 mg, 0.08 mmol), K2CO3 (476 mg, 3.45 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 75° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A100 (59 mg off-white solid) LC-MS (ES+): m/z 445 [M+H]+
Into a 100-mL round-bottom flask, was placed A41-2 (314 mg, 0.76 mmol), 2-Bromo-4-fluorophenylboronic acid (252 mg, 1.15 mmol), Pd(dppf)Cl2 (58.5 mg, 0.08 mmol), K2CO3 (476 mg, 3.45 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 75° C. for 2 hours. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A102 (53 mg off-white solid). LC-MS (ES+): m/z 506,508 [M+H]+
1H NMR (500 MHz, DMSO-d6) δ 11.41 (s, 1H), 8.76 (d, J=87.2 Hz, 2H), 8.19 (s, 2H), 7.60 (dd, J=126.7, 61.0 Hz, 6H).
Into a 100-mL round-bottom flask, was placed A41-2 (314 mg, 0.76 mmol), 4-fluoro-2-methoxyphenylboronic acid (196 mg, 1.15 mmol), Pd(dppf)Cl2 (58.5 mg, 0.08 mmol), K2CO3 (476 mg, 3.45 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 75° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A104 (57 mg off-white solid). LC-MS (ES+): m/z 458 [M+H]+
To a solution of A105-1 (2.54 g, 10.0 mmol) and 6-(2H-1,2,3-triazol-2-yl)-5-(trifluoromethyl)pyridin-3-amine (2.52 g, 11.0 mmol) in dichloromethane (25 mL), was added pyridine (1.19 g, 15.0 mmol) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (4.59 g, 30.0 mmol). The reaction was stirred at room temperature for 1 hour, and quenched by water, extracted with dichloromethane (250 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A105-2 (2.5 g). LC-MS (ES+): m/z 464 [M+H]+
Into a 100-mL round-bottom flask, was placed A105-2 (232 mg, 0.5 mmol), 2-(2-Chloro-4-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (154 mg, 0.6 mmol), Pd(dppf)Cl2 (40 mg, 0.05 mmol), K2CO3 (414 mg, 3.0 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A105 (40 mg), LC-MS (ES+): m/z 514 [M+H]+
1H NMR (500 MHz, DMSO-d6) δ 11.60 (s, 1H), 9.12 (d, J=2.4 Hz, 1H), 8.89 (d, J=2.4 Hz, 1H), 8.21 (s, 2H), 7.86 (d, J=9.1 Hz, 1H), 7.74 (d, J=6.3 Hz, 1H), 7.69 (dd, J=8.9, 2.7 Hz, 1H), 7.60 (dd, J=8.6, 6.1 Hz, 1H), 7.41 (td, J=8.4, 2.6 Hz, 1H).
Into a 100-mL round-bottom flask, was placed A105-2 (115 mg, 0.25 mmol), 2-methoxyphenylboronic acid (45.6 mg, 0.3 mmol), Pd(dppf)Cl2, (18.3 mg, 0.025 mmol), K2CO3 (207 mg, 1.5 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A67 (18 mg). LC-MS (ES+): m/z 492 [M+H]+
Into a 100-mL round-bottom flask, was placed A105-2 (116 mg, 0.25 mmol), 2-aminophenylboronic acid (41 mg, 0.3 mmol), Pd(dppf)Cl2, (18.3 mg, 0.025 mmol), K2CO3 (207 mg, 1.5 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A69 (24 mg), LC-MS (ES+): m/z 477 [M+H]+
To a solution of A110-1 (2.53 g, 10.0 mmol) and 6-(2H-1,2,3-triazol-2-yl)-5-(trifluoromethyl)pyridin-3-amine (2.52 g, 11.0 mmol) in dichloromethane (25 mL) was added pyridine (1.19 g, 15.0 mmol) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (4.59 g, 30.0 mmol). The reaction was stirred at room temperature for 1 hour, and quenched by water, extracted with dichloromethane (250 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A110-2 (1.9 g). LC-MS (ES+): m/z 464,466 [M+H]+
Into a 100-mL round-bottom flask, was placed A110-2 (215 mg, 0.5 mmol), 2-aminophenylboronic acid (82.2 mg, 0.6 mmol), Pd(dppf)Cl2 (36.6 mg, 0.05 mmol), K2CO3 (414 mg, 3.0 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EtOAc=1/1) to give compound A110 (55 mg). LC-MS (ES+): m/z 477[M+H]+
To a solution of 5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-amine (1.95 g) and 4-bromo-5-fluoro-2-methylbenzoic acid (2.33 g) in 20 mL of DCM, was added pyridine (1.6 g) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then POCl3 (3.1 g) was added dropwise. The reaction was stirred at room temperature for 2 hours and quenched by water, extracted with dichloromethane (100 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A126-1 (3.7 g, white solid). LC-MS (ES+): m/z 410,412[M+H]+
Into a 100-mL round-bottom flask, was placed A126-1 (41 mg), (3-Chloropyridin-4-yl)boronic acid (16 mg), dioxane (1 mL), water (0.2 mL), Pd(dppf)Cl2 (5 mg) and K2CO3 (30 mg). The resulting mixture was stirred at 90° C. and stirred for 16 hours under nitrogen. Removing of the solvent and purifying by column chromatography (DCM/MeOH=40/1) to give compound A126 (21 mg, white solid). LC-MS (ES+): m/z 443[M+H]+
Into a 100-mL round-bottom flask, was placed A126-1 (41 mg), (2-Amino-4-fluorophenyl)boronic acid (16 mg), dioxane (1 mL), water (0.2 mL), Pd(dppf)Cl2 (5 mg) and K2CO3 (30 mg). The resulting mixture was stirred at 90° C. for 16 hours under nitrogen. Removing of the solvent and purifying by column chromatography (DCM/MeOH=40/1) to give compound A113 (29 mg, white solid). LC-MS (ES+): m/z 441[M+H]+
A113: 1H NMR (500 MHz, DMSO-d6) δ 11.19 (d, J=22.7 Hz, 1H), 8.86 (dd, J=19.1, 2.3 Hz, 1H), 8.68 (dd, J=18.5, 2.2 Hz, 1H), 8.18 (d, J=1.9 Hz, 2H), 7.83-7.25 (m, 2H), 7.17-6.45 (m, 2H), 2.43 (d, J=18.8 Hz, 3H).
Into a 100-mL round-bottom flask, was placed A126-1 (41 mg), (2-bromo-4-fluorophenyl)boronic acid (26 mg), dioxane (1 mL), water (0.2 mL), Pd(dppf)Cl2 (5 mg) and K2CO3 (30 mg). The resulting mixture was stirred at 90° C. for 16 hours under nitrogen. Removing of the solvent and purifying by column chromatography (DCM/MeOH=40/1) to give compound A114 (19 mg, white solid). LC-MS (ES+): m/z 504,506[M+H]+ A114:
1H NMR (500 MHz, DMSO-d6) δ 11.20 (s, 1H), 8.86 (d, J=2.3 Hz, 1H), 8.69 (d, J=2.2 Hz, 1H), 8.19 (s, 2H), 7.79 (dd, J=8.6, 2.6 Hz, 1H), 7.62 (d, J=9.6 Hz, 1H), 7.50 (dd, J=8.6, 6.1 Hz, 1H), 7.42 (td, J=8.4, 2.6 Hz, 1H), 7.37 (d, J=7.1 Hz, 1H), 2.45 (s, 3H).
To a solution of 5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-amine (1.95 g) and 4-bromo-2-chloro-5-fluorobenzoic acid (2.53 g) in dichloromethane (20 mL) was added pyridine (1.6 g) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (3.1 g). The reaction was stirred at room temperature for 2 hours and quenched by water, extracted with dichloromethane (50 mL×2). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel (PE/EA=2/1) to afford compound A130-1 4-bromo-2-chloro-N-(5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-yl)-5-fluorobenzamide (3.9 g). LC-MS (ES+): m/z 430 [M+H]+
Into a 100-mL round-bottom flask, was placed A130-1 (43 mg), (4-aminopyrimidin-5-yl)boronic acid (14 mg), Pd(dppf)Cl2 (5 mg), K2CO3 (30 mg), dioxane (1 mL) and H2O (0.2 mL). The resulting mixture was stirred at 90° C. for 16 hours under nitrogen. The reaction solution was diluted with EA (5 mL) washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain A130 (23 mg) as a white solid. LC-MS (ES+): m/z 445 [M+H]+
Into a 100-mL round-bottom flask, was placed A130-1 (43 mg), (4-amino-2-oxo-1,2-dihydropyrimidin-5-yl)boronic acid (16 mg), Pd(dppf)Cl2 (5 mg), K2CO3 (30 mg), dioxane (1 mL) and H2O (0.2 mL). The resulting mixture was stirred at 90° C. for 16 hours under nitrogen. The reaction solution was diluted with EA (5 mL) washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain A133 (22 mg) as a white solid. LC-MS (ES+): m/z 461 [M+H]+
To a solution of A147-1 (2.64 g, 10.0 mmol) and 6-(2H-1,2,3-triazol-2-yl)-5-(trifluoromethyl)pyridin-3-amine (2.15 g, 11.0 mmol) in dichloromethane (25 mL) was added pyridine (1.19 g, 15.0 mmol) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (4.59 g, 30.0 mmol). The reaction was stirred for 1 hour and quenched by water, extracted with dichloromethane (125 mL×2). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A147-2 (2.6 g). LC-MS (ES+): m/z 475 [M+H]+
Into a 100-mL round-bottom flask, was placed A147-2 (950 mg, 2 mmol), (2-amino-4-fluorophenyl)boronic acid (388 mg, 2.5 mmol), Pd(dppf)Cl2 (18.3 mg, 0.025 mmol), K2CO3 (207 mg, 1.5 mmol), dioxane (3 mL) and H2O (0.5 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography to give compound A147-3 (700 mg). LC-MS (ES+): m/z 506 [M+H]+
To a solution of A147-3 (700 mg, 2 mmol) in ethyl acetate (20 mL) was added Pd/C (10%, 100 mg) under nitrogen atmosphere in a 100 mL round bottom flask. The flask was then vacuumed and flushed with hydrogen. The reaction mixture was hydrogenated at room temperature for 2 hours under hydrogen atmosphere using a hydrogen balloon, then filtered through a Celite pad and concentrated under reduced pressure. The residue was used without further purification and compound A147-4 (500 mg) was obtained. LC-MS (ES+): m/z 476 [M+H]+
To a solution of A147-4 (95 mg, 0.2 mmol) in acetonitrile (5 mL) was added p-toluenesulfonic acid (172 mg, 1 mmol) at 0° C. An aqueous solution of sodium nitrite (69 mg, 1 mmol, dissolved in 1 mL of water) was added to the resulting mixture and stirred for 0.5 hour and then potassium iodide (166 mg, 1 mmol, dissolved in 1 mL water) was added. The mixture was stirred at 25° C. for 1 hour, then raised to 100° C. and continued for 0.5 hour. The reaction was quenched by aqueous sodium thiosulfate solution and extracted by Eethyl acetate (50 mL×2). The organic layer was concentrated and purified by column chromatography to obtain compound A147 (22 mg). LC-MS (ES+): m/z 698 [M+H]+
Step 1: Synthesis of Compound A157-1
To a solution of 5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-amine (1.95 g) and 4-bromo-5-chloro-2-fluorobenzoic acid (2.53 g) in dichloromethane (20 mL) was added pyridine (1.6 g) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (3.1 g). The reaction was stirred for 2 hour at room temperature and quenched by water, extracted with dichloromethane (50 mL×2). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel (PE/EA=2/1) to afford compound A157-1 4-Bromo-5-chloro-N-(5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-yl)-2-fluorobenzamide (4.1 g) as a white solid. LC-MS (ES+): m/z 430 [M+H]+
Into a 50-mL round-bottom flask, was placed A157-1 (430 mg), (2-amino-4-fluorophenyl)boronic acid (155 mg), Pd(dppf)Cl2 (50 mg), K2CO3 (300 mg), dioxane (10 mL) and H2O (2 mL). The resulting mixture was stirred at 90° C. for 16 hours under nitrogen. Removing of the solvent and purifying by column chromatography (DCM/MeOH=40/1) to give compound A120 (345 mg) as a white solid. LC-MS (ES+): m/z 461 [M+H]+
1H NMR (500 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.86 (d, J=2.3 Hz, 1H), 8.67 (d, J=2.3 Hz, 1H), 8.19 (s, 2H), 7.98 (d, J=6.5 Hz, 1H), 7.42 (d, J=10.2 Hz, 1H), 6.93 (dd, J=8.4, 6.7 Hz, 1H), 6.54 (dd, J=11.8, 2.6 Hz, 1H), 6.42 (td, J=8.5, 2.6 Hz, 1H), 5.20 (s, 2H).
A120 (92 mg) was dissolved in concentrated hydrochloric acid (5 mL). At 0° C., an aqueous solution of sodium nitrite (20 mg, dissolved in 1 mL water) was added to the resulting mixture and stirred for 1 hour and then potassium iodide (34 mg, dissolved in 1 mL water) was added. The mixture was stirred at room temperature for 2 hours. The reaction was quenched by aqueous sodium thiosulfate solution and extracted by ethyl acetate. The organic layer was concentrated and purified by column chromatography (DCM/MeOH=40/1) to obtain compound A157 (65 mg) as a white solid. LC-MS (ES+): m/z 572 [M+H]+
Into a 10-mL round-bottom flask, was placed A157-1 (43 mg), (3-aminopyridin-4-yl)boronic acid (15 mg), Pd(dppf)Cl2 (5 mg), K2CO3 (30 mg), dioxane (1 mL) and H2O (0.2 mL). The resulting mixture was stirred at 90° C. for 16 hours under nitrogen. Removing of the solvent and purifying by column chromatography (DCM/MeOH=40/1) to give compound A124 (26 mg) as a white solid. LC-MS (ES+): m/z 444 [M+H]+
To a solution of 5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-amine (1.95 g) and 4-bromo-2-chloro-5-(fluorotrifluoromethyl)benzoic acid (3.03 g) in dichloromethane (20 mL) was added pyridine (1.6 g) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (3.1 g). The reaction was stirred at room temperature for 2 hours and quenched by water, extracted with dichloromethane (50 mL×2). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A220-1 4-bromo-2-chloro-N-(5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-yl)-5-(fluorotrifluoromethyl)benzamide (4.1 g) as a white solid. LC-MS (ES+): m/z 480 [M+H]+
Into a 10-mL round-bottom flask, was placed A220-1 (48 mg), (2-amino-4-fluorophenyl)boronic acid (15 mg), Pd(dppf)Cl2 (5 mg), K2CO3 (30 mg), dioxane (1 mL) and H2O (0.2 mL). The resulting mixture was stirred at 90° C. for 16 hours under nitrogen. Removing of the solvent and purifying by column chromatography (DCM/MeOH=40/1) to give compound A220 (25 mg) as a white solid. LC-MS (ES+): m/z 541 [M+H]+
Into a 50-mL round-bottom flask, was placed A274-1 (1.23 g, 5 mmol), 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (1.68 g, 10 mmol), Pd(PPh3)4 (578 mg, 0.5 mmol), Na2CO3 (1.59 g, 15 mmol), dioxane (12 mL) and H2O (3 mL). The resulting mixture was stirred at 85° C. for 3 hours under nitrogen. Removing of the solvent and purifying by column chromatography to give compound A274-2 (800 mg, 3.83 mmol) as a yellow solid.
Into a flask, A274-2 (800 mg, 3.83 mmol), NaOH (459 mg, 11.5 mmol), methanol (8 mL) and H2O (4 mL) were added and reacted at room temperature for 16 hours. The end of the reaction were monitored by LCMS. Adjusted the pH to 3 with concentrated hydrochloric acid under an ice bath, solid precipitated, filtered, and dried to obtain a yellow solid A274-3 (500 mg).
To a solution of A274-3 (500 mg, 2.56 mmol) in methanol (8 mL) was added Pd/C (10%, 250 mg) under nitrogen atmosphere in a round bottom flask. The flask was then vacuumed and flushed with hydrogen. The reaction mixture was hydrogenated at room temperature for 5 hours under hydrogen atmosphere using a hydrogen balloon, then filtered through a Celite pad and concentrated under reduced pressure. The residue was used without further purification and compound A274-4 (450 mg) was obtained.
To a solution of A274-4 (450 mg, 2.28 mmol) and 5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-amine (585 mg, 3 mmol) in dichloromethane (5 mL) was added pyridine (540 mg, 6.84 mmol) at room temperature. The mixture was cooled to 0° C. in an ice bath, and then phosphorus oxychloride was added dropwise (459 mg, 3 mmol). The reaction was stirred for 1 hour and quenched by water, extracted with dichloromethane (25 mL×2). The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by flash column chromatography over silica gel to afford compound A274-5 (600 mg, 1.60 mmol) as a reddish brown solid.
At 0° C., to a solution of A274-5 (600 mg, 1.60 mmol) in 2N HCl (6 mL, 2 mol·L−1 HCl), an aqueous solution of sodium nitrite (138 mg, 2 mmol, dissolved in 2 mL of water) was added to the resulting mixture and stirred for 15 minutes and then potassium iodide (332 mg, 2 mmol, dissolved in 2 mL water) was added. The mixture was stirred at 0° C. for 15 minutes, 4° C. for 10 minutes, 100° C. for 10 minutes, and then stirred at 0° C. for 5 minutes. The reaction was quenched by aqueous sodium thiosulfate solution and extracted by Eethyl acetate (25 mL×2). The organic layer was concentrated and purified by column chromatography to obtain compound A274-6 (200 mg, 0.41 mmol) as a reddish brown solid.
Into a round-bottom flask, was placed A274-6 (200 mg, 0.41 mmol), (2-amino-6-fluoropyridin-3-yl)boronic acid (94 mg, 0.6 mmol), Pd(dppf)Cl2 (29 mg, 0.04 mmol), K2CO3 (170 mg, 1.23 mmol), dioxane (2 mL) and H2O (0.5 mL). The resulting mixture was stirred at 75° C. for 2 hours under nitrogen. Removing of the solvent and purifying by column chromatography to give compound A274 (20 mg). LC-MS (ES+): m/z 470 [M+H]+
According to the synthesis method of the above example, select appropriate raw materials and/or marketed example intermediates to synthesize the example compounds in the table below. The raw materials and reagents used are all commercially available.
1H NMR (500 MHz, DMSO-d6) δ 11.44 (s, 1H), 8.85 (d, J=2.3 Hz, 1H), 8.67 (d, J=2.3 Hz, 1H), 8.19 (s, 2H), 7.77 (d, J=9.2 Hz, 1H), 7.60 (d, J=6.4 Hz, 1H), 7.05 (dd, J=8.4, 6.6 Hz, 1H), 6.66 (dd, J=11.5, 2.7 Hz, 1H), 6.59-6.44 (in, 1H).
1H NMR (500 MHz, DMSO-d6) δ 11.50 (s, 1H), 9.12 (d, J=2.4 Hz, 1H), 8.90 (d, J=2.4 Hz, 1H), 8.21 (s, 2H), 7.78 (d, J=9.2 Hz, 1H), 7.59 (d, J=6.5 Hz, 1H), 7.00 (dd, J=8.5, 6.7 Hz, 1H), 6.56 (dd, J=11.7, 2.6 Hz, 1H), 6.47-6.34 (m, 1H).
1H NMR (500 MHz, DMSO-d6) δ 11.34 (s, 1H), 9.17 (d, J=2.4 Hz, 1H), 8.90 (d, J=2.4 Hz, 1H), 8.21 (s, 2H), 8.00 (d, J=6.5 Hz, 1H), 7.43 (d, J=10.2 Hz, 1H), 6.93 (dd, J=8.4, 6.7 Hz, 1H), 6.54 (dd, J=11.7, 2.6 Hz, 1H), 6.42 (td, J=8.5, 2.6 Hz, 1H), 5.21 (s, 2H).
1H NMR (500 MHz, DMSO-d6) δ 11.42 (s, 1H), 8.82 (d, J=2.3 Hz, 1H), 8.66 (d, J=2.3 Hz, 1H), 8.20 (d, J=7.0 Hz, 3H), 7.58 (s, 1H), 7.12 (td, J=8.3, 7.9, 1.6 Hz, 1H), 6.84 (d, J=7.5 Hz, 1H), 6.76 (dd, J=8.2, 1.1 Hz, 1H), 6.61 (td, J=7.4, 1.1 Hz, 1H), 4.78 (s, 2H).
1H NMR (500 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.85 (d, J=2.3 Hz, 1H), 8.68 (d, J=2.3 Hz, 1H), 8.50 (s, OH), 8.18 (s, 2H), 7.74-7.53 (m, 2H), 7.54-7.37 (m, 2H), 7.27 (td, J=8.4, 2.5 Hz, 1H), 2.45 (s, 3H).
1H NMR (500 MHz, DMSO-d6) δ 11.53 (s, 1H), 8.95 (s, 1H), 8.81 (s, 1H), 8.68 (d, J=28.1 Hz, 2H), 8.19 (s, 2H), 8.03-7.67 (m, 2H), 7.58 (s, 1H).
Into a 50-mL round-bottom flask, was placed 4-bromo-2-chloro-N-(5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-yl)-5-fluorobenzamide (M1) (43 mg), (4-Fluoro-2-(hydroxymethyl)phenyl)boronic acid (25 mg), Pd(dppf)Cl2 (3 mg), K2CO3 (27 mg), dioxane (1 mL) and H2O (0.2 mL). The resulting mixture was stirred at 90° C. under nitrogen for 8 hours. The reaction was diluted with EA (5 mL), washed with brine. The organic layer was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A281 (18 mg) as white solid. LC-MS (ES+): m/z 476[M+H]+
Into a 50-mL round-bottom flask, was placed 5-bromo-4-chloropyridin-3-amine (413 mg), ethynyltrimethylsilane (300 mg), Pd(PPh3)2Cl2 (3 mg), CuI (5 mg), TEA (1 mL) and dioxane (10 mL). The reaction mixture was stirred at 90° C. under nitrogen for 8 hours. The mixture was diluted with EA (100 mL), washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) over silica gel to afford compound A282-1 (216 mg) as colorless oil. LC-MS (ES+): m/z 225[M+H]+
The mixture of A282-1 (216 mg), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxaborolane alkane) (500 mg), Pd(dppf)Cl2 (30 mg), K2CO3 (270 mg), dioxane (10 mL) and H2O (2 mL) was stirred at 100° C. under nitrogen for 8 hours in a 50-mL round-bottom flask. Removing of the solvent and purifying by column chromatography (DCM/MeOH=40/1) to afford compound A282-2 (88 mg) as a red oil. LC-MS (ES+): m/z 235[M+H]+
A mixture of 4-bromo-2-chloro-N-(5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-yl)-5-fluorobenzamide (M1) (43 mg), A282-2 (38 mg), Pd(dppf)Cl2 (3 mg), K2CO3 (27 mg), dioxane (1 mL) and H2O (0.2 mL) was stirred at 90° C. for 8 hours under nitrogen. The mixture was cooled to room temperature, diluted with EA (5 mL), and washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A282 (12 mg) as yellow solid. LC-MS (ES+): m/z 468[M+H]+
The mixture of 4-bromo-2-chloro-N-(5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-yl)-5-fluorobenzamide (M1) (43 mg), (4-fluoro-2-vinylphenyl)boronic acid (24 mg), Pd(dppf)Cl2 (3 mg), K2CO3 (27 mg), dioxane (1 mL) and H2O (0.2 mL) was stirred at 90° C. for 8 hours under nitrogen. The mixture was cooled to room temperature, diluted with EA (5 mL), and washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A286 (21 mg) as white solid. LC-MS (ES+): m/z 472[M+H]+
The mixture of 4-bromo-2-chloro-N-(5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin 3-yl)-5-fluorobenzamide (M1) (43 mg), 5-fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) aniline (36 mg), Pd(dppfCl2 (3 mg), K2CO3 (27 mg), dioxane (1 mL) and H2O (0.2 mL) was stirred at 90° C. for 8 hours under nitrogen. The mixture was cooled to room temperature, diluted with EA (5 mL) and washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound M2 (33 mg) as white solid. LC-MS (ES+): m/z 461[M+H]+
To a solution of M2 (23 mg), in DCM (1 mL) was added TEA (5 mg) and 1-chloro-2-methoxyethane (6 mg). The reaction mixture was stirred at room temperature for 2 hours and diluted with EA (5 mL), washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A296 (13 mg) as white solid. LC-MS (ES+): m/z 519 [M+H]+
To a solution of M2 (23 mg) in DCM (mL) was added TEA (5 mg) and 3-Chloropropyl-1-yne (5 mg). The reaction mixture was stirred at room temperature for 2 hours and diluted with EA (5 mL), washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A298 (18 mg), as yellow solid. LC-MS (ES+): m/z 499 [M+H]+
The mixture of M1 (43 mg), (2-(aminomethyl)-4-fluorophenyl)boronic acid (26 mg), Pd(dppf)Cl2 (3 mg), K2CO3 (27 mg), dioxane (1 mL) and H2O (0.2 mL) was stirred 90° C. for 8 hours under nitrogen. The mixture was cooled to room temperature, diluted with EA (5 mL) and washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A300 (12 mg) as yellow solid. LC-MS (ES+): m/z 475[M+H]+
To a solution of M2 (23 mg) in DCM (1 mL) was added TEA (5 mg) and acryloyl chloride (5 mg) at 0° C. The reaction mixture was stirred at room temperature for 2 hours and diluted with EA (5 mL), washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A298 (18 mg) as white solid. LC-MS (ES+): m/z 515[M+H]+
The mixture of S14 (443 mg), S12 (300 mg), Pd(PPh3)2Cl2 (3 mg), CuI (5 mg), TEA (1 mL) and dioxane (10 mL) was stirred at 90° C. for 8 hours under nitrogen. The mixture was cooled to room temperature, and diluted with EA (100 mL) and washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A308-1 (240 mg) as colorless oil. LC-MS (ES+): m/z 242[M+H]+
The mixture of A308-1 (240 mg), S13 (500 mg), Pd(dppf)Cl2 (30 mg), K2CO3 (270 mg), dioxane (10 mL) and H2O (2 mL) was stirred at 110° C. for 8 hours under nitrogen. The mixture was cooled to room temperature, diluted with EA (100 mL) and washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A308-2 (101 mg) as yellow oil. LC-MS (ES+): m/z 252[M+H]+
The mixture of M1 (43 mg), A308-2 (37 mg), Pd(dppf)Cl2 (3 mg), K2CO3 (27 mg), dioxane (1 mL) and H2O (0.2 mL) was stirred at 90° C. for 8 hours under nitrogen. The mixture was cooled to room temperature, diluted with EA (5 mL) and washed with brine. The organic phase was concentrated and purified by flash column chromatography (DCM/MeOH=40/1) to give compound A308 (19 mg) as yellow solid. LC-MS (ES+): m/z 485[M+H]+
To a solution of A310-4 (3.33 g, 10.0 mmol) in dioxane (35 mL) was added CuI (0.38 g, 2.0 mmol), Et3N (3.03 g, 30.0 mmol), Pd(PPh3)2Cl2 (0.73 g, 1 mmol) and ethyltrimethylsilane (2.04 g, 20.0 mmol). The reaction mixture was stirred at room temperature under nitrogen for 5 hours and quenched by water, extracted with dichloromethane (250 mLxa). The organic layer was washed by brine, dried over sodium sulfate, filtrated and evaporated in vacuo. The residue was purified by column chromatography to give compound A310-5 (2.5 g) as yellow solid. LC-MS (ES+): m/z 305[M+H]+
To a solution of A310-5 (2.5 g, 8.3 mmol) in methanol (30 mL) was added K2CO3 (3.45 g, 24.9 mmol). The reaction mixture was stirred at room temperature under nitrogen for 2 hours. Removing the solvent and purifying by column chromatography to give compound A310-6 (1.7 g) as Off-white solid. LC-MS (ES+): m/z 233[M+H]+
To a solution of A310-1 (2.52 g, 10 mmol) in DCM (30 mL) was added pyridine (2.37 g, 30 mmol) and 5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-amine (1.95 g, 10 mmol). Then POCl3 (1.68 g, 11 mmol) was added dropwise. The reaction was stirred at room temperature under nitrogen for 2 hours. The reaction was quenched by water and extracted by DCM (100 mL×2). The organic layer was concentrated and purified by column chromatography to give compound A310-2 (3.9 g) as off-white solid. LC-MS (ES+): m/z 430[M+H]+
The mixture of A310-2 (3.9 g, 9.0 mmol), potassium acetate (2.65 g, 27.0 mmol) Pd(dppf)Cl2 (658 mg, 0.9 mmol), and pinacol diborate (3.71 g, 14.5 mmol) in toluene (50 mL), was stirred at 75° C. under nitrogen for 2 hours. Removing the solvent and purifying by column chromatography to give compound A310-3 (3.7 g) as off-white solid LC-MS (ES+): m/z 478[M+H]+
The mixture of A310-3 (95 mg, 0.2 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), A310-6 (46 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction was concentrated and purified by column chromatography to give compound A310 (35 mg) as off-white solid. LC-MS (ES+): m/z 504[M+H]+
To a solution of A315-2 (2.52 g, 10 mmol) in DCM (30 mL), was added pyridine (2.37 g, 30 mmol) and 6-(2H-1,2,3-triazol-2-yl)-5-(trifluoromethyl)pyridin-3-amine (2.29 g, 10 mmol). Then POCl3 (1.68 g, 11 mmol) was added dropwise. The reaction was stirred at room temperature for 2 hours. The reaction was quenched by water and extracted by DCM (200 mL×2), the organic layer was concentrated in vacuum and purified by column chromatography to afford compound A315-2 (3.9 g) as off-white solid. LC-MS (ES+): m/z 464[M+H]+
The mixture of A310-2 (3.9 g, 8.4 mmol), potassium acetate (2.48 g, 25.2 mmol), Pd(dppf)Cl2 (585 mg, 0.8 mmol) and pinacol diborate (3.28 g, 12.8 mmol) in toluene (50 mL) was stirred at 75° C. under nitrogen for 2 hours. Removing the solvent and purifying by column chromatography to afforded compound A315-3 (3.7 g) as off-white solid. LC-MS (ES+): m/z 512[M+H]+
The mixture of A310-3 (102 mg, 0.2 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), 1-bromo-4-fluoro-2-vinylbenzene (40 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction was concentrated and purified by column chromatography to give compound A315 (30 mg) as off-white solid. LC-MS (ES+): m/z 504[M+H]+
The mixture of A315-3 (102 mg, 0.2 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), 1-bromo-2-ethynyl-4-fluorobenzene (40 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction was concentrated and purified by column chromatography to give compound A316 (30 mg) as off-white solid. LC-MS (ES+): m/z 504[M+H]+
The mixture of A310-3 (95 mg, 0.2 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), 4-bromo-3-ethynylpyridine (36 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction was concentrated and purified by column chromatography to give Compound A318 (36 mg) as off-white solid. LC-MS (ES+): m/z 453[M+H]+
To a solution of A310-1 (2.52 g, 10 mmol) in DCM (30 mL), was added pyridine (2.37 g, 30 mmol) and 5-(trifluoromethyl)pyridin-3-amine (1.62 g, 10 mmol). Then POCl3 (1.68 g, 11 mmol) was added dropwise. The reaction was stirred at room temperature for 2 hours. The reaction was quenched by water and extracted by DCM (100 mL×2), the organic layer was concentrated and purified by column chromatography to give compound A319-2 (3.1 g) as off-white solid. LC-MS (ES+): m/z 397[M+H]+
The mixture of A319-2 (3.1 g, 7.8 mmol), potassium acetate (2.30 g, 23.5 mmol), Pd(dppf)Cl2 (585 mg, 0.8 mmol), and pinacol diborate (3.00 g, 11.7 mmol) in toluene (35 mL), was stirred at 75° C. under nitrogen for 2 hours. The reaction was concentrated and purified by column chromatography to give compound A319-3 (3.7 g) as off-white solid. LC-MS (ES+): m/z 445[M+H]+
The mixture of A319-3 (90 mg, 0.2 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), 1-bromo-2-ethynyl-4-fluorobenzene (40 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction was concentrated in vacuum and purified by column chromatography to give compound A319 (33 mg) as off-white solid. LC-MS (ES+): m/z 437[M+H]+
To a solution of A323-1 (2.26 g, 10 mmol) in acetonitrile (30 mL) was added potassium carbonate (4.14 g, 30 mmol) and tetrahydrofuran-3-amine (1.75 g, 20 mmol). The reaction was stirred at 75° C. under nitrogen for 2 hours. The reaction was concentrated and purified by column chromatography to give compound A323-2 (2.3 g) as off-white solid. LC-MS (ES+): m/z 278[M+H]+
To a solution of A323-2 (2.26 g, 8.2 mmol) in methanol (30 mL) was added Pd/C (10%, 435 mg) under nitrogen atmosphere in a 100 mL round bottom flask. The flask was then vacuumed and flushed with hydrogen. The reaction mixture was hydrogenated at 75° C. for 2 hours under hydrogen atmosphere using a hydrogen balloon, then filtered through a Celite pad and concentrated under reduced pressure. The residue was purified by column chromatography to afford compound A323-3 (1.8 g) as off-white solid. LC-MS (ES+): m/z 248[M+H]+
To a solution of A310-1 (1.8 g, 7.2 mmol) in DCM (30 mL) was added pyridine (1.71 g, 21.6 mmol), N2-(tetrahydrofuran-3-yl)-3-(trifluoromethyl)pyridine-2,5-diamine (1.41 g, 7.2 mmol) and POCl3 (1.21 g, 7.2 mmol). The reaction was stirred at room temperature for 2 hours. The reaction was quenched by water and extracted by DCM (100 mL×2), the organic layer was concentrated and purified by column chromatography to give compound A323-4 (3.1 g) as off-white solid. LC-MS (ES+): m/z 482[M+H]+
The mixture of A323-4 (3.1 g, 6.4 mmol), potassium acetate (2.47 g, 15.2 mmol), Pd(dppf)Cl2 (439 mg, 0.6 mmol) and pinacol diborate (2.45 g, 9.6 mmol) in toluene (35 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction was concentrated and purified by column chromatography to give A323-5 (3.1 g) as off-white solid. LC-MS (ES+): m/z 530[M+H]+
The mixture of A323-5 (106 mg, 0.2 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), 4-bromo-3-ethynylpyridine (36 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction solution was concentrated and purified by column chromatography to give compound A323 (37 mg) as off-white solid. LC-MS (ES+): m/z 505[M+H]+
The mixture of A327-1 (3.01 g, 10.0 mmol), CuI (0.38 g, 2.0 mmol), Et3N (3.03 g, 30.0 mmol), Pd(PPh3)2Cl2 (0.73 g, 1 mmol), dioxane (35 mL) and ethyltrimethylsilane (2.04 g, 20.0 mmol) was stirred at room temperature under nitrogen for 5 hours. The reaction was quenched by water, extracted with dichloromethane (250 mL×2). The organic phase was dried over sodium sulfate, concentrated in vacuum and purified by column chromatography to give compound A327-2 (2.5 g) as yellow solid. LC-MS (ES+): m/z 272[M+H]+
The mixture of A315-3 (102 mg, 0.2 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), A327-2 (54 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction solution was concentrated in vacuum and purified by column chromatography to give compound A327 (38 mg) as off-white solid. LC-MS (ES+): m/z 505[M+H]+
The mixture of A315-3 (95 mg, 0.2 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), 4-bromo-3-ethynylpyridine (36 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction solution was concentrated in vacuum and purified by column chromatography to give compound A331 (37 mg) as off-white solid. LC-MS (ES+): m/z 487[M+H]+
To a solution of A332-1 (2.72 g, 10 mmol) in DCM (30 mL) was added pyridine (2.37 g, 30 mmol), 5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-amine (1.95 g, 10 mmol) and POCl3 (1.68 g, 11 mmol). The reaction mixture was stirred at room temperature for 2 hours, quenched by water and extracted by DCM (100 mL×2). The organic layer was concentrated and purified by column chromatography to give compound A332-2 (3.9 g) as off-white solid. LC-MS (ES+): m/z 450[M+H]+
The mixture of A310-2 (3.9 g, 8.6 mmol), potassium acetate (2.53 g, 25.8 mmol), Pd(dppf)Cl2 (658 mg, 0.9 mmol), pinacol diborate (3.31 g, 12.9 mmol) and toluene (50 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction solution was concentrated and purified by column chromatography to give Compound A332-3 (3.7 g) as off-white solid. LC-MS (ES+): m/z 501[M+H]+
To a solution of A332-4 (1.89 g, 10.0 mmol), in THF (30 mL) was added sodium bicarbonate (2.52 g, 30 mmol) and propargyl chloride (0.89 g, 10.0 mmol). The reaction was stirred at room temperature for 2 hours. The reaction was quenched by water and extracted by EA (100 mL×2), the organic layer was concentrated and purified by column chromatography to obtain compound A332-5 (1.7 g) as off-white solid. LC-MS (ES+): m/z 242[M+H]+
The mixture of A332-3 (100 mg, 0.2 mmol), K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), A332-5 (48 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) was stirred at 75° C. under nitrogen for 2 hours. The reaction was concentrated in vacuum and purified by column chromatography to give compound A332 (35 mg) as off-white solid. LC-MS (ES+): m/z 513[M+H]+
To a solution of A343-1 (1.91 g, 10.0 mmol) in methanol (25 mL) was added sodium methylmercaptide (1.40 g, 20 mmol). The reaction was stirred at 75° C. for 2 hours. The reaction solution was concentrated and purified by column chromatography to give off-white solid A343-2 (1.5 g). LC-MS (ES+): m/z 204[M+H]+
To a solution of A343-2 (1.50 g, 7.3 mmol) in dichloromethane (20 mL) was added m-CPBA (1.73 g, 10 mmol). The reaction was stirred at room temperature for 2 hours. Monitored by LCMS, then concentrated and purified by column chromatography to give off-white solid A343-3 (1.1 g). LC-MS (ES+): m/z 236[M+H]+
A343-3 (1.10 g, 4.7 mmol) was added to the reaction flask, and then palladium on carbon (212 mg, 2 mmol) and methanol (15 mL) were added. The reaction was stirred at room temperature under hydrogen for 2 hours. The end of the reaction were monitored by LCMS. The reaction solution was filtered, evaporated to obtain off-white solid 900 mg. LC-MS (ES+): m/z 206[M+H]+.
A332-1 (2.72 g, 10 mmol) was added to the reaction flask, and then DCM (30 mL), pyridine (2.37 g, 30 mmol) and 3-chloro-4-methylsulfonylanilide (2.06 g, 10 mmol) were added. Then POCl3 (1.68 g, 11 mmol) was added dropwise. The reaction was carried out at room temperature for 2 hours. The end of the reaction were monitored by LCMS. The reaction solution was purified by column chromatography to obtain off-white solid product 3.9 g. LC-MS (ES+): m/z 460[M+H]+
A343-5 (3.9 g, 8.5 mmol) was added to the reaction flask, and then potassium acetate (2.50 g, 25.5 mmol), Pd(dppf)Cl2 (658 mg, 0.9 mmol), toluene (50 mL), pinacol diborate (3.31 g, 12.9 mmol) were added. The reaction was carried out at 75° C. under nitrogen for 2 hours. The end of the reaction were monitored by LCMS. The reaction solution was purified by column chromatography to obtain off-white solid product 3.7 g. LC-MS (ES+): m/z 508[M+H]+
A343-6 (114 mg, 0.2 mmol) was added to the reaction flask, and then K2CO3 (83 mg, 0.6 mmol), Pd(dppf)Cl2 (15 mg, 0.02 mmol), 4-bromo-3-ethynylpyridine (36 mg, 0.2 mmol), dioxane (1.5 mL) and H2O (0.3 mL) were added. The reaction was carried out at 75° C. under nitrogen for 2 hours. The end of the reaction were monitored by LCMS. The reaction solution was purified by column chromatography to obtain off-white solid product 39 mg. LC-MS (ES+): m/z 483[M+H]+
4-bromo-2,3,5,6-tetrafluorobenzoic acid (92 mg), 3-chloro-4-((trifluoromethyl)thio) aniline (72 mg), pyridine (180 mg) and DCM (10 mL) were added to the 50 mL reaction flask, the solution was cooled to 0° C. then POCl3 (200 mg) was added dropwise. the reaction was carried out at room temperature for 2 hour The reaction solution was poured into ice water, and dichloromethane (50 mL) was added to the above solution. The reaction solution was washed with saturated NaCl solution. The organic phase was purified by silica gel column (PE/EA=1/1) to obtain product A344-1 (55 mg), LC-MS (ES+): m/z 481[M+H]+
A344-1 (55 mg), (3-ethynylpyridin-4-yl)boronic acid (40 mg), Pd(dppf)Cl2 (5 mg), K2CO3 (80 mg), dioxane (10 mL) and H2O (1 mL) were added to a 25 mL round flask and replaced with nitrogen. The reaction mixture was heated to 90° C. and reacted for 4 hours. The end of the reaction were monitored by LCMS. The reaction solution was diluted with EA (5 mL), washed with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A344 (16 mg), LC-MS (ES+): m/z 505[M+H]+.
2,3-dichloro-5-nitropyridine (400 mg), tetrahydrofuran-3-ol (160 mg), potassium carbonate (552 mg) and ethiperidine (20 mL) were added to a 25 mL round flask and heated to 80° C. and reacted for 3 hours. The reaction solution was cooled to room temperature and filtered. The reaction solution was diluted with EA (200 mL), washed with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A361-1 (110 mg), LC-MS (ES+): m/z 279 [M+H]+
A361-1 (110 mg), ethanol (10 mL) and Pd/C (40 mg) were added to a 50 mL round flask and replaced with hydrogen three times. The reaction was stirred at room temperature under hydrogen for 1 hours. The reaction solution was filtered and concentrated to obtain product A361-2 (60 mg), LC-MS (ES+): m/z 249[M+H]+
A361-2 (50 mg), 2′-amino-5-chloro-2,4′-difluoro-[1,1′-biphenyl]-4-carboxylic acid (48 mg), pyridine (90 mg) and DCM (10 mL) were added to a 50 mL reaction flask, and cooled to 0° C. And POCl3 (100 mg) was added dropwise. the reaction was carried out at room temperature for 2 hour. The reaction solution was poured into ice water. The reaction solution was diluted with DCM (50 mL), washed with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A361 (13 mg), LC-MS (ES+): m/z 514[M+H]+
2,3-dichloro-5-nitropyridine (400 mg), 2-oxo-7-azaspiro[4.4]nonane (250 mg), potassium carbonate (552 mg) and acetonitrile (20 mL) were added to a 50 mL reaction flask, and heated to 80° C. and reacted for 3 hours. The reaction solution was cooled to room temperature and filtered. The reaction solution was diluted with EA (200 mL), washed with saturated NaCl solution twice. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A371-1 (300 mg), LC-MS (ES+): m/z 284 [M+H]+
A371-1 (160 mg), ethanol (10 mL) and Pd/C (50 mg) were added to a 50 mL reaction flask and replaced with hydrogen three times. The reaction was stirred at room temperature under hydrogen for 1 hours. The reaction solution was filtered and concentrated to obtain product A371-2 (60 mg), LC-MS (ES+): m/z 254[M+H]+
A371-2 (50 mg), 2-chloro-4-(3-ethynylpyridin-4-yl)-5-fluorobenzoic acid (76 mg), pyridine (100 mg) and DCM (10 mL) were added to a 50 mL reaction flask, and stirred and cooled to 0° C. And POCl3 (105 mg) was added dropwise. the reaction was carried out at room temperature for 2 hour. The reaction solution was poured into ice water to quench the reaction. The reaction solution was diluted with DCM (50 mL), washed with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A371 (23 mg), LC-MS (ES+): m/z 511[M+H]+
2,3-dichloro-5-nitropyridine (400 mg), 7-azabicyclo[2.2.1]heptane-1-carbonitrile (212 mg), potassium carbonate (552 mg) and acetonitrile (20 mL) were added to a 50 mL reaction flask, and heated to 80° C. and reacted for 3 hours. The reaction solution was cooled to room temperature and filtered. The reaction solution was diluted with EA (200 mL), washed with saturated NaCl solution twice. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A373-1 (120 mg), LC-MS (ES+): m/z 279 [M+H]+
A373-1 (120 mg), ethanol (10 mL) and Pd/C (40 mg) were added to a 50 mL reaction flask and replaced with hydrogen three times. The reaction was stirred at room temperature under hydrogen for 1 hours. The reaction solution was filtered and concentrated to obtain product A373-2 (40 mg), LC-MS (ES+): m/z 249[M+H]+
A373-2 (40 mg), 2-chloro-4-(3-ethynylpyridin-4-yl)-5-fluorobenzoic acid (45 mg), pyridine (70 mg), and DCM (10 mL) were added to a 50 mL reaction flask, and cooled to 0° C. And POCl3 (75 mg) was added dropwise. the reaction was carried out at room temperature for 2 hour. The reaction solution was poured into ice water The reaction solution was diluted with DCM (50 mL), washed with saturated NaCl solution twice. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A373 (7 mg), LC-MS (ES+): m/z 506[M+H]+
2,3-Dichloro-5-nitropyridine (400 mg), (7-azabicyclo[2.2.1]heptane (196 mg), potassium carbonate (552 mg) and acetonitrile (20 mL) were added to a 50 mL reaction flask, and heated to 80° C. and reacted for 3 hours. The reaction solution was cooled to room temperature and filtered. The filtrate was diluted with EA (200 mL), washed with saturated NaCl solution twice. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A374-1 (150 mg), LC-MS (ES+): m/z 254 [M+H]+
A374-1 (150 mg), ethanol (10 mL) and Pd/C (50 mg) were added to a 50 mL reaction flask and replaced with hydrogen three times. The reaction was stirred at room temperature under hydrogen for 1 hours. The reaction solution was filtered and concentrated to obtain product A374-2 (60 mg), LC-MS (ES+): m/z 224[M+H]+
A374-2 (40 mg), 2-chloro-4-(3-ethynylpyridin-4-yl)-5-fluorobenzoic acid (55 mg), pyridine (70 mg) and DCM (10 mL) were added to a 50 mL reaction flask, and stirred and cooled to 0° C. And POCl3 (90 mg) was added dropwise. the reaction was carried out at room temperature for 2 hour. The reaction solution was poured into ice water The reaction solution was diluted with DCM (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A374 (6 mg), LC-MS (ES+): m/z 481[M+H]+
2,3-dichloro-5-nitropyridine (400 mg), (3aR,6aS)-5,5-difluorooctahydropenta[c]pyrrole (294 mg), potassium carbonate (552 mg) and acetonitrile (20 mL) were added to a 50 mL reaction flask, and heated to 80° C. and reacted for 3 hours. The reaction solution was cooled to room temperature and filtered. The reaction solution was diluted with EA (200 mL), washed with saturated NaCl solution twice. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A377-1 (180 mg), LC-MS (ES+): m/z 304 [M+H]+
A377-1 (150 mg), ethanol (10 mL) and Pd/C (50 mg) were added to a 50 mL reaction flask and replaced with hydrogen three times. The reaction was stirred at room temperature under hydrogen for 1 hours. The reaction solution was filtered and concentrated to obtain product A377-2 (70 mg), LC-MS (ES+): m/z 274[M+H]+
A377-2 (50 mg), 2-chloro-4-(3-ethynylpyridin-4-yl)-5-fluorobenzoic acid (55 mg), pyridine (70 mg) and DCM (10 mL) were added to a 50 mL reaction flask, and stirred and cooled to 0° C. And POCl3 (90 mg) was added dropwise. the reaction was carried out at room temperature for 2 hour. The reaction solution was poured into ice water The reaction solution was diluted with DCM (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A377 (11 mg), LC-MS (ES+): m/z 531[M+H]+
2,3-dichloro-5-nitropyridine (400 mg), 2,4,6,7-tetrahydro-5H-[1,2,3]triazolyl[4,5-c]pyridine-5-tert-butyl carboxylate (450 mg), potassium carbonate (552 mg) and acetonitrile (20 mL) were added to a 50 mL reaction flask, and heated to 80° C. and reacted for 3 hours. The reaction solution was cooled to room temperature and filtered. The reaction solution was diluted with EA (200 mL), washed with saturated NaCl solution twice. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A381-1 (406 mg), LC-MS (ES+): m/z 381 [M+H]+
A381-1 (200 mg), ethanol (10 mL) and Pd/C (100 mg) were added to a 50 mL reaction flask and replaced with hydrogen three times. The reaction was stirred at room temperature under hydrogen for 1 hour. The reaction solution was filtered and concentrated to obtain product A381-2 (110 mg), LC-MS (ES+): m/z 351[M+H]+
A381-2 (45 mg), 2-chloro-4-(3-ethynylpyridin-4-yl)-5-fluorobenzoic acid (50 mg), pyridine (70 mg) and DCM (10 mL) were added to a 50 mL reaction flask, and stirred and cooled to 0° C. And POCl3 (80 mg) was added dropwise. the reaction was carried out at room temperature for 2 hour. The reaction solution was poured into ice water The reaction solution was diluted with DCM (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A381 (7 mg), LC-MS (ES+): m/z 508 [M+H]+
2,3-dichloro-5-nitropyridine (400 mg), 4,5,6,7-tetrahydro-2H-indazole (245 mg), potassium carbonate (552 mg) and acetonitrile (20 mL) were added to a 50 mL reaction flask, and heated to 80° C. and reacted for 3 hours. The reaction solution was cooled to room temperature and filtered. The reaction solution was diluted with EA (200 mL), washed with saturated NaCl solution twice. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to obtain product A383-1 (310 mg), LC-MS (ES+): m/z 279 [M+H]+
A383-1 (310 mg), ethanol (10 mL) and Pd/C (100 mg) were added to a 50 mL reaction bottle and replaced with hydrogen three times. The reaction was stirred at room temperature under hydrogen for 1 hour. The reaction solution was filtered and concentrated to obtain product A383-2 (160 mg), LC-MS (ES+): m/z 249[M+H]+
A383-2 (45 mg, 0.18 mmol), 2-chloro-4-(3-ethynylpyridin-4-yl)-5-fluorobenzoic acid (50 mg, 0.18 mmol), pyridine (70 mg, 0.89 mmol) and DCM (10 mL) were added to a 50 mL reaction flask, and stirred and cooled to 0° C. And POCl3 (80 mg, 0.52 mmol) was added dropwise. After the dripping, the reaction was stirred at room temperature for 2 hours. The reaction solution was poured into ice water to quench the reaction. The reaction solution was diluted with DCM (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A383 (11 mg, 0.022 mmol), LC-MS (ES+): m/z 506 [M+H]+
2,3-dichloro-5-nitropyridine (400 mg, 2.08 mmol), 2-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine (400 mg, 2.08 mmol), potassium carbonate (552 mg, 4 mmol) and acetonitrile (20 mL) were added to a 50 mL reaction flask, and heated to 80° C. and reacted for 3 hours. The reaction solution was cooled to room temperature and filtered. The reaction solution was diluted with EA (200 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A388-1 (506 mg, 1.45 mmol), LC-MS (ES+): m/z 349 [M+H]+
A388-1 (480 mg, 1.38 mmol), MeOH (10 mL) and Pd/C (100 mg) were added to a 50 mL reaction bottle and replaced with hydrogen three times. Hydrogen was added and stirred for 1 hour at room temperature. The reaction solution was filtered and concentrated to obtain product A388-2 (300 mg, 0.94 mmol), LC-MS (ES+): m/z 319[M+H]+
A388-2 (50 mg, 0.16 mmol), 2-chloro-4-(3-ethynylpyridin-4-yl)-5-fluorobenzoic acid (43 mg, 0.16 mmol), pyridine (62 mg, 0.78 mmol) and DCM (10 mL) were added to a 50 mL reaction flask, and stirred and cooled to 0° C. And POCl3 (80 mg, 0.52 mmol) was added dropwise. After the dripping, the reaction was stirred at room temperature for 2 h. The reaction solution was poured into ice water to quench the reaction. The reaction solution was diluted with DCM (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A388 (20 mg, 0.035 mmol), LC-MS (ES+): m/z 576 [M+H]+
A392-1 (253 mg, 1 mmol), A392-2 (161 mg, 1.1 mmol), PdCl2(dppf)CH2Cl2 (10 mg, 0.012 mol), K2CO3 (276 mg, 2 mmol), dioxane (5 mL) and H2O (1 mL) were added to a 50 mL reaction bottle and replaced with nitrogen. The reaction mixture was heated to 90° C. and reacted for 4 hours. The end of the reaction were monitored by LCMS and stopped heating. The reaction solution was diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A392-3 (210 mg, 0.76 mmol), LC-MS (ES+): m/z 276[M+H]+.
A392-4 (202 mg, 1 mmol), A392-5 (158 mg, 1.2 mmol), HATU (570 mg, 1.5 mmol), DIEA (258 mg, 2 mmol), DMF (5 mL) were added to a 50 mL reaction flask, and heated to 80° C. and reacted for 3 hours. Then the reaction solution was stopped heating and cooled to room temperature. The reaction solution was diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A392-6 (187 mg, 0.59 mmol), LC-MS (ES+): m/z 317[M+H]+
A392-6 (158 mg, 2 mmol) and ethanol (5 mL), H2O (3 ml) were added to a 50 mL reaction flask and stirred. Then Fe powder (280 mg, 5 mmol) and NH4Cl (265 mg, 5 mmol) was added in batches. After the addition was completed, heating to 70° C. and reacting for 2 hours. Then the reaction solution was stopped heating and cooled to room temperature. The reaction solution was filtered and concentrated. The leftover was separated by silica gel column (DCM/MeOH=20/1) to obtain product A392-7 (103 mg, 0.36 mmol), LC-MS (ES+): m/z 287[M+H]+
A392-7 (50 mg, 0.17 mmol), A392-3 (48 mg, 0.17 mmol), pyridine (67 mg, 0.85 mmol) and DCM (5 mL) were added to a 50 mL reaction flask, and stirred at 0° C. And POCl3 (80 mg, 0.52 mmol) was added dropwise. After the dripping, the reaction was stirred at room temperature for 2 hours. The reaction solution was diluted with DCM (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A392 (38 mg, 0.15 mmol), LC-MS (ES+): m/z 544[M+H]+
A393-1 (202 mg, 1 mmol), A393-2 (124 mg, 1.1 mmol), HATU (570 mg, 1.5 mmol), DIEA (258 mg, 2 mmol) and DMF (5 mL) were added to a 50 mL reaction flask, and heated to 80° C. for 3 hours. Then the reaction solution was stopped heating and cooled to room temperature. The reaction solution was diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A393-3 (170 mg, 0.57 mmol), LC-MS (ES+): m/z 298[M+H]+
A393-3 (150 mg, 0.51 mmol) and ethanol (5 mL), H2O (3 ml) were added to a 50 mL reaction flask and stirred. Then Fe powder (280 mg, 5 mmol) and NH4Cl (265 mg, 5 mmol) was added in batches. After the addition was completed, heating to 70° C. and reacting for 2 hours. Then the reaction solution was stopped heating and cooled to room temperature. The reaction solution was filtered- and concentrated. The leftover was separated by silica gel column (DCM/MeOH=20/1) to obtain product A393-4 (105 mg, 0.39 mmol), LC-MS (ES+): m/z 268[M+H]+
A393-4 (47 mg, 0.18 mmol), A392-3 (48 mg, 0.16 mmol), pyridine (67 mg, 0.85 mmol) and DCM (5 mL) were added to a 50 mL reaction flask, and stirred and cooled to 0° C. Then POCl3 (78 mg, 0.52 mmol) was added dropwise. After the dripping, the reaction was stirred at room temperature for 2 hours. The reaction solution was diluted with DCM (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A393 (30 mg, 0.057 mmol), LC-MS (ES+): m/z 525[M+H]+
A395-1 (202 mg, 1 mmol), A395-2 (133 mg, 1.1 mmol), HATU (570 mg, 1.5 mmol), DIEA (258 mg, 2 mmol) and DMF (5 mL) were added to a 50 mL reaction flask, and heated to 80° C. for 3 hours. Then the reaction solution was stopped heating and cooled to room temperature. The reaction solution was diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A395-3 (150 mg, 0.49 mmol), LC-MS (ES+): m/z 306[M+H]+
A395-3 (150 mg, 0.49 mmol) and ethanol (5 mL), H2O (3 ml) were added to a 50 mL reaction flask and stirred. Then Fe powder (270 mg, 5 mmol), H2O (3 ml) was added in batches. After the addition was completed, heating to 70° C. for 2 hours. Then the reaction solution was stopped heating and cooled to room temperature. The reaction solution was filtered and concentrated. The leftover was separated by silica gel column (DCM/MeOH=20/1) to obtain product A395-4 (100 mg, 0.36 mmol), LC-MS (ES+): m/z 276[M+H]+
A395-4 (48 mg, 0.17 mmol), A392-3 (48 mg, 0.16 mmol), pyridine (67 mg, 0.85 mmol) and DCM (5 mL) were added to a 50 mL reaction flask, and stirred at 0° C. And POCl3 (78 mg, 0.51 mmol) was added dropwise. After the dripping, the reaction was stirred at room temperature for 2 hours. The reaction solution was diluted with DCM (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and separated by silica gel column (PE/EA=1/1) to obtain product A395 (22 mg, 0.041 mmol), LC-MS (ES+): m/z 533 [M+H]+
Into a 100-mL round-bottom flask, was placed A398-1 (192 mg), A398-2 (193 mg), DIEA (258 mg) and DMF (5 mL), and The resulting mixture was stirred at 100° C. for 3 hours. Then the reaction solution was cooled to room temperature, diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to give compound A398-3 (220 mg), LC-MS (ES+): m/z 333[M+H]+
To a solution of A398-3 (165 mg) in ethanol (5 mL) was added Fe (270 mg) in batches. The reaction was stirred at 70° C. for 2 hours. The reaction solution was filtered, removing of the solvent and purified by silica gel column (DCM/MeOH=20/1) to give compound A398-4 (120 mg), LC-MS (ES+): m/z 303[M+H]+
To a solution of A398-4 (52 mg), A392-3 (48 mg) in DCM (5 mL) was added pyridine (67 mg) at room temperature. The mixture was cooled to 0° C. in an ice bath. Then POCl3 (78 mg) was added dropwise. The reaction was stirred for 1 hour at room temperature and quenched by water, extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by silica gel column (PE/EA=1/1) to give compound A398 (40 mg), LC-MS (ES+): m/z 560 [M+H]+
To a solution of A399-1 (202 mg) in DMF (5 mL) was added A399-2 (193 mg), HATU (570 mg), DIEA (258 mg). The reaction was stirred at 80° C. for 3 hours. The reaction solution was diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to give compound A399-3 (136 mg), LC-MS (ES+): m/z 361[M+H]+
To a solution of A399-3 (180 mg) in ethanol (5 mL) was added Fe (270 mg) in batches. The reaction was stirred at 70° C. 2 hours. The reaction solution was filtered, removing of the solvent and purified by silica gel column (DCM/MeOH=20/1) to give compound A399-4 (122 mg), LC-MS (ES+): m/z 331[M+H]+
To a solution of A399-4 (57 mg) and A392-2 (48 mg) in DCM (5 mL) was added pyridine (67 mg) at room temperature. The mixture was cooled to 0° C. in an ice bath. Then POCl3 (78 mg) was added dropwise. The reaction was stirred for 2 hour at room temperature and quenched by water, extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by silica gel column (PE/EA=1/1) to give compound A399 (34 mg), LC-MS (ES+): m/z 588 [M+H]+
Step 1: Synthesis of Compound A402-3 Into a 100-mL round-bottom flask, was placed A402-1 (340 mg), A402-2 (268 mg), PdCl2(dppf)CH2Cl2 (10 mg), K2CO3 (552 mg), dioxane (10 mL) and H2O (1 mL). The resulting mixture was stirred at 100° C. for 2 hours under nitrogen.
Removing of the solvent and purified by silica gel column (PE/EA=3/1) to give compound A402-3 (230 mg), LC-MS (ES+): m/z 289[M+H]+.
Step 2: Synthesis of Compound A402 To a solution of A402-3 (50 mg), A402-4 (39 mg), pyridine (67 mg) in DCM (5 mL) was added pyridine (67 mg) at room temperature. The mixture was cooled to 0° C. in an ice bath. Then POCl3 (78 mg) was added dropwise. The reaction was stirred for 2 hour at room temperature and quenched by water, extracted with dichloromethane.
The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by silica gel column (PE/EA=1/1) to give compound A402 (28 mg), LC-MS (ES+): m/z 500 [M+H]+
Into a 100-mL round-bottom flask, was placed A404-1 (340 mg), A404-2 (98 mg), Pd(PPh3)2Cl2 (10 mg), CuI (10 mg), TEA (552 mg), dioxane (10 mL). The resulting mixture was stirred at 90° C. for 4 hours under nitrogen. The reaction solution was diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=1/1) to give compound A404-3 (160 mg), LC-MS (ES+): m/z 311[M+H]+.
Into a 100-mL round-bottom flask, was placed A404-3 (155 mg), A404-4 (110 mg), PdCl2(dppf)CH2Cl2 (10 mg), K2CO3 (138 mg), dioxane (5 mL) and H2O (1 mL). The resulting mixture was stirred at 90° C. for 4 hours under nitrogen. The reaction solution was diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=3/1) to give compound A404-5 (110 mg), LC-MS (ES+): m/z 334[M+H]+.
To a solution of A404-5 (50 mg), A404-6 (29 mg) in DCM (5 mL) was added pyridine (67 mg) at room temperature. The mixture was cooled to 0° C. in an ice bath. Then POCl3 (78 mg) was added dropwise. The reaction was stirred for 2 hour at room temperature and quenched by water, extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by silica gel column (PE/EA=1/1) to give compound A404 (17 mg), LC-MS (ES+): m/z 439 [M+H]+
Into a 100-mL round-bottom flask, was placed A405-1 (340 mg), A405-2 (86 mg), PdCl2(dppf)CH2Cl2 (10 mg), K2CO3 (276 mg), dioxane (5 mL) and H2O (1 mL). The resulting mixture was stirred at 90° C. for 4 hours under nitrogen. The reaction solution was diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=5/1) to give compound A405-3 (110 mg), LC-MS (ES+): m/z 254,256[M+H]+.
Into a 100-mL round-bottom flask, was placed A405-3 (110 mg), A405-4 (95 mg), PdCl2(dppf)CH2Cl2 (10 mg), K2CO3 (130 mg), dioxane (5 mL) and H2O (1 mL). The resulting mixture was stirred at 90° C. for 4 hours under nitrogen. The reaction solution was diluted with EA (50 mL), washed twice with saturated NaCl solution. The organic phase was concentrated and purified by silica gel column (PE/EA=3/1) to give compound A405-5 (60 mg), LC-MS (ES+): m/z 278 [M+H]+.
To a solution of A405-5 (50 mg), A405-6 (35 mg), pyridine (70 mg) in DCM (5 mL) was added pyridine (70 mg) at room temperature. The mixture was cooled to 0° C. in an ice bath. Then POCl3 (82 mg) was added dropwise. The reaction was stirred for 2 hour at room temperature and quenched by water, extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum. The residue was purified by silica gel column (PE/EA=1/1) to give compound A405 (33 mg), LC-MS (ES+): m/z 455 [M+H]+.
According to the synthesis method of the above example, select appropriate raw materials and/or marketed example intermediates to synthesize the example compounds in the table below. The raw materials and reagents used are all commercially available.
Under an ice-water bath, POCl3 (0.31 g, 2.0 mmoL) was added dropwise to a DCM (10 mL) solution containing SM1 (0.34 g, 1.0 mmoL), SM2 (0.20 g, 1.0 mmoL) and pyridine (0.84 g, 10 mmoL). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was diluted with DCM (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product 605-1 (0.46 g, 0.89 mmoL) as yellow solid. LC-MS (ES+): m/z 517[M+H]+
SM3 (0.1 g, 1.0 mmoL), 605-1 (0.26 g, 0.5 mmoL), CuI (2.0 mg, 0.01 mmoL), Pd(PPh3)2Cl2 (3.5 mg, 0.005 mmoL) and TEA (0.3 g, 3.0 mmoL) were added in sequence to a single-neck bottle containing THF (10 mL) and a stirrer. The reaction liquid container were replaced with nitrogen. The reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product 605-2 (0.21 g, 0.4 mmoL) as white solid. LC-MS (ES+): m/z 488 [M+H]+
SM4 (0.14 g, 0.6 mmoL), 605-2 (0.21 g, 0.4 mmoL), Pd(dppf)Cl2 (14.6 mg, 0.02 mmoL) and K2CO3 (0.12 g, 0.8 mmoL) were added in sequence to a single-neck bottle containing dioxane (4 mL), H2O (1 mL) and a stirrer. The reaction liquid container were replaced with nitrogen. The reaction mixture was stirred in a 90° C. oil bath for 8 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product A409 (0.11 g, 0.24 mmoL) as white solid. LC-MS (ES+): m/z 447 [M+H]+
For the synthesis method of 677-2, please refer to the synthesis of A310-3.
3-bromo-5-fluoro-2-iodoaniline (0.16 g, 0.5 mmoL), 677-2 (0.12 g, 0.3 mmoL), Pd(dppf)Cl2 (14.6 mg, 0.02 mmoL) and K2CO3 (0.12 g, 0.8 mmoL) were added in sequence to a single-neck bottle containing dioxane (4 mL), H2O (1 mL) and a stirrer. The reaction liquid container were replaced with nitrogen. The reaction mixture was stirred in a 90° C. oil bath for 8 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product 586-1 (0.065 g, 0.12 mmoL) as white solid. LC-MS (ES+): m/z 538/540 [M+H]+
ethynyltrimethylsilane (0.06 g, 0.6 mmoL), 586-1 (0.065 g, 0.12 mmoL), CuI (2.0 mg, 0.01 mmoL), Pd(PPh3)2Cl2 (3.5 mg, 0.005 mmoL) and TEA (0.3 g, 3.0 mmoL) were added in sequence to a single-neck bottle containing THF (4 mL) and a stirrer. The reaction liquid container were replaced with nitrogen. The reaction mixture was stirred at room temperature for 5 hours, and then K2CO3 (0.42 g, 3 mmoL) and MeOH (10 mL) were added to the reaction mixture and stirring was continued for 2 hours.
After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product A412 (0.05 g, 0.1 mmoL) as white solid. LC-MS (ES+): m/z 485[M+H]+
Under an ice-water bath, POCl3 (0.31 g, 2.0 mmoL) was added dropwise to a DCM (10 mL) solution containing 4-bromo-2-chloro-5-fluorobenzoic acid (0.25 g, 1.0 mmoL), 6-(2H-1,2,3-triazol-2-yl)-5-(trifluoromethyl)pyridine-3-amine (0.20 g, 1.0 mmoL) and pyridine (0.84 g, 10 mmoL). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was diluted with DCM (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product 550-1 (0.42 g, 0.9 mmoL) as white solid. LC-MS (ES+): m/z 463/465[M+H]+
(2-chloro-6-formylphenyl)boronic acid (0.092 g, 0.5 mmoL), 550-1 (0.092 g, 0.3 mmoL), Pd(dppf)Cl2 (14.6 mg, 0.02 mmoL) and K2CO3 (0.12 g, 0.8 mmoL) were added in sequence to a single-neck bottle containing dioxane (4 mL), H2O (1 mL) and a stirrer. The reaction liquid container were replaced with nitrogen. The reaction mixture was stirred in a 90° C. oil bath for 8 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product 550-2 (0.11 g, 0.21 mmoL) as white solid. LC-MS (ES+): m/z 524[M+H]+
(1-dizo-2-oxopropyl)phosphonate dimethyl ester (0.11 g, 0.5 mmoL), 550-2 (0.11 g, 0.21 mmoL) and K2CO3 (0.12 g, 0.8 mmoL) were added in sequence to a single-neck bottle containing MeOH (4 mL) and a stirrer. The reaction mixture was stirred at room temperature for 4 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product A413 (0.08 g, 0.15 mmoL) as white solid. LC-MS (ES+): m/z 520[M+H]+
(4-fluoro-2-formylphenyl)boronic acid (0.09 g, 0.5 mmoL), 605-2 (0.21 g, 0.4 mmoL), Pd(dppf)Cl2 (14.6 mg, 0.02 mmoL) and K2CO3 (0.12 g, 0.8 mmoL) were added in sequence to a single-neck bottle containing dioxane (4 mL), H2O (1 mL) and a stirrer. The reaction liquid container were replaced with nitrogen. The reaction mixture was stirred in a 90° C. oil bath for 8 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product 597-1 (0.09 g, 0.2 mmoL) as white solid. LC-MS (ES+): m/z 460[M+H]+
(1-dizo-2-oxopropyl)phosphonate dimethyl ester (0.11 g, 0.5 mmoL), 597-1 (0.09 g, 0.2 mmoL) and K2CO3 (0.12 g, 0.8 mmoL) were added in sequence to a single-neck bottle containing MeOH (4 mL) and a stirrer. The reaction mixture was stirred at room temperature for 4 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product A415 (0.06 g, 0.15 mmoL) as white solid. LC-MS (ES+): m/z 456[M+H]+
propyne (0.1 g, 2.5 mmoL), 605-1 (0.26 g, 0.5 mmoL), CuI (2.0 mg, 0.01 mmoL), Pd(PPh3)2Cl2 (3.5 mg, 0.005 mmoL) and TEA (0.3 g, 3.0 mmoL) were added in sequence to a single-neck bottle containing THF (10 mL) and a stirrer. The reaction liquid container were replaced with nitrogen. The reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product 651-1 (0.13 g, 0.3 mmoL) as white solid. LC-MS (ES+): m/z 430/432[M+H]+
5-fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) aniline (0.12 g, 0.5 mmoL), 651-1 (0.13 g, 0.3 mmoL), Pd(dppf)Cl2 (14.6 mg, 0.02 mmoL) and K2CO3 (0.12 g, 0.8 mmoL) were added in sequence to a single-neck bottle containing dioxane (4 mL), H2O (1 mL) and a stirrer. The reaction liquid container were replaced with nitrogen. The reaction mixture was stirred in a 90° C. oil bath for 8 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product A423 (0.09 g, 0.2 mmoL) as white solid. LC-MS (ES+): m/z 461[M+H]+
Vinyl magnesium bromide (2.0 mL, 2.0 mmoL) was added dropwise to a THF (10 mL) solution containing 605-1 (0.52 g, 1.0 mmoL) at −70° C. The reaction mixture was stirred at −70° C. for 2 hours. After the reaction was completed, the reaction solution was quenched saturated ammonium chloride solution (30 mL) and extracted with DCM (50 mL). The organic phase was washed with saturated brine (3*50 mL) and dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product 662-1 (0.28 g, 0.7 mmoL) as white solid. LC-MS (ES+): m/z 418/420[M+H]+
5-fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) aniline (0.14 g, 0.6 mmoL), 662-1 (0.17 g, 0.4 mmoL), Pd(dppf)Cl2 (14.6 mg, 0.02 mmoL) and K2CO3 (0.12 g, 0.8 mmoL) were added in sequence to a single-neck bottle containing dioxane (4 mL), H2O (1 mL) and a stirrer. The reaction liquid container were replaced with nitrogen. The reaction mixture was stirred in a 90° C. oil bath for 8 hours. After the reaction was completed, the reaction solution was diluted with EA (50 mL), washed with saturated brine (3*50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated and separated using a Flash silica gel column (PE/EA=2/1) to obtain product A425 (0.12 g, 0.3 mmoL) as white solid. LC-MS (ES+): m/z 449 [M+H]+.
Into a 25-mL round-bottom flask, was placed A558 (100 mg, 0.17 mmol), ethynyltrimethylsilane (16.62 mg, 0.17 mmol), Pd(PPh3)2Cl2 (11.86 mg, 0.017 mmol), TEA (34.24 mg, 0.0 mmol), DMF (2 mL). The resulting mixture was stirred at 80° C. for 16 hours under nitrogen. Removing of the solvent and purifying by column chromatography (PE/EA=2/1) to give compound 711-4-P1 (LC-MS (ES+): m/z 470[M+H]) and 711-4-P2 (LC-MS (ES+): m/z 652/654 [M+H]) mixture (40 mg) as white solid.
To a solution of 711-4-P1 and 711-4-P2 (40 mg) in MeOH (5.0 mL) was added KF (3.56 mg, 0.06 mmol). The resulting mixture was stirred at 50° C. for 2 hours, solvent was removed in vacuo and the mixture was purified by pre-HPLC to give compound A511 (1.1 mg) (LC-MS (ES+): m/z 526 [M+H]+) and A510 (1.3 mg) (LC-MS (ES+): m/z 579/581 [M+H]0) as white solids.
Into a 250-mL three-neck flask, 2,2,6,6-tetramethylpiperidine (3.98 g, 28.18 mmol) was dissolved in THF (20 mL) under nitrogen, and n-butyl lithium (2.5 mol/L in THF, 10.9 ml 27.27 mmol) was dropwised at −50° C. Keeping −50° C. for half an hour after dropwise, a solution of 5-bromo-6-fluoronicotinic acid (2.00 g, 9.09 mmol) in THF (20 mL) was dropwised, slowly raising the temperature to −20° C., keeping at −20° C. for 1 h, cooling the reaction to −60° C., 12 (3.46 g, 13.63 mmol) dissolved in THF (20.0 mL) was dropwised. After the reaction completed, it is diluted with 100 mL of water and extracted with EA (3*30 mL). Keep the water phase, adjust the pH to 3 with 3N HCl, then extract with EA (3*50 mL), The organic phase wash with saturated brine (3*30 mL), dry with anhydrous sodium sulfate and concentrate to obtain the product 002-40 (1.5 g, 4.35 mmol) as a red solid. LC-MS (ES+): m/z 345/347 [M+H]+
To a solution of 002-40 (1.5 g, 4.35 mmol) in toluene (15.0 mL) and tert-butanol (15 mL) was added DPPA (1.794 g, 6.52 mmol) and TEA (1.318 g, 13.05 mmol). The resulting mixture was stirred at 110° C. for 2 hours under nitrogen, solvent was removed in vacuo and the mixture was separated on a silica gel column with PE:EA=96:4 to give compound 002-41 (1.5 g, 3.59 mmol) as a brown solid. LC-MS (ES+): m/z 416/418 [M+H]+
To 002-41 (0.6 g, 1.44 mmol) in 1,4-dioxane (6.0 mL) and H2O (1 mL) was added A315-3 (1.104 g, 2.16 mmoL), Pd(dppf)Cl2 (105.29 mg, 0.15 mmoL) and K2CO3 (0.39 g, 2.88 mmoL). The resulting mixture was stirred at 80° C. for 16 hours under nitrogen. Water was added, and the organic layer was extracted with EtOAc (3*20 mL). The organic layers were combined, washed with brine (3*50 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure to give compound 002-42 (0.13 g, 0.19 mmoL) as white solid. LC-MS (ES+): m/z 674/676 [M+H]+
To 002-42 (70 mg, 0.1 mmol) in THF (2.0 mL) was added ethynyltrimethylsilane (12.25 mg, 0.12 mmol), Pd(PPh3)2Cl2 (7.3 mg, 0.01 mmol) and TEA (20.99 mg, 0.21 mmol). The resulting mixture was stirred at 80° C. for 3 hours under nitrogen. Water was added, and the organic layer was extracted with EtOAc (3*20 mL). The organic layers were combined, washed with brine (3*50 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure and the mixture was separated on a silica gel column with PE:EA=2/1 to give compound 002-43 (85 mg, 0.12 mmol) as yellow oil. LC-MS (ES+): m/z 692 [M+H]+
To 002-43 (85 mg, 0.12 mmol) in DCM (2.0 mL) was added TFA (0.14 mL). The resulting mixture was stirred at room temperature for 2 hours, after the reaction completed, it is diluted with EA (20 mL). The mixture was adjusted the pH to 9 with saturated NaHCO3 solution, then extract with EA (2*30 mL), The organic phase wash with saturated brine (3*30 mL), dry with anhydrous sodium sulfate and filtered. The filter was under reduced pressure to give compound 002-44 (70 mg, 0.12 mmol) as yellow solid. LC-MS (ES+): m/z 592 [M+H]+
To 002-44 (60 mg, 0.1 mmol) in MeOH (2.0 mL) was added K2CO3 (19.60 mg, 0.14 mmol). The resulting mixture was stirred at 50° C. for 10 minutes, solvent was removed in vacuo and the mixture was separated on pre-HPLC to give compound A520 (12 mg, 0.023 mmol) as white solid. LC-MS (ES+): m/z 520 [M+H]+
To A558 (60.0 mg, 0.102 mmol) in toluene (10.0 mL) and H2O (1.0 mL) was added tert-butyldimethyl ((4,4,5,5-tetramethyl-1,3,2-dioxobenzaldehyde-2-yl)ethynyl)silane (50.0 mg, 0.36 mmol), [PdCl2(dppf)]CH2Cl2 (30 mg, 0.037 mmol) and K2CO3 (0.39 g, 2.88 mmol). The resulting mixture was stirred at 95° C. for 16 hours under nitrogen. Water was added, and the organic layer was extracted with EtOAc (3*20 mL). The organic layers were combined, washed with brine (3*50 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure to give compound A553-1 (35 mg, 0.054 mmol) as white solid. LC-MS (ES+): m/z 650.25 [M+H]+
To A553-1 (200 mg, 0.308 mmol) in THF (10.0 mL) was added TBAF (0.1M in THF, 1.0 ml). The resulting mixture was stirred at room temperature for 30 minutes, solvent was removed in vacuo and the mixture was separated on pre-HPLC to give compound A553 (92 mg, 0.0.172 mmol) as white solid. LC-MS (ES+): m/z 536.10 [M+H]+.
1H NMR (500 MHz, DMSO-d6) δ 11.57 (s, 1H), 9.16 (d, J=2.4 Hz, 1H), 8.91 (d, J=2.4 Hz, 1H), 8.22 (s, 2H), 7.90-7.84 (m, 2H), 7.70 (d, J=6.1 Hz, 1H), 5.59 (s, 2H), 4.33 (s, 1H).
To A555-1 (1.00 g, 5.73 mmol) in DMF (10.0 mL) was added sodium methoxide (5.16 g, 30% wt, 28.65 mmol). The resulting mixture was stirred at 100° C. for 2.5 hours. Water was added, and the organic layer was extracted with EtOAc (3*20 mL). The organic layers were combined, washed with brine (3*50 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure to give compound A555-2 (0.90 g, 92.34% yield) as a yellow powder. LC-MS (ES+): m/z 171.0 [M+H]+.
To A555-2 (0.500 g, 2.94 mmol) in glacial acetic acid (10.0 mL) was added a solution of bromine (0.17 mL, 3.23 mmol) in glacial acetic acid (5 mL) slowly at room temperature. The resulting mixture was stirred at room temperature for 16 hours. Water was added, the mixture is filtered, collect the filter cake and dry to give compound A555-3 (465 mg, 63.53% yield) as a yellow solid. LC-MS (ES+): m/z 249/251 [M+H]+.
To A555-3 (115 mg, 0.46 mmol) in acetonitrile (2.0 mL) was added phosphorus oxychloride (0.22 mL, 2.31 mmol) at room temperature. The resulting mixture was stirred at 85° C. for 2 hours. Water was added, the mixture is filtered, collect the filter cake and dry to give compound A555-4 (55 mg, 44.53% yield) as a yellow solid. LC-MS (ES+): m/z 249/251 [M+H]+.
To A555-4 (200 mg, 0.75 mmol) in DMF (5.0 mL) was added potassium iodide (1.24 g, 7.48 mmol). The resulting mixture was stirred at 100° C. for 5 hours. Water was added, and the organic layer was extracted with EtOAc (3*20 mL). The organic layers were combined, washed with brine (3*50 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure the mixture was separated on a silica gel column with PE:EA=1/1 to give compound A555-5 (78 mg, 29.06% yield) as a yellow solid. LC-MS (ES+): m/z 359/361 [M+H]+.
To A555-5 (240 mg, 0.67 mmol) in EtOH/H2O (5 mL/1 mL) was added Fe(187 mg, 3.34 mmol) and NH4Cl (179 mg, 3.34 mmol). The resulting mixture was stirred at 60° C. for 4 hours. The mixture was filtered. The filter was under reduced pressure the mixture was separated on a silica gel column with PE:EA=3/2 to give compound A555-6 (185 mg, 71.49% yield) as a yellow solid. LC-MS (ES+): m/z 329/331 [M+H]+.
To A558 (60.0 mg, 0.102 mmol) in 1,4-dioxane/water (5 mL/1 mL) was added A315-3 (418 mg, 0.97 mmoL), Pd(dppf)Cl2 (60 mg, 0.07 mmoL) and K2CO3 (134 mg, 0.97 mmoL). The resulting mixture was stirred at 80° C. for 2 hours under nitrogen. Water was added, and the organic layer was extracted with EtOAc (3*20 mL). The organic layers were combined, washed with brine (3*50 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure and the mixture separated on a silica gel column with PE:EA=0/1 to give compound A555-7 (120 mg, 42.05% yield) as a yellow solid. LC-MS (ES+): m/z 586/588 [M+H]+
To A555-7 (100 mg, 0.17 mmol) in 1,4-dioxane/water (2 mL/0.2 mL) was added A555-8 (68 mg, 0.26 mmoL), Pd(dppf)Cl2·CH2Cl2 (28 mg, 0.03 mmoL) and K2CO3 (71 mg, 0.51 mmoL). The resulting mixture was stirred at 80° C. for 6 hours under nitrogen. Water was added, and the organic layer was extracted with EtOAc (3*20 mL). The organic layers were combined, washed with brine (3*50 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure and the mixture separated on a silica gel column with DCM/MeOH=20/1 to give compound A555-9 (25 mg, 22.71% yield) as a yellow solid. LC-MS (ES+): m/z 646 [M+H]+
To A555-9 (25 mg, 0.04 mmol) in THF (1.0 mL) was added TBAF (0.1M in THF, 1.0 ml). The resulting mixture was stirred at room temperature for 60 minutes, solvent was removed in vacuo and the mixture was separated on pre-HPLC to give compound A555 (6.6 mg, 32.07%) as a yellow solid. LC-MS (ES+): m/z 532[M+H]+.
1H NMR (500 MHz, DMSO-d6) δ 11.50 (s, 1H), 9.17 (d, J=2.4 Hz, 1H), 8.89 (d, J=2.4 Hz, 1H), 8.22 (s, 2H), 7.89 (d, J=9.0 Hz, 2H), 7.74 (d, J=6.0 Hz, 1H), 5.82 (s, 2H), 4.08 (s, 1H), 3.83 (s, 3H).
To 5-bromo-2,4-dichloro-3-nitropyridine (2 g, 7.35 mmol) in DMF (20.0 mL) was added KI (3.67 g, 22.05 mmol). The resulting mixture was stirred at 100° C. for 16 hours. Water was added, the mixture is filtered, the filter cake wash with ice water (3*10 mL), collect the filter cake and dry to give compound 771-1 (2.7 g, 7.43 mmol) as a gray solid. LC-MS (ES+): m/z 362/364 [M+H]+.
To 711-1 (2.7 g, 7.43 mmoL) in EtOH (20.0 mL) and H2O (20.0 mL) was added Fe2.08 g, 37.2 mmoL) and NH4Cl (2.0 g, 37.2 mmol). The resulting mixture was stirred at 40° C. for 3 hours. The mixture was filtered. The filter was extracted with EtOAc (3*50 mL). The organic layers were combined, washed with saturated sodium bicarbonate solution (100 ml) and brine (100 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure and the mixture separated on a silica gel column with PE/EA=90/10 to give compound 711-2 (1.2 g, 3.6 mmoL) as a white solid. LC-MS (ES+): m/z 332/334 [M+H]+.
To A315-3 (2.76 g, 5.4 mmoL) in 1,4-dioxane/water (30 mL/3.0 mL) was added 711-2 (1.2 g, 3.6 mmoL), Pd(dppf)Cl2 (26.35 mg, 0.036 mmoL) and K2CO3 (0.99 g, 7.2 mmoL). The resulting mixture was stirred at 80° C. for 16 hours under nitrogen. Water was added, and the organic layer was extracted with EtOAc (3*20 mL). The organic layers were combined, washed with brine (3*50 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure and the mixture separated on a silica gel column with DCM/EA=3/1 to give compound A558 (0.48 g, 0.8 mmoL) as white solid. LC-MS (ES+): m/z 589/591 [M+H]+.
To A558 (100 mg, 0.17 mmol) in DMSO (2.0 mL) was added CSF (51.40 mg, 0.34 mmol). The resulting mixture was stirred at 130° C. for 16 hours. Water was added, and the organic layer was extracted with EtOAc (3*20 mL). The organic layers were combined, washed with brine (3*50 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure to give compound A560 (15 mg, 0.026 mmol) as white solid. LC-MS (ES+): m/z 569/571 [M+H]+.
To A560 (15 mg, 0.17 mmol) in THF (2.0 mL) was added ethynyltrimethylsilane (2.6 mg, 0.026 mmol), Pd(PPh3)2Cl2 (1.8 mg, 0.0026 mmol) and TEA (5.3 mg, 0.05 mmol). The resulting mixture was stirred at 80° C. for 16 hours under nitrogen.
Water was added, and the organic layer was extracted with EtOAc (3*10 mL). The organic layers were combined, washed with brine (3*10 mL), dried over Na2SO4, and filtered. The filter was under reduced pressure to give compound 717-1 (20 mg, 0.03 mmol) as white solid. LC-MS (ES+): m/z 588 [M+H]+.
To 717-1 (20 mg, 0.03) in MeOH (1.0 mL) was added K2CO3 (9.40 mg, 0.07 mmol). The resulting mixture was stirred at 50° C. for 2 hours, solvent was removed in vacuo and the mixture was separated on pre-HPLC to give compound A559 (2.0 mg) as a white solid. LC-MS (ES+): m/z 516 [M+H]+.
According to the synthesis method of the above example, select appropriate raw materials and/or marketed example intermediates to synthesize the example compounds in the table below. The raw materials and reagents used are all commercially available.
1H NMR (500 MHz, DMSO-d6) δ 11.65 (s, 1H), 9.17 (d, J=2.4 Hz, 1H), 8.93 (d, J=2.4 Hz, 1H), 8.25-8.15 (m, 4H), 7.91 (d, J=9.0 Hz, 1H), 7.71 (d, J=6.0 Hz, 1H), 5.76 (s, 2H), 4.50 (s, 1H).
1H NMR (500 MHz, DMSO-d6) δ 11.57 (s, 1H), 9.15 (d, J=2.4 Hz, 1H), 8.91 (d, J=2.4 Hz, 1H), 8.21 (d, J=1.7 Hz, 2H), 7.95-7.83 (m, 2H), 7.69 (d, J=6.0 Hz, 1H), 5.73 (s, 2H).
1H NMR (500 MHz, DMSO-d6) δ 11.60 (s, 1H), 9.14 (d, J=2.4 Hz, 1H), 8.91 (d, J=2.4 Hz, 1H), 8.21 (s, 2H), 7.88 (d, J=9.0 Hz, 1H), 7.75-7.66 (m, 2H), 5.29 (s, 2H), 4.50 (s, 1H).
Test compounds of the present invention (in 100% DMSO) were spotted into a 384-well dilution plate and serially diluted to the required concentrations with DMSO. Then, 0.2 μL of the test compounds were transferred into a new 384-well reaction plate by Echo following centrifugation. The MALT enzyme and its substrate, AMC-labeled peptide, were prepared in 1× protease buffer to yield 2× enzyme solution and 2× peptide solution. Subsequently, 10 μL of 2× enzyme solution was added into the 384-well reaction plate, incubating with the test compounds for 15 minutes at 25° C. After that, 10 μL of 2× peptide solution was added into the plate, continuing to incubate for 90 minutes at 25° C. The plate was then placed on an Envision multifunctional microplate reader to collect raw data, which would be converted into inhibition rate:
Inhibition rate %=(max−sample)/(max−min)*100%.
Curve fitting was done with Graphpad Prism software to determine the IC50 values. The specific experimental operations are as follows:
58.82 g Na Citrate·2H2O powder was added into 140 mL ddH2O and mixed in a 37° C. water bath. Once there were no visible particles, the remaining solutions were added successively with continuous stirring for 10 minutes. The final volume was adjusted to 200 mL. The buffer was stored at room temperature. DTT was added before use.
The results are expressed with IC50, wherein “A” stands for “IC50≤10 nM”, and “B” stands for “10 nM<IC50≤100 nM”, and “C” stands for “100 nM<IC50≤500 nM”, and “D” stands for “IC50>500 nM”. The IC50 data for MALT1 inhibition activity of representative compounds are shown in Table 1.
According to Table 1, the compounds of the present invention exhibit strong inhibitory activities on MALT1.
12.5*103 Jurkat cells were seeded on a 96-well plate and incubated with various concentrations of compound solution for 30 min. Then, 500×PMA/Ionomycin stimulating factor was diluted with culture medium and added to the cell plate. The final concentrations of PMA/Ionomycin were 81 nM and 1.34 M respectively. The final concentrations of the compounds were 10000, 3333.3, 1111.1, 370.4, 123.5, 41.2, 13.7, 4.6, 1.5, 0 nM (The final concentration of DMSO is 0.5%). After incubation for 20 h, the supernatant was collected via centrifugation. The final IL-2 concentration was detected by ELISA method (IL-2 Duoset, R&D Systems, DY202). The signal value of each sample at a wavelength of 450 nm was collected by EnVision multifunctional microplate reader and converted to the IL-2 concentration via the ELISACalc.exe software. The IL-2 secretion inhibitory activity IC50 was fitted by GraphPad Prism software. The cell viability was tested simultaneously using the Cell-Titer Glo kit.
The compounds of the present invention show strong inhibitory effect on IL-2 secretion of Jurkat cells (PMA/Ionomycin inducing). The IC50 data of representative compounds for inhibiting IL-2 secretion of Jurkat cells are shown in Table 2.
1900 mg MgCl2 was dissolved into a final volume of 400 mL ultrapure water. 17.42 g K2HPO4 and 13.65 g KH2PO4 were dissolved respectively into ultrapure water with a final volume of 1000 mL.
The K2HPO4 and KH2PO4 stock solutions above were mixed with pH adjusted to 7.30±0.10 to obtain 100 mM potassium phosphate (K-PBS) buffer.
The stop solution was acetonitrile containing 1 ng/mL labetalol and 1 ng/mL glyburide, and stored at 4° C.
The verapamil (positive control) and test sample stock solution were diluted to 50 μM and 200 μM respectively with MeOH/ACN/H2O solution (1:1:2, volume ratio).
The T1/2 and CL was calculated using the first-order kinetic equation:
C
t
=C
0
*e−Kt
C
t=(½)*C0
T
1/2=ln 2/k=0.693/k
Slope k was determined from natural logarithmic linear regression of percent parent drug remaining versus incubation time.
The in vitro half-life (in vitro T1/2) was calculated from the slope value:
The in vitro intrinsic clearance rate (in vitro CLint, in μL/min/mg proteins) was calculated according to the following formula (repeated average):
Test positive control: verapamil.
Any compound value not within the specified range would be excluded and repeat experiment.
The compounds of the present invention had moderate or slow metabolism in three species: humans, rats, and mice, and the compounds of the present invention had good stability in liver microsomes in vitro of humans, rats.
The female BALB/c mice (20-30 g) was purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. to study the pharmacokinetic characteristics in mice (intravenous administration of 1 mg/kg and intragastric administration of 5 mg/kg).
The blood was collected at 5 min (intravenous administration only), 15 min, 30 min, 1 h, 2 h, 4 h, 7 h and 24 h after administration through the orbital venous plexus into a centrifuge tube containing EDTA anticoagulant, and 100 μL of whole blood was collected at each time point. After the centrifugation, the plasma was separated and stored in a −80° C. refrigerator until analysis.
The above samples were processed by the protein precipitation method. The standard curve and other samples were also prepared in the same way. The precipitated samples were centrifuged at 3200 rpm for 10 minutes at 4° C. The supernatant was aspirated and mixed with water 1:1, and then analyzed by LC-MS/MS. The quantitative limit of the standard curve of this compound in BALB/c mouse plasma was 1.00-1000 ng/mL.
The non-compartmental model of Phoenix WinNonlin software was used to calculate the pharmacokinetic parameters of animals after drug administration.
The compound of the present invention had higher exposure and higher bioavailability after oral administration in female BALB/c mice.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/104458 | Jul 2021 | WO | international |
| PCT/CN2021/117357 | Sep 2021 | WO | international |
| 202210383102.0 | Apr 2022 | CN | national |
| 202210715786.X | Jun 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/101146 | 6/24/2022 | WO |
