Patent Application
20220402867
The disclosure belongs to the technical field of chemical medicines, in particular to a sulfo-substituted biaryl compound with RORγt agonistic activity, a preparation method thereof, a pharmaceutical composition containing the compound, and use of the sulfo-substituted biaryl compound in the preparation of a medicament for treating diseases related to RORγt.
Tumor immunotherapy has recently attracted much attention and has become the focus of the field of tumor therapy. Tumor immunotherapy is to control and kill tumor cells by mobilizing the body's immune system and enhancing the anti-tumor immunity of the tumor microenvironment. It targets the human immune system rather than the tumor directly. In recent years, good news of tumor immunotherapy keeps coming, showing strong anti-tumor activity so far in the treatment of some tumor types such as melanoma and non-small cell lung cancer, and there have been tumor-immune monoclonal antibody agents approved by FDA for clinical use. Tumor immunotherapy was named “The Most Important Scientific Breakthrough” of the year by the journal Science in 2013 due to its excellent efficacy and innovativeness. Due to their original discoveries in the field of tumor immunity, American scientist James P. Allison and Japanese scientist Tasuku Honjo were awarded the 2018 Nobel Prize in Physiology or Medicine. Therefore, tumor immunotherapy is expected to become an innovation in the field of tumor treatment, following surgery, chemotherapy, radiotherapy and targeted therapy.
Helper T cells 17 (Th17) is a newly discovered subtype of helper T cells, mainly secreting interleukin-17 (IL-17), and has been found to play an important role in the development of autoimmunity and inflammation. At present, researches have found that Th17 cells widely exist in tumor tissues, but nothing is known about the function of Th17 cells in them. In 2009, Professor Dong Chen published an article in Immunity (Immunity 2009, 31, 787-798), mainly analyzing that Th17 cells can promote the activation of cytotoxic T cells to exert tumor immune function. Their research found that mice deficient in IL-17A were more prone to develop pulmonary melanoma. If T-cell therapy is administered to mice, treatment with IL-17A-secreting T cells can effectively prevent tumor development. More importantly, with the aid of IL-17A, Th17 cells showed stronger therapeutic efficacy than Th1 cells. What is even more surprising is that, the use of Th17 cell therapy can also effectively activate tumor-specific CD8+ T cells, wherein, CD8+ T cells are essential cells for anti-tumor. Th17 cells can recruit dendritic cells into tumor tissue, and can induce CD8α+ dendritic cells to accumulate into tumor tissue. In addition, Th17 cells activate the chemokine CCL20 in tumor tissue. Overall, Th17 cells can effectively promote the activity of tumor-specific CD8+ T cells. These new findings broaden the horizons of tumor immunotherapy.
Retinoid acid receptor-related orphan receptor (ROR), also known as NF1R, is a member of the ligand-dependent transcription factor nuclear receptor (NR) superfamily. The RORs subfamily mainly includes three members: RORα, RORβ and RORγ. RORγ mainly includes two subtypes, RORγ1 (RORγ) and RORγ2 (RORγt), in which RORγ is distributed in skeletal muscle, thymus, testis, pancreas, prostate, heart and liver, etc., while RORγt is only expressed in some immune cells. Researches have reported that Th17 cells specifically express RORγt, and the activation of RORγt can promote the differentiation of Th17 cells and produce the pro-inflammatory cytokine IL-17. Therefore, it is theoretically possible to increase the differentiation of Th17 cells by activating RORγt, thereby promoting the activity of tumor-specific CD8+ T cells and exerting tumor immune function (ACS Chem. Biol. 2016, 11, 1012-1018).
On Jun. 9, 2015, Celgene reached an agreement with Lycera for an anti-tumor T-cell drug at an upfront payment of $82.5 million plus a recent payment of $22.5 million. Lycera announced that substantial evidence has been achieved that its oral RORγt agonist can improve T cell efficacy, increase IL-17 production and promote Tc cell expression, thereby stimulating immune responses against cancer cells, resulting in durable tumor cell-killing effects (ONCOIMMUNOLOGY, 2016, 5, e1254854-1˜e1254854-13). So far, Lycera's RORγt agonist small molecule compound LYC-55716 has entered phase 2 clinical trials, which fully demonstrates the great potential of RORγt agonists for small molecule tumor immunotherapy.
In 2018, Journal of Medicinal Chemistry published a review of small molecule RORγt agonists, detailing the structural types and characteristics of some RORγt agonists (J. Med. Chem. 2018, 61, 5794-5804). Most of these small-molecule compounds have defects in activity or druggability. So far, only one compound, LYC-55716, has entered clinical trials. Therefore, it is of great significance to find high-quality small-molecule RORγt agonists for this potential tumor immunotherapy target and apply them to virological infection and cancer treatment.
In one aspect, the present disclosure provides compounds of formula (I) or stereoisomers, tautomers, stable isotopic derivatives, pharmaceutically acceptable salts or solvates thereof:
wherein,
R1 is selected from optionally substituted C1-C6alkyl, optionally substituted C3-C6 cycloalkyl and optionally substituted 3-6 membered heterocycloalkyl, each of the substitutents is independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, mercapto, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio and C3-C6 cycloalkyl;
R2, R3, R4 are each independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, mercapto, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy and an optionally substituted C1-C6 alkylthio group, each of the substitutents is independently selected from hydrogen, halogen, hydroxyl, mercapto, nitro and cyano;
R5 and R5′ are each independently selected from hydrogen, hydroxy, halogen, nitro, cyano, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C1-C6 alkylthio and optionally substituted C3-C6 cycloalkyl, each of the substitutents is independently selected from hydrogen, halogen, nitro, cyano, hydroxyl, mercapto, C1-C6 alkoxy and C1-C6 alkylthio;
R6 is selected from hydrogen, halogen, cyano, hydroxyl, mercapto, nitro, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy and optionally substituted C1-C6 alkylthio, each of the substituents is independently selected from hydrogen, halogen, nitro, cyano, hydroxyl and mercapto;
R7 is selected from optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 3-6 membered heterocycloalkyl, and an amino optionally substituted by C1-C6 alkyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl, each of the substitutents is independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, mercapto, C1-C6 alkoxy, C1-C6 alkylthio, C3-C6 cycloalkyl, and an amino optionally substituted with C1-C6 alkyl; and X is selected from CH and N.
In some preferred embodiments, R1 is selected from optionally substituted C1-C6 alkyl and optionally substituted C3-C6 cycloalkyl, each of the substituents is independently selected from halogen, C3-C6 cycloalkyl and C1-C6 alkoxy, such as halogen substituted C1-C6 alkyl.
Preferably, R1 is selected from C1-C6 alkyl and C3-C6 cycloalkyl, such as but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
More preferably, R1 is selected from C1-C3 alkyl and C3-C5 cycloalkyl.
More preferably, R1 is selected from methyl, ethyl, propyl and cyclopropyl.
In some preferred embodiments, R2 is selected from H and halogen, such as fluorine, chlorine, bromine, iodine; preferably, R2 is H.
In some preferred embodiments, R3 and R4 are each independently selected from hydrogen, halogen, cyano, C1-C6 alkyl optionally substituted with halogen and C1-C6 alkoxy optionally substituted with halogen; such as but not limited to fluorine, chlorine, bromine, iodine, cyano, and 1, 2 or 3 halogens (preferably fluorine)-substituted methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy or isopropoxy.
More preferably, R3 and R4 are each independently selected from hydrogen, halogen, methyl, trifluoromethyl, trifluoromethoxy and difluoromethoxy; further preferably, R3 and R4 are each independently selected from halogen; most preferably, both R3 and R4 are chlorine.
In some preferred embodiments, R5 and R5′ are each independently selected from hydrogen, substituted C1-C3 alkyl, and substituted C3-C6 cycloalkyl, each of the substituents is independently selected from hydroxy, C1-C3 alkoxy and halogen, such as halogen substituted (C1-C3 alkoxy C1-C3 alkyl); such as but not limited to hydroxy substituted methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl; halogen and/or methoxy or ethoxy substituted methyl, ethyl, cyclopropyl or cyclobutyl; preferably, R5 is selected from hydrogen, —CH2OH, —CH2OCH3,
and R5′ is H; more preferably, R5 is selected from hydrogen and —CH2OCH3, and R5′ is H.
In some preferred embodiments, R6 is selected from hydrogen and halogen, preferably, R6 is hydrogen.
In some preferred embodiments, R7 is selected from C1-C6 alkyl; C1-C6 alkyl substituted with hydroxy, C1-C6 alkoxy or C3-C6 cycloalkyl; C3-C6 cycloalkyl; and C3-C6 cycloalkyl substituted with hydroxy, C1-C6 alkyl or C1-C6 alkoxy.
Preferably, R7 is selected from C1-C6 alkyl, C1-C6 alkyl substituted with hydroxy or C3-C6 cycloalkyl, and C3-C6 cycloalkyl; such as but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hydroxy; methyl, ethyl or propyl substituted with cyclopropyl or cyclobutyl; cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
More preferably, R7 is selected from methyl, ethyl, cyclopropylmethyl and —CH2CH2—OH, most preferably methyl, ethyl and —CH2CH2—OH.
In some embodiments of the disclosure, the compound has the structure of formula (II):
wherein: R1, R3, R4, R5, R7 and X are as defined above for formula (I).
Most preferably, the compound of formula (I) or (II) of the present disclosure is selected from the following specific compound examples:
In a second aspect, the present disclosure also provides a process for the preparation of compounds of formula (I). General synthetic schemes for synthesizing the compounds of the present disclosure are exemplified below. For each reaction step, appropriate reaction conditions are known to those skilled in the art or can be routinely determined. The starting materials and reagents used in the preparation of these compounds are generally commercially available unless otherwise specified, or can be prepared by the methods below, the methods analogous to those given below, or the methods known in the art. If necessary, the starting materials and intermediates in the synthetic schemes can be separated and purified by conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography and the like. The materials can be characterized using conventional methods including physical constants and spectral data.
In one embodiment, the process includes the steps of:
wherein R1, R2, R3, R4, R5, R5′, R6, R7 and X are as defined above for formula (I), and Z is Br or I.
Step 1: a compound of formula (I-1) or formula (I-2) is reacted with a compound of formula (I-3) by coupling reaction such as Suzuki coupling reaction in the presence of a catalyst such as a palladium catalyst, to obtain compound of formula (I-4).
In Step 1, the palladium catalyst is a well-known palladium catalyst for Suzuki coupling in the art, including but not limited to PdCl2(dtbpf), Pd(dppf)Cl2 etc.; the reaction is preferably carried out in a suitable organic solvent which can be selected from tetrahydrofuran, ethers (such as diethyl ether, ethylene glycol monomethyl ether, etc.), N-methylpyrrolidone, N,N-dimethylformamide, dimethylacetamide, dichloromethane, 1,4-dioxane, dimethyl sulfoxide and any combination thereof, preferably 1,4-dioxane; the reaction is preferably carried out in the presence of a suitable base which can be selected from sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate and cesium carbonate, preferably, the base is potassium carbonate; the reaction is preferably carried out at a suitable temperature, such as 50-200° C., 80-150° C., preferably 90-120° C.
Step 2: the compound of formula (I-4) is reacted with a carboxylic acid of formula (I-5) in the presence of a condensing agent, or the compound of formula (I-4) is reacted with an acyl chloride of formula (I-6) under the action of a base, to obtain a compound of formula (I).
In Step 2, the condensing agent is one well known in the art for coupling carboxylic acid with amine, including but not limited to EDC, DCC, HATU, etc.; the reaction is preferably carried out in a suitable organic solvent which can be selected from tetrahydrofuran, ethers (such as diethyl ether, ethylene glycol monomethyl ether, etc.), N-methylpyrrolidone, N,N-dimethylformamide, dimethylacetamide, dichloromethane, 1,4-dioxane, dimethyl sulfoxide and any combination thereof; the reaction is preferably carried out in the presence of a suitable base including but not limited to sodium carbonate, potassium carbonate, cesium carbonate, N,N-diisopropyl ethylamine, triethylamine, HOBt or pyridine, preferably, the base is N,N-diisopropyl ethylamine; the reaction is preferably carried out at a suitable temperature, such as 0-200° C., 10-100° C. or 20-50° C., preferably room temperature (20-25° C.).
In another embodiment, the process includes the steps of:
wherein R1, R2, R3, R4, R5, R5′, R6, R7 and X are as defined above for formula (I), and Z is Br or I.
Step 1: a compound of formula (II-1) is condensed with a carboxylic acid of formula (II-2) in the presence of a condensing agent to obtain a compound of formula (II-3).
In Step 1, the condensing agent is one well known in the art for coupling carboxylic acid with amine, including but not limited to EDC, DCC, HATU, etc.; the reaction is preferably carried out in a suitable organic solvent which can be selected from tetrahydrofuran, ethers (such as diethyl ether, ethylene glycol monomethyl ether, etc.), N-methylpyrrolidone, N,N-dimethylformamide, dimethylacetamide, dichloromethane, 1,4-dioxane, dimethyl sulfoxide and any combination thereof, the reaction is preferably carried out in the presence of a suitable base including but not limited to sodium carbonate, potassium carbonate, cesium carbonate, N,N-diisopropyl ethylamine, triethylamine, HOBt or pyridine, preferably, the base is N,N-diisopropyl ethylamine; the reaction is preferably carried out at a suitable temperature, such as 0-200° C., 10-100° C. or 20-50° C., preferably room temperature (20-25° C.).
Step 2: the compound of formula (II-3) is reacted with a thiol of formula (II-4) under the action of a catalyst such as a palladium catalyst, to obtain a compound of formula (II-5).
In Step 2, the palladium catalyst includes but is not limited to Pd2(dba)3; the reaction is preferably carried out in a suitable organic solvent which can be selected from tetrahydrofuran, ethers (such as diethyl ether, ethylene glycol monomethyl ether, etc.), N-methylpyrrolidone, N,N-dimethylformamide, dimethylacetamide, dichloromethane, 1,4-dioxane, dimethyl sulfoxide and any combination thereof, preferably 1,4-dioxane; the reaction is preferably carried out in the presence of a suitable base which can be selected from N,N-diisopropyl ethylamine, triethylamine, HOBt and pyridine, preferably, the base is N,N-diisopropyl ethylamine; the reaction is preferably carried out in the presence of xantphos, and preferably at a suitable temperature, such as 50-200° C. or 80-150° C., preferably 90-120° C.
Step 3: The compound of formula (II-5) is oxidized to give a compound of formula (I).
In Step 3, the oxidizing agent that can be used for the above-mentioned oxidation is well known in the art, including but not limited to m-chloroperoxybenzoic acid, peroxyacetic acid or hydrogen peroxide; the reaction is preferably carried out in a suitable organic solvent selected from tetrahydrofuran, ethers (such as diethyl ether, ethylene glycol monomethyl ether, etc.), N-methylpyrrolidone, N,N-dimethylformamide, dimethylacetamide, dichloromethane, 1,4-dioxane, dimethyl sulfoxide and any combination thereof, preferably dichloromethane; the reaction is preferably carried out at a suitable temperature, such as −50-100° C., 0-80° C. or 20-50° C., preferably room temperature (20-25° C.).
The above synthetic schemes only exemplify the preparation of some compounds of the present disclosure. The compounds of the present disclosure, or stereoisomers, tautomers, stable isotopic derivatives, pharmaceutically acceptable salts or solvates thereof can be prepared by a variety of methods, including the methods given above, the methods given in the Examples or a method analogous to them, by those of ordinary skill in the art on the basis of the above-mentioned synthetic schemes in combination with conventional techniques in the art.
In the third aspect, the present disclosure provides a compound of formula (I) or (II) as defined above, or a stereoisomer, tautomer, stable isotopic derivatives, pharmaceutically acceptable salts or solvates, for use as a medicament, in particle as a RORγt agonist.
In the fourth aspect, the present disclosure also provides a pharmaceutical composition comprising the compound of formula (I) or (II) as described above or a stereoisomer, tautomer, stable isotopic derivative, pharmaceutically acceptable salt or solvate thereof as the active ingredient, and one or more pharmaceutically acceptable carriers.
In the fifth aspect, the present disclosure provides the use of a compound of formula (I) or (II) as described above or a stereoisomer, tautomer, stable isotope derivative, pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament with RORγt receptor agonistic activity.
The present disclosure preferably provides, in the fifth aspect, the use of a compound of formula (I) or (II) as described above or a stereoisomer, tautomer, stable isotopic derivative, pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition comprising the same in the manufacture of a medicament for the treatment or prevention of a disease associated with RORγt, such as a disease treatable or preventable by activation of RORγt, wherein the above compound or pharmaceutical composition is optionally combined with one or more chemotherapies or immunotherapies.
In the sixth aspect, the present disclosure also provides a method of preventing or treating a disease associated with RORγt (eg, a disease treatable or preventable by activation of RORγt), comprising administering to an individual in need thereof an effective dose of the compound of formula (I) or (II) as described above or its stereoisomers, tautomers, stable isotopic derivatives, pharmaceutically acceptable salts or solvates thereof or pharmaceutical compositions comprising them.
In the seventh aspect, the present disclosure provides a pharmaceutical combination, comprising the concurrent or sequential administration of a compound of formula (I) or (II) as described above or a stereoisomer, tautomer, stable isotope derivative, pharmaceutically acceptable salts or solvates thereof or pharmaceutical compositions comprising them, and one or more other agents that act by the same or a different mechanism of action. Such other agents are those which, when used in combination with the compounds of the present disclosure, produce a combined beneficial effect.
For the various aspects of the disclosure described above, the disease associated with RORγt is selected from tumors and cancers, including but not limited to colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, lung cancer, leukemia, bladder cancer, stomach cancer, cervical cancer, testicular cancer, skin cancer, rectal cancer, thyroid cancer, kidney cancer, uterine cancer, pemphigus cancer, liver cancer, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, eye cancer.
For the above-mentioned compounds of the disclosure for use as medicaments, prevention or treatment methods of the disclosure, pharmaceutical compositions, pharmaceutical combinations or uses, the respective preferred embodiments of the compounds of formula (I) or (II) as defined above are preferred, more preferrably the specific compounds listed above, namely Compound 1 to Compound 44.
Unless otherwise defined herein, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including those variations or substitutions of equivalent techniques that would be apparent to those skilled in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better illustrate the present disclosure.
The term “halogen” as used herein refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
The term “hydroxyl” as used herein refers to —OH.
The term “alkyl” as used herein refers to a straight or branched chain saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms. Alkyl groups have 1 to 10, such as 1 to 6, such as 1 to 4, or 1 to 3 carbon atoms. For example, as used herein, the term “C1-C6 alkyl” refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl or tert-butyl), pentyl (including n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylpentyl, etc.; the alkyl group can be unsubstituted or substituted by one or more substituents, including but not limited to alkyl, alkoxy, alkylthio, cyano, hydroxyl, mercapto, carbonyl, carboxyl, aryl, heteroaryl, amino, halogen, sulfonyl, sulfinyl, phosphoryl.
The term “alkoxy” as used herein, refers to an alkyl group as defined above which is attached to the parent molecular moiety via an oxygen atom, eg, C1-C6 alkoxy or C1-C3 alkoxy. Representative examples of alkoxy include but are not limited to methoxy, ethoxy, propoxy (including n-propoxy, isopropoxy), butoxy (including n-butoxy, isobutoxy, tert-butoxy), pentyloxy (including n-pentyloxy, isopentyloxy, neopentyloxy), hexyloxy (including n-hexyloxy, isohexyloxy) and the like.
The term “alkylthio” as used herein refers to a —S-alkyl group, wherein the alkyl group is as defined above for “alkyl”.
The term “halogen-substituted C1-C6 alkyl” as used herein refers to the C1-C6 alkyl groups described above wherein one or more (eg. 1, 2, 3, 4 or 5) hydrogen atoms are replaced by halogens. It will be understood by those skilled in the art that when there is more than one halogen substituent, the halogens may be the same or different, and may be located on the same or different C atoms. Examples of “halogen substituted C1-C6 alkyl” are eg. —CH2F, —CHF2, —CF3, —CCl3, —C2F5, —C2Cl5, —CH2CF3, —CH2Cl or —CH2CH2CF3, etc.
The term “halogen-substituted C1-C6 alkoxy” as used herein refers to the C1-C6alkoxy groups described above wherein one or more (eg. 1, 2, 3, 4 or 5) hydrogen atoms are replaced by halogens. It will be understood by those skilled in the art that when there is more than one halogen substituent, the halogens may be the same or different, and may be located on the same or different C atoms. Examples of “halogen substituted C1-C6 alkoxy” are eg. —OCH2F, —OCHF2, —OCF3, —OCCl3, —OC2F5, —OC2Cl5, —OCH2CF3, —OCH2Cl or —OCH2CH2CF3, etc.
Similarly, the term “halogen substituted (C1-C3 alkoxy C1-C3 alkyl)-” refers to a C1-C3 alkoxy C1-C3 alkyl group wherein one or more (eg. 1, 2, 3, 4 or 5) hydrogen atoms of the C1-C3alkoxy or C1-C3 alkyl moiety, preferably the C1-C3 alkyl moiety, are replaced by halogen, preferably fluorine.
The term “cycloalkyl” as used herein refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (eg. monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclic, including spiro, fused or bridged systems (such as bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, spiro[3.4]octyl, bicyclo[3.1.1]hexyl, etc.). The cycloalkyl group has 3 to 10 carbon atoms, such as 3 to 8 carbon atoms, 3 to 7 carbon atoms, 3 to 6 carbon atoms or 3 to 5 carbon atoms. For example, as used herein, the term “C3-C6 cycloalkyl” refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon rings having 3 to 6 ring carbon atoms, preferably the term “C3-C6 cycloalkyl” refers herein to saturated monocyclic rings having 3 to 6 ring carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
The term “heterocycloalkyl” as used herein refers to a monocyclic or polycyclic non-aromatic group having 2, 3, 4, 5, 6, 7, 8, 9 carbon atoms and one or more (eg. 1, 2, 3 or 4) groups selected from C(═O), O, S, S(═O), S(═O)2, N and NR (R represents hydrogen atom or substituents such as but not limited to alkyl or cycloalkyl) in the ring. As used herein, the term “3-6 membered heterocycloalkyl” means a heterocyclyl group containing 3-6 ring atoms, wherein 1 or more ring atoms are selected from N, O (optionally oxidized) and S (optionally oxidized) heteroatoms, this term includes 3, 4, 5 or 6 membered heterocycloalkyl, examples of which include but are not limited to oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, piperazino, morpholino, piperidino, etc.
The term “aryl” as used herein refers to an all-carbon monocyclic or fused ring having a fully conjugated π-electron system, and typically having 6-14 carbon atoms, preferably 6-12 carbon atoms, most preferably 6 carbon atoms. Aryl groups may be unsubstituted or substituted with one or more substituents including, but not limited to alkyl, alkoxy, cyano, hydroxyl, carbonyl, carboxyl, aryl, aralkyl, amino, halogen, sulfonyl, sulfinyl, phosphoryl. Examples of unsubstituted aryl groups include but are not limited to phenyl, naphthyl and anthracenyl.
The term “heteroaryl” as used herein refers to a monocyclic or fused ring having 5-12 ring atoms, preferably a monocyclic ring of 5-6 ring atoms, containing 1-4 (eg. 1, 2, 3 or 4) heteroatoms selected from N, O (optionally oxidized), and S (optionally oxidized), the remaining ring atoms being C, and having a fully conjugated π-electron system, including but not limited to pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, quinolinyl, isoquinolinyl, triazolyl, tetrahydropyrrolyl. Heteroaryl groups can be unsubstituted or substituted, and the substituents include but are not limited to alkyl, alkoxy, aryl, aralkyl, amino, halogen, hydroxy, cyano, nitro, carbonyl, and hetero-aliphatic ring group.
If a substituent is described as “optionally substituted”, the substituent may be (1) unsubstituted or (2) replaced by one or more (eg., 1, 2, 3, 4, or 5, preferably 1) of the list of substituents. Unless specifically defined, groups used in the definitions of compounds herein have meanings well known to those skilled in the art.
The term “pharmaceutically acceptable salts” as used herein includes conventional salts formed with pharmaceutically acceptable inorganic or organic acids, or inorganic or organic bases. Illustrative examples of suitable salts include but are not limited to organic salts derived from amino acids such as glycine and arginine, ammonia, primary amines, secondary amines and tertiary amines, and cyclic amines such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
Exemplary acid addition salts include, for example, inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate (eg, phosphate, hydrogen phosphate or dihydrogen phosphate), carbonate, bicarbonate or perchlorate; organic acid salts such as acetate, propionate, butyrate, valerate, caproate, heptanoate, octanoate, cyclopentane propionate, undecanoate, lactate, malate, oxalate, fumarate, tartrate, maleate, citrate, nicotinate, benzoate, salicylate or ascorbate; sulfonates such as mesylate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-toluenesulfonate, 2-naphthalenesulfonate, 3-phenylsulfonate or camphorsulfonate; acidic amino acid salts such as aspartate or glutamate.
The present disclosure also includes all pharmaceutically acceptable isotopic compounds that are identical to the compounds of the present disclosure, except that one or more atoms are replaced by atoms that have the same atomic number but an atomic mass or mass number different from the atomic mass or mass number that predominates in nature. Examples of isotopes suitable for inclusion in the compounds of the present disclosure include but are not limited to isotopes of hydrogen (eg., 2H, 3H); isotopes of carbon (eg., 11C, 13C, and 14C); isotopes of chlorine (eg., 36Cl); isotopes of fluorine (eg., 18F); isotopes of iodine (eg. 123I and 125I); isotopes of nitrogen (eg. 13N and 15N); isotopes of oxygen (eg. 15O, 17O and 18O); isotopes of phosphorus (eg. 32P); and isotopes of sulfur (eg. 35S).
The term “stereoisomer” as used herein refers to isomers formed due to at least one asymmetric center. In compounds having one or more (eg, 1, 2, 3 or 4) asymmetric centers, it can give rise to racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Certain individual molecules may also exist as geometric isomers (cis/trans). Similarly, the compounds of the present disclosure may exist as mixtures of two or more different structural forms in rapid equilibrium (often referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers etc. For example, a nitroso-oxime can exist in solution in equilibrium in the following tautomeric forms:
It is to be understood that the scope of this disclosure covers all such isomers or mixtures thereof in any ratio (eg. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).
Unless otherwise indicated, compounds of the present disclosure are intended to be available as stereoisomers (which include cis and trans isomers, optical isomers (eg, R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers and mixtures thereof). The compounds of the present disclosure may exhibit more than one type of isomerism and consist of mixtures thereof (eg., racemic mixtures and pairs of diastereomers).
The compounds of the present disclosure may exist in the form of solvates, preferably hydrates, wherein the compounds of the present disclosure comprise a polar solvent as a structural element of the compound crystal lattice, in particular for example water, methanol or ethanol. The amount of polar solvent, especially water, may be present in stoichiometric or non-stoichiometric ratios.
The term “treatment” as used herein refers to any treatment of a disease in a mammal, including: (1) preventing the disease, i.e. causing symptoms of the clinical disease not to develop; (2) inhibiting the disease, i.e. preventing the development of the clinical symptoms; (3) alleviating the disease, that is, causing the subsidence of clinical symptoms.
The term “cancer” or “tumor” as used herein refers to the growth and proliferation of neoplastic cells, either malignant or benign, and all precancerous cells and cancer cells and tissues. For the compounds, methods, pharmaceutical compositions, pharmaceutical combinations and uses of the present disclosure, the cancer or tumor includes but is not limited to colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, lung cancer, leukemia, bladder cancer, stomach cancer, cervical cancer, testicular cancer, skin cancer, rectal cancer, thyroid cancer, kidney cancer, uterine cancer, pemphigus cancer, liver cancer, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, eye cancer.
The term “effective dose” as used herein refers to the amount of a compound that, when administered, alleviates to some extent one or more symptoms of the disorder being treated.
The term “individual” as used herein includes human or non-human animals. Exemplary human subjects include human subjects with a disease (eg, a disease described herein) (referred to as patients) or normal subjects. “Non-human animals” in the present disclosure include all vertebrates such as non-mammals (eg. birds, amphibians, reptiles) and mammals such as non-human primates, livestock and/or domesticated animals (eg. sheep, dogs, cats, cows, pigs, etc.).
The “pharmaceutical composition” in the present disclosure refers to a composition comprising one or more compounds of formula (I) or (II) or their stereoisomers, tautomers, stable isotopic derivatives, pharmaceutically acceptable salts or solvates and carriers generally accepted in the art for the delivery of biologically active compounds to organisms such as humans.
The “pharmaceutically acceptable carrier” as used herein refers to a carrier with which the therapeutic agent is administered and which, within the scope of sound medical judgment, is suitable for contact with human and/or other animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications corresponding to a reasonable benefit/risk ratio, including but not limited to any glidants, sweetening agents, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, disintegrating agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers, such as including but not limited to calcium carbonate, calcium phosphate, various sugars and various starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
The pharmaceutical compositions of the present disclosure can be formulated by techniques known to those skilled in the art, such as those disclosed in Remington's Pharmaceutical Sciences 20th Edition, such as conventional mixing, dissolving, granulating, methods for preparing dragees, grinding, emulsification, freeze-drying, etc., and can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc. These pharmaceutical compositions may contain conventional ingredients in pharmaceutical formulations, such as diluents, carriers, pH adjusters, sweeteners, disintegrants, and the like.
The administration routes of the compounds of the present disclosure or their pharmaceutically acceptable salts or their pharmaceutical compositions include but are not limited to oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration. The preferred route of administration is oral administration.
For oral administration, the pharmaceutical compositions can be formulated by admixing the active compound with pharmaceutically acceptable carriers well known in the art. These carriers enable the compounds of this disclosure to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions and the like. For example, for pharmaceutical compositions for oral administration, tablets may be obtained by combining the active ingredient with one or more solid carriers, granulating the resulting mixture if desired, and adding minor amounts of excipients if desired, to process into mixtures or granules to form tablets or tablet cores. The tablet core can be associated with an optional enteric coating material and processed into a coated formulation that is more conducive to absorption by an organism (eg, a human).
Dosage regimens can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is noted that dosage amounts may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the specific dosing regimen should be adjusted over time according to the needs of the individual and the professional judgment of the person administering or supervising the administration of the composition.
The compounds of the present disclosure may be administered as the sole active ingredient or, as appropriate, in combination with additional drugs or therapies that may have or produce the same or different pharmacological effects, with the proviso that, when used in combination the compounds of the present disclosure, do not result in undesired reductions in activity, adverse interactions or side effects.
When a compound of formula (I) or (II) is administered in combination with other drugs, the dosage of the other drugs co-administered will of course depend on the type of co-drug used, the specific drug used, the condition to be treated, the general health of the patient, the judgment of the physician or the veterinarian.
The drugs used in combination with the present compounds, i.e., co-agents, may be administered simultaneously, separately or sequentially with the compounds of the present disclosure by the same or different route of administration. They may be contained in the same pharmaceutical composition, or may be in separate form, such as a combination product, preferably in the form of a kit. They may be prepared and/or formulated by the same or different manufacturers. Furthermore, the compound of the present disclosure and the additional drug may be added to the combination therapy (i) prior to delivering the combination product to a physician (eg., in the case of a kit comprising the compound of the present disclosure and the additional drug); (ii) immediately prior to administration by the physician himself (or under the direction of the physician); (iii) by the patient himself, eg. during sequential administration of the compound of the disclosure and the additional drug.
The amount of the compound of the disclosure administered will depend on the individual being treated, severity of the disorder or condition, rate of administration, disposition of the compound, and judgment of the prescribing physician. In general, an effective dose is about 0.0001 to about 5000 mg/kg body weight/day, eg, about 0.01 to about 1000 mg/kg/day (single or divided administration). For a 70 kg person, this would add up to about 0.007 mg/day to about 7000 mg/day, eg. about 0.7 mg/day to about 1500 mg/day. In some cases, dose levels not higher than the lower limit of the foregoing ranges may be sufficient, while in other cases larger doses may be employed without causing any deleterious side effects, provided that the larger dose is first divided into several smaller doses to be administered throughout the day.
The content or amount of the compound of the present disclosure in the pharmaceutical composition can be about 0.01 mg to about 1000 mg, suitably 0.1-500 mg, preferably 0.5-300 mg, more preferably 1-150 mg, particularly preferably 1-50 mg, such as 1.5 mg, 2 mg, 4 mg, 10 mg, 25 mg, etc.
The present disclosure provides a class of sulfo-substituted biaryl compounds with the characteristic structural formula (I). It has been found through research that such compounds can effectively activate RORγt protein receptor, thereby regulating the differentiation of Th17 cells and increasing the production of IL-17, so can be used as an immunomodulator for the treatment of diseases related to Th17 cell differentiation.
The compounds of the present disclosure have the following beneficial effects:
The technical solutions of the present disclosure are illustrated below with reference to specific embodiments, but the protection scope of the present disclosure is not limited to these embodiments. All modifications or equivalent substitutions that do not depart from the concept of the present disclosure are included in the protection scope of the present disclosure.
The experimental methods without specific conditions in the following examples are generally in accordance with the conventional conditions of this type of reaction, or in accordance with the conditions suggested by manufacturers. Percentages and parts are weight percentages and parts unless otherwise specified. Unless otherwise stated, ratios of liquids are by volume.
Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources, prepared according to methods in the prior art, or prepared according to methods analogous to those disclosed in this application.
Abbreviations used herein have the meanings commonly understood in the art unless clearly defined otherwise in the specification. The meanings of the abbreviations used in the specification are listed below:
PdCl2 (dtbpf): 1,1′-di-tert-butylphosphinoferrocene palladium dichloride
Pd(dppf)Cl2: [1,1′-bis(diphenylphosphino)ferrocene] palladium dichloride
xantphos: 4,5-Bisdiphenylphosphine-9,9-dimethylxanthene
pd2(dba)3: Tri(dibenzylideneacetone) dipalladium
HATU: 2-(7-Azobenzotriazole)-N,N,N′,N′,-tetramethylurea hexafluorophosphate
DIEA: N,N-diisopropylethylamine
DMF: N,N-dimethylformamide
DCM: dichloromethane
EA: Ethyl acetate
PE: Petroleum ether
rt: room temperature
LC-MS: Liquid Chromatography Mass Spectrometry
ESI: Electrospray ionization
m/z: mass-to-charge ratio
DTT: Dithiothreitol
EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
DCC: Dicyclohexylcarbodiimide
HOBt: 1-Hydroxybenzotriazole
TLC: Thin Layer Chromatography
In the preparation of the target compounds provided by the present disclosure, column chromatography was carried out with silica gel (300-400 mesh) produced by Rushan Sun Desiccant Co., Ltd.; thin-layer chromatography was carried out with GF254 (0.25 mm); nuclear magnetic resonance spectroscopy (NMR) was carried out with Varian-400 nuclear magnetic resonance spectrometer; liquid mass spectrometry (LC/MS) was carried out with Agilent TechnologiESI 6120 liquid mass spectrometer.
In addition, all operations involving easily oxidizable or easily hydrolyzed raw materials are carried out under nitrogen protection. Unless otherwise specified, the raw materials used in the present disclosure are all commercially available which can be used directly without further purification, and the temperatures used in the present disclosure are all in degrees Celsius ° C.
Where the structure of the compound of the present disclosure is inconsistent with the name thereof, the structural formula generally controls, unless it can be determined from the context that the name of the compound is correct.
The title compound was synthesized according to the following scheme:
4-Ethylsulphonylbenzeneboronic acid (514 mg, 2.4 mmol), 4-bromo-3,5-dichloroaniline (482 mg, 2 mmol), PdCl2(dtbpf) (65 mg, 0.1 mmol), potassium carbonate (816 mg, 6 mmol) were added to 1,4-dioxane/water (5 mL/1 mL). The resulting mixture was stirred at 110° C. for 3 hours under nitrogen protection, the solvent was removed under reduced pressure, and the residue was separated and purified by silica gel column (PE:EA=3:2) to obtain the target compound (320 mg, yield 48.5%, white solid). LC-MS (ESI) m/z: 330.0 [M+H]+.
2,6-Dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-amine (30 mg, 0.091 mmol), 2-(4-(ethylsulfonyl)phenyl)acetic acid (24.9 mg, 0.109 mmol), HATU (41.4 mg, 0.109 mmol), DIEA (35.3 mg, 0.273 mmol) were added sequentially to DMF (1.5 mL). The reaction mixture was stirred at room temperature for 2.5 hours. Water (10 mL) was added to the reaction mixture, which was extracted with EA (10 mL×2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. Separation and purification by preparative plate (silica gel, EA:PE=1.5:1) gave the target compound (13.3 mg, yield 27.0%, white solid). LC-MS (ESI) m/z: 538.17 [M−H]−. 1H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 7.99 (d, J=8.4 Hz, 2H), 7.91-7.82 (m, 4H), 7.62 (d, J=8.3 Hz, 2H), 7.57 (d, J=8.4 Hz, 2H), 3.87 (s, 2H), 3.38 (q, J=7.4 Hz, 2H), 3.30-3.26 (m, 2H), 1.17-1.08 (m, 6H).
The title compound was synthesized according to the following scheme:
4-Methylsulfonylphenylboronic acid (240 mg, 1.2 mmol), 4-bromo-3,5-dichloroaniline (241 mg, 1 mmol), PdCl2 (dtbpf) (33 mg, 0.05 mmol) and potassium carbonate (408 mg, 3 mmol) were added to a mixture of 1,4-dioxane/water (5 mL/1 mL). The resulting mixture was stirred at 110° C. for 3 hours under nitrogen protection. The reaction was completed, and was cooled to room temperature, added with water (20 mL), extracted with ethyl acetate (20 mL×3), and the solvent was removed under reduced pressure. The residue was separated and purified on a silica gel column (PE:EA=3:2) to obtain the target compound (168 mg, yield 53.1%, yellow solid). LC-MS (ESI) m/z: 317.9[M+H]+.
2,6-Dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-amine (58 mg, 0.18 mmol), HATU (84 mg, 0.22 mmol), 2-(4-(ethylsulfonyl)phenyl)acetic acid (50 mg, 0.22 mmol) and DIEA (70 mg, 0.54 mmol) were successively added to DCM (3 mL). The reaction solution was reacted at room temperature overnight. TLC showed that the raw materials had been run out of and the reaction was completed. The reaction was washed with saturated aqueous sodium bicarbonate solution (20 mL) and saturated sodium chloride (20 mL) successively, and the crude product was separated via an automated chromatographic system using silica gel column (PE:EA=1:4), to obtain the target product (70 mg, 71.4% yield, white solid). LC-MS (ESI) m/z: [M+H]+/[M+18]+ 526.0/543.0.
1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.04 (d, J=8.2 Hz, 2H), 7.87 (d, J=8.2 Hz, 2H), 7.85 (s, 2H), 7.62 (d, J=8.2 Hz, 2H), 7.57 (d, J=8.2 Hz, 2H), 3.87 (s, 2H), 3.31 (s, 3H), 3.31-3.25 (m, 2H), 1.11 (t, J=7.3 Hz, 3H).
The title compound was synthesized according to the following scheme:
4-Isopropanesulfonylbenzeneboronic acid (252 mg, 1.2 mmol), 4-bromo-3,5-dichloroaniline (241 mg, 1 mmol), PdCl2 (dtbpf) (33 mg, 0.05 mmol) and potassium carbonate (408 mg, 3 mmol) were added to a mixture of 1,4-dioxane/water (5 mL/1 mL). The resulting mixture was stirred at 110° C. for 3 hours under nitrogen protection. The reaction was completed, and was cooled to room temperature, added with water (20 mL), and extracted with EA (20 mL×3). The solvent was removed under reduced pressure, and the residue was separated and purified by silica gel column (PE:EA=3:2) to obtain the target compound (230 mg, yield 66.8%, yellow solid). LC-MS (ESI) m/z: 344.0[M+H]+.
2,6-Dichloro-4′-(isopropylsulfonyl)-[1,1′-biphenyl]-4-amine (62 mg, 0.18 mmol), HATU (84 mg, 0.22 mmol), 2-(4-(ethylsulfonyl)phenyl)acetic acid (50 mg, 0.22 mmol) and DIEA (70 mg, 0.54 mmol) were successively added in DCM (3 mL). The reaction solution was reacted at room temperature overnight. TLC showed that the raw materials had been run out of and the reaction was completed. The reaction was washed with saturated aqueous sodium bicarbonate solution (20 mL) and saturated sodium chloride (20 mL) successively, and the crude product was separated by chromatography using silica gel column (PE:EA=1:2) to obtain the target product (70 mg, 70.0% yield, white solid). LC-MS (ESI) m/z: 571.0[M+18]+.
1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 7.96 (d, J=8.2 Hz, 2H), 7.91-7.81 (m, 4H), 7.62 (d, J=8.2 Hz, 2H), 7.58 (d, J=8.2 Hz, 2H), 3.87 (s, 2H), 3.55-3.46 (m, 1H), 3.31-3.25 (m, 2H), 1.19 (d, J=6.8 Hz, 6H), 1.11 (t, J=7.3 Hz, 3H).
The title compound was synthesized according to the following scheme:
2,6-Dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-amine (30 mg, 0.091 mmol), 2-(4-(methylsulfonyl)phenyl)acetic acid (23.4 mg, 0.109 mmol), 1-propylphosphoric anhydride (50% in ethyl acetate, 139 mg, 0.218 mmol), triethylamine (33.2 mg, 0.328 mmol) were successively added to EA (1.5 mL). The reaction mixture was stirred at room temperature for 2 hours. Water (10 mL) and EA (10 mL) were added to the reaction mixture. The aqueous phase was separated and extracted with EA (10 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. Separation and purification by preparative plate (silica gel, dichloromethane:methanol=30:1) to obtain the target compound (30.0 mg, yield 62.6%, white solid). LC-MS (ESI) m/z: 543.0 [M+18]+.
1H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 7.99 (d, J=8.2 Hz, 2H), 7.91 (d, J=8.2 Hz, 2H), 7.85 (s, 2H), 7.61 (d, J=8.2 Hz, 2H), 7.57 (d, J=8.2 Hz, 2H), 3.86 (s, 2H), 3.38 (q, J=14.7, 7.3 Hz, 2H), 3.21 (s, 3H), 1.14 (t, J=7.3 Hz, 3H).
The title compound was synthesized according to the following scheme:
2,6-Dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-amine (120 mg, 0.35 mmol), HATU (160 mg, 0.42 mmol), 2-(5-Bromopyridin-2-yl)acetic acid (91 mg, 0.42 mmol) and DIEA (135 mg, 1.05 mmol) were successively added in DCM (3 mL). The reaction solution was reacted at room temperature overnight. TLC showed that the raw materials had been run out of and the reaction was completed. The reaction solution was washed with saturated aqueous sodium bicarbonate solution (20 mL) and saturated sodium chloride (20 mL) successively, and the crude product (200 mg) was directly used in the next step.
2-(5-Bromopyridin-2-yl)-N-(2,6-dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-yl)acetamide crude (200 mg) synthesized in the previous step, DIEA (135 mg, 1.05 mmol), xantphos (9.8 mg, 0.017 mmol), Pd2(dba)3 (16 mg, 0.017 mmol) and 1,4-dioxane (3 mL) were added to a microwave reaction tube, bubbled with nitrogen for 5 minutes, and then reacted under microwave at 120° C. for 2 hours. TLC showed that the reaction was completed, and the reaction was separated via an automated chromatographic system using silica gel column (PE:EA=1:2) to obtain the target product (130 mg, 70.3% over two steps, yellow solid). LC-MS (ESI) m/z: 509.0[M+H]+.
At room temperature, N-(2,6-dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(5-(ethylthio)pyridine-2-yl)acetamide (130 mg, 0.27 mmol), m-chloroperoxybenzoic acid (107 mg, 0.53 mmol, 85% content) were added to DCM (5 mL), and the reaction solution was reacted at room temperature for 1 hour. The reaction was completed, and was washed once with 2N sodium carbonate solution (10 mL), and separated via an automated chromatographic system using silica gel column (PE:EA=1:3) to obtain the target product (42 mg, yield 28.8%, yellow solid). LC-MS (ESI) m/z: 541.0 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.98 (d, J=2.3 Hz, 1H), 8.29 (dd, J=8.2, 2.4 Hz, 1H), 7.99 (d, J=8.3 Hz, 2H), 7.85 (s, 2H), 7.73 (d, J=8.2 Hz, 1H), 7.58 (d, J=8.3 Hz, 2H), 4.08 (s, 2H), 3.45-3.35 (m, 4H), 1.17-1.12 (m, 6H).
The title compound was synthesized according to the following scheme:
Potassium tert-butoxide (1.48 g, 13.2 mmol) was dissolved in dimethyl sulfoxide (5 mL), and 4-bromobenzenethiol (1.64 mL, 13.2 mmol) was added dropwise to the reaction solution at room temperature. Upon the dropwise addition was completed, the reaction mixture was stirred at room temperature for reaction for 15 minutes, and then bromocyclopropane (3.18 mL, 39.7 mmol) was added to the reaction solution. The tube was sealed and the reaction mixture was stirred at 80° C. for reaction for 24 hours. TLC showed that the raw materials was reacted completely, and a new main spot was formed (Rf=0.7, PE:EA=10:1). The reaction solution was poured into ice water (50 mL) and extracted with methyl tert-butyl ether (50 mL×2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound (2.8 g, yield 92.4%, brown liquid).
1-Bromo-4-cyclopropylthiobenzene (6-3) (3.00 g, 13.1 mmol) was dissolved in DCM (50 mL) and m-chloroperoxybenzoic acid (5.65 g, 32.7 mmol) was slowly added to the reaction solution under ice bath. After the addition was completed, the ice bath was removed and the reaction mixture was stirred at room temperature overnight. TLC showed that the raw materials were reacted completely, and a new main spot was formed (Rf=0.5, PE:EA=10:1). The reaction solution was filtered, the filtrate was poured into saturated aqueous sodium bicarbonate solution (50 mL), the organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was separated and purified on a silica gel column (PE:EA=100:1-30:1) to obtain the target compound (1.50 g, 43.9% yield, yellow solid).
1-Bromo-4-cyclopropylsulfonylbenzene (6-4) (500 mg, 1.91 mmol), Bis(pinacolato)diboron (729 mg, 2.87 mmol), potassium acetate (564 mg, 5.74 mmol)) and Pd(dppf)Cl2 (140 mg, 0.192 mmol) were added to 1,4-dioxane (5 mL). Under nitrogen protection, the reaction mixture was stirred at 90° C. overnight. Then the reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain crude product, which was separated and purified by silica gel column (PE:EA=100:1-30:1) to obtain the target compound (200 mg, yield 3.39%, yellow liquid).
2-(4-(cyclopropylsulfonyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (200 mg, 0.649 mmol), 4-bromo-3,5-dichloroaniline (130 mg, 0.541 mmol), potassium carbonate (187 mg, 1.35 mmol) and PdCl2 (dtbpf) (59.1 mg, 0.081 mmol) were added to a mixed solvent of 1,4-dioxane (8 mL) and water (1 mL). Under nitrogen protection, the reaction mixture was stirred at 110° C. for 1.5 hours, and monitored by TLC for completion. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain crude product, which was separated and purified by silica gel column (PE:EA=8:1-4:1) to obtain the target compound (64.0 mg, yield 34.6%, brown solid). LC-MS (ESI) m/z 342.0 [M+H]+.
2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-amine (50.0 mg, 0.146 mmol), 2-[4-(ethylsulfonyl))phenyl]acetic acid (40.0 mg, 0.175 mmol), DIEA (0.0724 mL, 0.438 mmol) and HATU (83.3 mg, 0.219 mmol) were added to DMF (3 mL). The reaction mixture was stirred at room temperature overnight. The reaction solution was poured into water (20 mL) and extracted with EA (10 mL×2). The organic phases were combined, washed successively with saturated aqueous sodium bicarbonate solution (15 mL) and saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Crystallization from methanol gave the target compound (20.0 mg, 24.8% yield, white solid). LC-MS (ESI) m/z 550.0 [M−1]−.
1H NMR (400 MHz, CDCl3) δ 7.97 (d, J=8.3 Hz, 2H), 7.91 (d, J=8.2 Hz, 2H), 7.67 (s, 2H), 7.54 (d, J=8.2 Hz, 2H), 7.48 (s, 1H), 7.43 (d, J=8.3 Hz, 2H), 3.84 (s, 2H), 3.14 (q, J=7.5 Hz, 2H), 2.59-2.50 (m, 1H), 1.44-1.37 (m, 2H), 1.30 (t, J=7.5 Hz, 3H), 1.13-1.05 (m, 2H).
2,6-Dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-amine (64 mg, 0.2 mmol), HATU (91 mg, 0.24 mmol), 2-(4-(methylsulfonyl)phenyl)acetic acid (52 mg, 0.24 mmol) and DIEA (77 mg, 0.6 mmol) were successively added in DCM (3 mL), and the reaction solution was reacted at room temperature overnight. TLC showed that the raw materials had been run out of and the reaction was completed. The reaction solution was washed with saturated aqueous sodium bicarbonate solution (20 mL) and saturated sodium chloride (20 mL) successively, and the crude product was separated via automated chromatographic system using silica gel column (PE:EA=1:2) to obtain the target product (90 mg, 86.5% yield, white solid). LC-MS (ESI) m/z: 529.0 [M+18]+.
1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.04 (d, J=8.4 Hz, 2H), 7.91 (d, J=8.4 Hz, 2H), 7.85 (s, 2H), 7.62 (d, J=8.3 Hz, 2H), 7.57 (d, J=8.4 Hz, 2H), 3.86 (s, 2H), 3.32 (s, 3H), 3.22 (s, 3H).
The title compound was synthesized according to the following scheme:
2-(5-Bromopyridin-2-yl)-N-(2,6-dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl)acetamide (60.0 mg, 0.117 mmol), pd2(dba)3 (10.7 mg, 0.0117 mmol), xantphos (6.77 mg, 0.0117 mmol), triethylamine (0.0324 mL, 0.233 mmol) and ethanethiol (0.0437 mL, 0.584 mmol) were added to 1,4-dioxane (1.5 mL). Under nitrogen protection, the reaction mixture was reacted under microwave at 120° C. for 1.5 hours, and monitored by LC-MS for completion. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain crude product, which was separated and purified by silica gel column (PE:EA=4:1-2:1) to obtain the target compound (29.0 mg, yield 50.2%, yellow oil). LC-MS (ESI) m/z: 495.0 [M+H]+.
N-(2,6-Dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(6-(ethylthio)pyridin-3-yl) acetamide (8-3) (29.0 mg, 0.0585 mmol) was dissolved in DCM (2 mL) and m-chloroperoxybenzoic acid (23.8 mg, 0.117 mmol) was slowly added to the reaction under ice bath. Upon the addition was complete, the ice bath was removed, and the reaction was continued under stirring at room temperature for 1.5 hours, and monitored by LC-MS for completion. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 mL), extracted with DCM (10 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by a preparative plate (PE:EA=1:3) to obtain the target compound (10.0 mg, yield 32.4%, white solid). LC-MS (ESI) m/z 527.0 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 9.65 (s, 1H), 9.14 (d, J=2.1 Hz, 1H), 8.24 (dd, J=8.1, 2.3 Hz, 1H), 8.02 (d, J=8.2 Hz, 2H), 7.71 (s, 2H), 7.57 (d, J=8.1 Hz, 1H), 7.46 (d, J=8.2 Hz, 2H), 4.03 (s, 2H), 3.20 (q, J=7.4 Hz, 2H), 3.13 (s, 3H), 1.36 (t, J=7.4 Hz, 3H).
The title compound was synthesized according to the following scheme:
2-(5-Bromopyridin-2-yl)-N-(2,6-dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-yl)acetamide (100 mg, 0.189 mmol), methyl mercaptan (18.2 mg, 0.379 mmol), xantphos (10.9 mg, 0.019 mmol), Pd2(dba)3 (10.9 mg, 0.019 mmol) and DIEA (48.9 mg, 0.379 mmol) were dissolved in 1,4-dioxane (4 mL). The reaction mixture was stirred under microwave at 100° C. for 1 hour, and monitored by LC-MS for completion. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by a preparative plate (EA:PE=1:1) to obtain the target compound (36.0 mg, yield 38.4%, white solid). LC-MS (ESI) m/z: 495.0 [M+H]+.
N-(2,6-Dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(5-(methylthio)pyridin-2-yl)acetamide (9-1) (30.0 mg, 0.06 mmol) was dissolved in DCM (3 mL), and m-chloroperoxybenzoic acid (21.0 mg, 0.121 mmol) was slowly added to the reaction solution under ice bath. The ice bath was removed and the reaction mixture was stirred for an additional 1.5 hours at room temperature, and monitored by LC-MS for completion. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (15 mL), extracted with DCM (15 mL×2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain a crude product, which was separated and purified by a preparative plate (EA:PE=2:1) to obtain the target compound (10.0 mg, yield 31.3%, white solid). LC-MS (ESI) m/z: 527.0 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 9.60 (s, 1H), 9.19 (s, 1H), 8.28 (d, J=6.1 Hz, 1H), 7.98 (d, J=8.1 Hz, 2H), 7.71 (s, 2H), 7.57 (d, J=8.1 Hz, 1H), 7.45 (d, J=8.0 Hz, 2H), 4.03 (s, 2H), 3.27-3.11 (m, 5H), 1.33 (t, J=7.4 Hz, 3H).
2-(5-Bromopyridin-2-yl)-N-(2,6-dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl)acetamide (60.0 mg, 0.117 mmol), Pd2(dba)3 (10.7 mg, 0.0117 mmol), xantphos (6.77 mg, 0.0117 mmol), triethylamine (0.0487 mL, 0.350 mmol) and methyl mercaptan (281 mg, 0.584 mmol) were added to 1,4-dioxane (1.5 mL). Under nitrogen protection, the reaction mixture was stirred under microwave at 100° C. for 1.5 hours, and monitored by LC-MS for completion. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (PE:EA=4:1-2:1) to obtain the target compound (30.0 mg, yield 53.4%, yellow oil). LC-MS (ESI) m/z: 480.9 [M+H]+.
N-(2,6-Dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(5-(methylthio)pyridin-2-yl) acetamide (10-1) (30.0 mg, 0.0623 mmol) was dissolved in DCM (2 mL) and m-chloroperoxybenzoic acid (25.3 mg, 0.125 mmol) was slowly added to the reaction solution under ice bath. Upon the addition was complete, the ice bath was removed, and the reaction was continued under stirring at room temperature for 1.5 hours, and monitored by LC-MS for completion. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 mL), extracted with DCM (10 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by a preparative plate (PE:EA=1:3) to obtain the target compound (3.00 mg, yield 9.38%, white solid). LC-MS (ESI) m/z 513.0 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 9.61 (s, 1H), 9.19 (d, J=2.0 Hz, 1H), 8.28 (dd, J=8.1, 2.2 Hz, 1H), 8.02 (d, J=8.3 Hz, 2H), 7.71 (s, 2H), 7.58 (d, J=8.1 Hz, 1H), 7.46 (d, J=8.2 Hz, 2H), 4.03 (s, 2H), 3.15 (s, 3H), 3.13 (s, 3H).
2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-amine (70.0 mg, 0.205 mmol), 2-[4-(methylsulfonyl)phenyl]acetic acid (52.6 mg, 0.245 mmol), HATU (117 mg, 0.307 mmol) and DIEA (79.3 mg, 0.614 mmol) were added to DCM (3 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was poured into saturated aqueous ammonium chloride solution (20 mL) and extracted with DCM (15 mL×2). The organic phases were combined, washed successively with saturated aqueous sodium bicarbonate solution (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by a preparative plate (EA:PE=3:1) to obtain the target compound (14.5 mg, yield 13.2%, white solid). LC-MS (ESI) m/z 536.0 [M−H]−.
1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 7.96 (d, J=8.1 Hz, 2H), 7.90 (d, J=8.0 Hz, 2H), 7.68 (s, 2H), 7.53 (d, J=7.9 Hz, 2H), 7.42 (d, J=8.0 Hz, 2H), 3.82 (s, 2H), 3.07 (s, 3H), 2.61-2.49 (m, 1H), 1.43-1.37 (m, 2H), 1.17-1.06 (m, 2H).
N-(2,6-Dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(4-(ethylsulfonyl)phenyl)acetamide (450 mg, 0.83 mmol) and paraformaldehyde (25 mg, 0.83 mmol) were added to anhydrous dimethylsulfoxide (5 mL). To the reaction mixture was added sodium methoxide (1.78 mg, 0.03 mmol). The reaction mixture was stirred at room temperature overnight. EA (10 mL) and water (10 mL) were added. The organic phase was separated, the aqueous phase was extracted with EA (10 mL×2), the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by column chromatography (silica gel, EA/(DCM/PE=1/2)=0˜100%) to obtain the target compound (200 mg, yield 42.4%, white solid). LC-MS (ESI) m/z: 586.9 [M+NH4]+.
1H NMR (500 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.06 (d, J=8.4 Hz, 2H), 7.98-7.91 (m, 4H), 7.74 (d, J=8.4 Hz, 2H), 7.63 (d, J=8.4 Hz, 2H), 5.22 (t, J=5.2 Hz, 1H), 4.23-4.13 (m, 1H), 4.09-4.03 (m, 1H), 3.83-3.68 (m, 1H), 3.45 (q, J=7.3 Hz, 2H), 3.34 (q, J=7.4 Hz, 2H), 1.21 (t, J=7.3 Hz, 3H), 1.17 (t, J=7.3 Hz, 3H).
Dichloromethane (1 mL), 2,6-dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-amine (45 mg, 0.139 mmol), 2-(4-(ethylsulfonyl)phenyl)-3-hydroxypropionic acid (30 mg, 0.116 mmol), EDC (33 mg, 0.174 mmol), HOBt (24 mg, 0.174 mmol) and DIEA (45 mg, 0.348 mmol) were successively added to a 25 mL single-necked flask. The resulting reactant was stirred at room temperature for 48 hours under nitrogen protection, the solvent was then removed under reduced pressure, and the residue was purified by preparative chromatography to obtain the target compound (18.0 mg, yield 27.2%, white solid). LC-MS (ESI) m/z: 570.0[M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 7.93 (d, J=8.4 Hz, 2H), 7.85 (d, J=8.4 Hz, 2H), 7.65 (d, J=8.4 Hz, 2H), 7.50 (d, J=8.4 Hz, 2H), 6.78 (s, 2H), 6.53 (s, 1H), 3.92 (t, J=6.8 Hz, 1H), 3.78-3.73 (m, 1H), 3.44 (dd, J=12.2, 5.4 Hz, 1H), 3.39-3.34 (m, 2H), 3.29-3.25 (m, 2H), 1.16-1.07 (m, 6H).
To a 250 mL there-necked flask were added 4-ethylsulfonyl phenylacetic acid (2.50 g, 10.9 mmol) and tetrahydrofuran (35 mL), the reaction solution was cooled to −20° C. and then added dropwise isopropylmagnesium bromide (32.8 mL, 32.8 mmol, 1 mol/L). Upon the addition was complete, the reaction solution was reacted at room temperature for 6 hours, and then paraformaldehyde (2.95 g, 32.8 mmol) was added. The reaction continued at room temperature for 18 hours, then cooled to 0° C., quenched with saturated aqueous ammonium chloride solution (40 mL), and acidified to pH=2˜3 with hydrochloric acid (1 mol/L) at 0° C. The separated aqueous phase was extracted again with EA (50 mL×2), the organic layers were combined, and the solvent was rotary dried under reduced pressure to obtain a crude product. The crude product was separated with a silica gel column (dichloromethane:methanol=1:0-4:1) to obtain the target product (2.00 g, yield 71.0%, colorless oil). LC-MS (ESI) m/z 276.1 [M+18]+.
1H NMR (400 MHz, McOH-d4) δ 7.90-7.85 (m, 2H), 7.65-7.60 (m, 2H), 4.13-4.07 (m, 1H), 3.94-3.87 (m, 1H), 3.87-3.81 (m, 1H), 3.20 (q, J=7.4 Hz, 2H), 1.21 (t, J=7.4 Hz, 3H).
2-(4-(ethylsulfonyl)phenyl)-3-hydroxypropionic acid (1.88 g, 7.27 mmol) and 3 mol/L hydrogen chloride methanol solution (60 mL) were added to a 100 mL single-necked flask. The reaction was carried out at 60° C. for 18 hours, and rotary dried under reduced pressure to obtain a crude product. The crude product was subjected to silica gel column (EA:PE=0:1-1:1) to obtain the target product (1.30 g, yield 65.6%, white solid). LC-MS (ESI) m/z: 290.1 [M+18]+.
To a 50 mL single-necked flask were successively added methyl 2-(4-(ethylsulfonyl)phenyl)-3-hydroxypropionate (1.10 g, 4.03 mmol), acetonitrile (10 mL), silver oxide (1.21 g, 5.25 mmol), then cooled to 0° C. Methyl iodide (1.72 g, 12.1 mmol) was added, and the reaction solution was stirred at room temperature for 7 days. The solvent was removed under reduced pressure by rotary evaporation, and the crude product was separated by silica gel column (EA:PE=0:1-2:1) to obtain the target product (800 mg, yield 69.3%, oil). LC-MS (ESI) m/z: 287.0 [M+H]+.
To a 50 mL single-necked flask were added methyl 2-(4-(ethylsulfonyl)phenyl)-3-methoxypropionate (800 mg, 2.79 mmol), methanol (5 mL), water (5 mL), lithium hydroxide (200 mg, 8.38 mmol), and the reaction solution was stirred at 50° C. for 2 hours. The solvent was removed under reduced pressure by rotary evaporation, and the crude product was re-diluted with EA (20 mL) and water (20 mL). The separated aqueous phase was acidified with hydrochloric acid (1 mol/L) to pH=3, and then extracted with EA (20 mL×3). The organic phases from the last three extractions were combined, and the filtrate was rotary dried under reduced pressure to obtain a crude product. The crude product was prepared by high pressure liquid chromatography to obtain the target product (270 mg, yield 35.5%, white solid). LC-MS (ESI) m/z: 273.0 [M+H]+.
1H NMR (400 MHz, MeOH-d4) δ 7.90-7.84 (m, 2H), 7.65-7.60 (m, 2H), 4.04-3.98 (m, 1H), 3.97-3.92 (m, 1H), 3.73-3.68 (m, 1H), 3.34 (s, 3H), 3.20 (q, J=7.6 Hz, 2H), 1.21 (t, J=7.6 Hz, 3H).
To a 50 mL single-necked flask were added 2-(4-(ethylsulfonyl)phenyl)-3-methoxypropionic acid (12-5) (40 mg, 0.146 mmol), DCM (3 mL) and thionyl chloride (87 mg, 0.734 mmol), the reaction solution was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure by rotary evaporation, and the crude product was re-dissolved in DCM (3 mL), added with DIEA (113 mg, 0.881 mmol), 3,5-dichloro-4-(4-ethylsulfonylphenyl)aniline (58 mg, 0.176 mmol). The reaction solution was further stirred at room temperature for 4 hours. The crude product was obtained by rotary evaporation under reduced pressure, which was subject to high pressure liquid chramatography to obtain the target product (40 mg, yield 46.7%, white solid). LC-MS (ESI) m/z: 584.0 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 7.99 (d, J=8.4 Hz, 2H), 7.89 (d, J=8.4 Hz, 2H), 7.84 (s, 2H), 7.68 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.4 Hz, 2H), 4.17-4.11 (m, 1H), 4.00 (t, J=9.0 Hz, 1H), 3.66-3.60 (m, 1H), 3.38 (q, J=7.4 Hz, 2H), 3.31-3.26 (m, 5H), 1.18-1.06 (m, 6H).
2,6-Dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-amine (100 mg, 0.316 mmol), 2-(4-bromophenyl) acetic acid (81.6 mg, 0.379 mmol), DIEA (0.157 mL, 0.949 mmol) and HATU (180 mg, 0.474 mmol) were successively added to DCM (3 mL). The reaction mixture was stirred at room temperature for 2 hours and monitored by LC-MS for completion. The reaction solution was poured into water (20 mL) and extracted with DCM (15 mL×2). The organic phases were combined, washed successively with saturated ammonium chloride (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound (196 mg, crude product, white solid). LC-MS (ESI) m/z: 509.9 [M−H]−.
2-(4-Bromophenyl)-N-(2,6-dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl)acetamide (196 mg, 0.382 mmol), Pd2(dba)3 (35.0 mg, 0.0382 mmol), xantphos (22.1 mg, 0.0382 mmol), triethylamine (0.159 mL, 1.15 mmol) and 2-mercaptoethanol (0.0538 mL, 0.764 mmol) were added into 1,4-dioxane (3 mL). Under nitrogen protection, the reaction mixture was stirred at 100° C. overnight, and monitored by TLC for completion. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound (600 mg, crude, yellow oil).
N-(2,6-Dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(4-(2-hydroxyethylthio)phenyl)acetamide (600 mg, 1.18 mmol) was dissolved in DCM (10 mL), and m-chloroperoxybenzoic acid (507 mg, 2.94 mmol) was slowly added to the reaction under ice bath. Upon the addition was complete, the ice bath was removed, and the reaction was continued under stirring at room temperature for 2 hours, and monitored by LC-MS for completion. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (30 mL), extracted with DCM (20 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified on silica gel column (PE:EA=2:1-1:2) to obtain the target compound (30.0 mg, 17.5% yield over three steps, white solid). LC-MS (ESI) m/z: 541.9 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 8.02 (d, J=8.0 Hz, 2H), 7.94 (d, J=7.9 Hz, 2H), 7.66 (s, 2H), 7.60-7.53 (m, 3H), 7.45 (d, J=7.9 Hz, 2H), 4.06-4.00 (m, 2H), 3.84 (s, 2H), 3.42-3.33 (m, 2H), 3.14 (s, 3H).
2-[4-(Ethylsulfonyl)phenyl]-3-methoxypropionic acid (40.0 mg, 0.147 mmol) was dissolved in dry DCM (3 mL), and thionyl chloride (0.0533 mL, 0.735 mmol) was added dropwise to the reaction solution at room temperature. Upon the addition was complete, the reaction was continued under stirring at room temperature for 3 hours. The intermediate obtained by concentrating the reaction solution under reduced pressure was dissolved in dry DCM (3 mL), and 2,6-dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-amine (46.5 mg, 0.147 mmol) and DIEA (0.159 mL, 0.955 mmol) were added successively. The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was poured into water (20 mL) and extracted with DCM (15 mL×2). The organic phases were combined, washed successively with saturated ammonium chloride (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (PE:EA=1:9) to obtain the target compound (20.0 mg, yield 23.9%, white solid). LC-MS (ESI) m/z: 569.9 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 8.02 (d, J=8.1 Hz, 2H), 7.90 (d, J=8.1 Hz, 2H), 7.70 (s, 2H), 7.58 (d, J=7.9 Hz, 2H), 7.45 (d, J=8.1 Hz, 2H), 4.07-3.99 (m, 1H), 3.98-3.92 (m, 1H), 3.82-3.74 (m, 1H), 3.49 (s, 3H), 3.19-3.07 (m, 5H), 1.30 (t, J=7.4 Hz, 3H).
2-[4-(Ethylsulfonyl)phenyl]-3-methoxypropionic acid (40.0 mg, 0.147 mmol) was dissolved in dry DCM (3 mL), and thionyl chloride (0.0533 mL, 0.735 mmol) was added dropwise to the reaction solution at room temperature. Upon the addition was complete, the reaction was continued under stirring at room temperature for 3 hours. The intermediate obtained by concentrating the reaction solution under reduced pressure was dissolved in dry DCM (3 mL), and 2,6-dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-amine (50.3 mg, 0.147 mmol) and DIEA (0.158 mL, 0.955 mmol) were added successively. The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was poured into water (20 mL) and extracted with DCM (15 mL×2). The organic phases were combined, washed successively with saturated ammonium chloride (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (PE:EA=1:9) to obtain the target compound (27.0 mg, yield 30.8%, white solid). LC-MS (ESI) m/z: 596.1 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 7.98 (d, J=8.1 Hz, 2H), 7.87 (d, J=8.0 Hz, 2H), 7.82 (s, 2H), 7.66 (d, J=8.0 Hz, 2H), 7.53 (d, J=8.1 Hz, 2H), 4.15-4.08 (m, 1H), 3.98 (t, J=9.0 Hz, 1H), 3.65-3.57 (m, 1H), 3.29-3.25 (m, 5H), 2.98-2.89 (m, 1H), 1.18-1.13 (m, 2H), 1.11-1.04 (m, 5H).
Methyl 2-(4-bromophenyl)acetate (9.00 g, 39.3 mmol), pd2(dba)3 (2.88 g, 3.14 mmol), xantphos (1.82 g, 3.14 mmol), DIEA (13.0 mL, 78.6 mmol) and benzyl mercaptan (9.22 mL, 78.6 mmol) were added to 1,4-dioxane (120 mL). Under nitrogen protection, the reaction mixture was stirred at 100° C. overnight, and monitored by TLC for completion. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (PE:EA=20:1-10:1) to obtain the target compound (10.2 g, yield 95.3%, yellow oily).
Methyl 2-(4-(benzylthio)phenyl)acetate (10.2 g, 37.4 mmol) was dissolved in a mixed solvent of acetic acid (100 mL) and water (25 mL). Under an ice bath, N-chlorosuccinimide (20.0 g, 150 mmol) was slowly added to the reaction solution. After the addition was complete, the ice bath was removed, and the reaction was continued under stirring at room temperature for 2 hours, and monitored by TLC for completion. The reaction solution was poured into water (200 mL), extracted with EA (50 mL×2), the organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (PE:EA=10:1-5:1) to obtain the target compound (8.5 g, yield 91.3%, colorless oil).
Methyl 2-[4-(chlorosulfonyl)phenyl]acetate (5.00 g, 20.1 mmol) was dissolved in a mixed solvent of tetrahydrofuran (60 mL) and water (40 mL). Sodium bicarbonate (3.38 g, 40.2 mmol) and sodium sulfite (3.04 g, 24.1 mmol) were added to the reaction at room temperature. Upon the addition was complete, the reaction solution was heated to 70° C. and stirred for 2 hours, and monitored by LC-MS for completion. The reaction solution was concentrated under reduced pressure to obtain the intermediate, which was dissolved in anhydrous dimethylsulfoxide (90 mL), and added with (bromomethyl)cyclopropane (5.90 mL, 60.3 mmol). Under nitrogen protection, the reaction mixture was stirred at 100° C. for 3 hours, and monitored by TLC for completion. The reaction solution was poured into water (150 mL), extracted with EA (80 mL×2), the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (PE:EA=7:3) to obtain the target compound (2.80 g, yield 51.9%, light brown solid).
Methyl 2-(4-cyclopropylmethylsulfonylphenyl)acetate (58.0 mg, 0.216 mmol) was dissolved in methanol (3 mL). At room temperature, a 1M aqueous sodium hydroxide solution (1 mL) was added dropwise to the reaction solution. Upon the addition was complete, the reaction was continued under stirring at room temperature for 2 hours, and monitored by LC-MS for completion. The reaction solution was poured into water (10 mL) and adjusted to pH-4 with 1M dilute hydrochloric acid. The reaction was extracted with EA (15 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound (45.0 mg, crude product, colorless oil). LC-MS (ESI) m/z 507.1 [2M−H]−.
2,6-Dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-amine (56.0 mg, 0.177 mmol), 2-(4-cyclopropylmethylsulfonylphenyl)acetic acid (45.0 mg, 0.177 mmol), DIEA (0.0878 mL, 0.531 mmol) and HATU (101 mg, 0.266 mmol) were successively added to DCM (3 mL). The reaction mixture was stirred at room temperature for 2 hours and monitored by LC-MS for completion. The reaction solution was poured into water (20 mL) and extracted with DCM (15 mL×2). The organic phases were combined, washed successively with saturated ammonium chloride (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by reverse phase column (20% water/80% acetonitrile) to obtain the target compound (35.0 mg, 29.4% over two steps, white solid). LC-MS (ESI) m/z: 550.0 [M−H]−.
1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.02 (d, J=8.1 Hz, 2H), 7.85 (d, J=8.1 Hz, 2H), 7.82 (s, 2H), 7.59 (d, J=8.0 Hz, 2H), 7.55 (d, J=8.1 Hz, 2H), 3.85 (s, 2H), 3.29 (s, 3H), 3.23 (d, J=7.0 Hz, 2H), 0.86-0.76 (m, 1H), 0.48-0.39 (m, 2H), 0.14-0.06 (m, 2H).
Methyl 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetate (1000 mg, 3.73 mmol) and paraformaldehyde (302 mg, 3.35 mmol) were dissolved in anhydrous dimethylsulfoxide (10 mL). Sodium methoxide (8.05 mg, 0.149 mmol) was added to the reaction solution at room temperature. Upon the addition was complete, the reaction solution was stirred at room temperature overnight. The reaction solution was poured into ice water (25 mL), and neutralized with 1M dilute hydrochloric acid to pH-7. The resultant mixture was extracted with EA (15 mL×3), the combined organic phases was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (PE:EA=10:1-1:1) to obtain the target compound (765 mg, yield 65.7%, light brown solid), and the raw material (196 mg) was recovered. LC-MS (ESI) m/z: 297.1 [M−H]−.
Methyl 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)-3-hydroxypropanoate (490 mg, 1.64 mmol) and silver oxide (457 mg, 1.97 mmol) were dissolved in acetonitrile (4 mL) under a condition protected from light. Under an ice bath, iodomethane (0.153 mL, 2.46 mmol) was added dropwise to the reaction solution. Upon the addition was complete, the ice bath was removed and the reaction was stirred at room temperature for 3 days. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which wad separated and purified by silica gel column (PE:EA=10:1-1:1) to obtain the target compound (70.0 mg, yield 13.6%, pale yellow solid), and the raw material (255 mg) was recovered. LC-MS (ESI) m/z: 313.1 [M+H]+.
Methyl 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)-3-methoxypropanoate (70.0 mg, 0.224 mmol) was dissolved in methanol (2 mL). At room temperature, a 1M aqueous sodium hydroxide solution (2 mL) was added dropwise to the reaction solution. Upon the addition was complete, the reaction was continued under stirring at room temperature for 2 hours, and monitored by LC-MS for completion. The reaction solution was poured into water (10 mL) and adjusted to pH-4 with 1M dilute hydrochloric acid. The reaction was extracted with EA (10 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound (60.0 mg, crude product, pale yellow oil). LC-MS (ESI) m/z: 297.0 [M−H]−.
2-(4-((Cyclopropylmethyl)sulfonyl)phenyl)-3-methoxypropionic acid (60.0 mg, 0.201 mmol) was dissolved in dry DCM (3 mL). At room temperature, thionyl chloride (0.073 mL, 1.01 mmol) was added dropwise to the reaction solution. Upon the addition was complete, the reaction was continued under stirring at room temperature for 3 hours. The intermediate obtained by concentrating the reaction solution under reduced pressure was dissolved in dry DCM (3 mL), and 2,6-dichloro-4′-(methylsulfonyl)-[1,1′-biphenyl]-4-amine (63.6 mg, 0.201 mmol) and DIEA (0.216 mL, 1.31 mmol) was added successively. The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was poured into water (20 mL) and extracted with DCM (15 mL×2). The organic phases were combined, washed successively with saturated ammonium chloride (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by reverse phase column (20% water/80% acetonitrile) to obtain the target compound (26.0 mg, 19.5% yield over two steps, white solid). LC-MS (ESI) m/z: 594.1 [M−H]−.
1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.01 (d, J=8.1 Hz, 2H), 7.87 (d, J=8.1 Hz, 2H), 7.84 (s, 2H), 7.66 (d, J=8.0 Hz, 2H), 7.53 (d, J=8.1 Hz, 2H), 4.18-4.11 (m, 1H), 3.98 (t, J=8.9 Hz, 1H), 3.62-3.55 (m, 1H), 3.28 (s, 3H), 3.27 (s, 3H), 3.23 (d, J=7.1 Hz, 2H), 0.84-0.75 (m, 1H), 0.47-0.36 (m, 2H), 0.14-0.04 (m, 2H).
2-(4-Methylsulfonyl) phenylacetic acid (9.00 g, 42.0 mmol) was dissolved in methanol (100.0 mL), and thionyl chloride (15.0 g, 126 mmol) was added at 0° C. The reaction solution was warmed to room temperature and stirred for 1 hour, and then was stopped. The reaction solution was concentrated under reduced pressure, and the obtained residue was separated and purified by column chromatography (PE:EA=3:1-2:1) to obtain the target compound (8.60 g, yield 89.7%, yellow solid). MS (ESI) m/z: 229.0 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 7.91 (d, J=8.4 Hz, 2H), 7.49 (d, J=8.5 Hz, 2H), 3.73 (s, 2H), 3.72 (s, 3H), 3.05 (s, 3H).
To a solution of methyl 2-(4-methylsulfonyl)phenylacetate (9.40 g, 41.2 mmol) and paraformaldehyde (1.17 g, 39.2 mmol) in anhydrous dimethyl sulfoxide (100.0 mL) was added sodium methoxide (89.0 mg, 1.65 mmol), and the reaction was stirred at room temperature for 16 hours, and then was stopped. The reaction solution was poured into ice water (300.0 mL), 1.0M hydrochloric acid solution was added, and extracted with EA (400 mL×2). The organic phase was washed with saturated brine (60.0 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was separated and purified by column chromatography (PE:EA=3:1-1:1) to obtain the target compound (6.15 g, yield 57.8%, pale yellow oil). MS (ESI) m/z: 290.1 [M+NH4]+.
1H NMR (400 MHz, DMSO-d6) δ 7.87 (d, 2H), 7.57 (d, 2H), 5.08 (t, J=4.9 Hz, 1H), 3.95-3.90 (m, 2H), 3.72-3.65 (m, 1H), 3.61 (s, 3H), 3.18 (s, 3H).
Methyl 2-(4-(methylsulfonyl)phenyl)-3-hydroxypropanoate (6.10 g, 23.6 mmol) was dissolved in DCM (140 mL) and cooled to 0° C. with an ice-water bath. Fluoroboric acid (4.14 g, 23.6 mmol, 48% aqueous solution) and trimethylsilyl diazomethane (11.8 mL, 23.6 mmol, 2M) were successively added to the reaction solution under a condition protected from light. After 20 minutes, trimethylsilane diazomethane (11.8 mL, 23.6 mmol) was added again. After 20 minutes, trimethylsilane diazomethane (11.8 mL, 23.6 mmol) was added once again. After 20 minutes, trimethylsilane diazomethane (11.8 mL, 23.6 mmol) was added again. The reaction solution was poured into water (80.0 mL), extracted with DCM (200 mL×3), and allowed to stand to separate the layers. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was separated and purified by column chromatography (PE:EA=5:1-3:1) to obtain the target compound (4.62 g, yield 71.9%, colorless oil). MS (ESI) m/z: 273.2 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 7.90 (d, 2H), 7.53 (d, 2H), 4.00-3.92 (m, 2H), 3.71 (s, 3H), 3.69-3.65 (m, 1H), 3.35 (s, 3H), 3.04 (s, 3H).
Methyl 2-(4-(methylsulfonyl)phenyl)-3-methoxypropanoate (1.00 g, 3.67 mmol) was dissolved in the mixture of 1,4-dioxane (30.0 mL) and 6.0 M hydrochloric acid (30.0 mL). The reaction solution was stirred at 80° C. for 2 hours, and then was stopped. The reaction solution was cooled to room temperature, carefully poured into water (80.0 mL), extracted with EA (150 mL×2), and allowed to stand to separate the layers. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by reverse phase chromatography (acetonitrile/water containing 0.05% formic acid) to obtain the target compound (880 mg, yield 77.3%, white solid). MS (ESI) m/z: 276.0 [M+NH4]+.
1H NMR (400 MHz, CDCl3) δ 7.91 (d, J=8.3 Hz, 2H), 7.54 (d, J=8.3 Hz, 2H), 4.01-3.92 (m, 2H), 3.72-3.66 (m, 1H), 3.37 (s, 3H), 3.05 (s, 3H).
2-(4-(Methylsulfonyl)phenyl)-3-methoxypropionic acid (105 mg, 0.407 mmol) was dissolved in DCM (3.00 mL), and thionyl chloride (52.4 mg, 118.97 mmol) was added at 0° C. The reaction solution was stirred at room temperature for 2 hours, dried under reduced pressure to remove the solvent to obtain a crude product. The crude product was redissolved in DCM (3.0 mL) and carefully added dropwise to the ice-water bath cooled solution of DIEA (131 mg, 1.02 mmol) and 2,6-dichloro-4′-(ethylsulfonyl)-[1,1′-biphenyl]-4-amine (112 mg, 0.339 mmol) in DCM (2.00 mL). The reaction solution was stirred for 20 minutes in an ice-water bath. The crude product was obtained by drying under reduced pressure and removing the solvent, which was subject to high pressure liquid chromatography (acetonitrile/water containing 0.05% ammonia water) to obtain the target compound (51.3 mg, yield 26.6%, pale yellow solid). MS (ESI) m/z: 570.2 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 8.52 (s, 1H), 7.97 (d, J=8.4 Hz, 2H), 7.92 (d, J=8.2 Hz, 2H), 7.70 (s, 2H), 7.59 (d, J=8.3 Hz, 2H), 7.44 (d, J=8.4 Hz, 2H), 4.06-4.01 (m, 1H), 4.00-3.94 (m, 1H), 3.80-3.74 (m, 1H), 3.48 (s, 3H), 3.19 (q, J=7.4 Hz, 2H), 3.06 (s, 3H), 1.33 (t, J=7.4 Hz, 3H).
3-Methoxy-2-(4-methylsulfonyl) phenyl)propanoic acid (105 mg, 0.407 mmol) was dissolved in DCM (3.00 mL), and thionyl chloride (52.4 mg, 0.441 mmol) was added at 0° C. The reaction solution was stirred at room temperature for 2 hours, dried under reduced pressure to remove the solvent, the crude product was redissolved in DCM (3.0 mL), and carefully added dropwise to the ice-water bath cooled solution of DIEA (131 mg, 1.02 mmol) and 2,6-dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-amine (116 mg, 0.339 mmol) in DCM (2.0 mL). The reaction solution was stirred for 20 minutes in an ice-water bath. The crude product was obtained by drying under reduced pressure and removing the solvent, which was subject to high pressure liquid chromatography (acetonitrile/water containing 0.05% ammonia water) to obtain the target compound (52.9 mg, yield 26.9%, off-white solid). MS (ESI) m/z: 582.0 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 7.97 (d, J=8.0 Hz, 2H), 7.93 (d, J=7.6 Hz, 2H), 7.72 (s, 2H), 7.61 (d, J=7.7 Hz, 2H), 7.43 (d, J=8.0 Hz, 2H), 4.08-3.98 (m, 2H), 3.79-3.74 (m, 1H), 3.48 (s, 3H), 3.06 (s, 3H), 2.57-2.50 (m, 1H), 1.43-1.38 (m, 2H), 1.13-1.05 (m, 2H).
2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-amine (150 mg, 0.438 mmol), 2-(5-bromopyridine-2-yl)acetic acid (114 mg, 0.526 mmol), DIEA (0.217 mL, 1.31 mmol) and HATU (250 mg, 0.658 mmol) were added to DCM (5 mL). The reaction mixture was stirred at room temperature for 2 hours and monitored by LC-MS for completion. The reaction solution was poured into water (20 mL) and extracted with DCM (10 mL×2). The organic phases were combined, washed successively with saturated ammonium chloride (15 mL) and saturated aqueous sodium bicarbonate solution (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound (283 mg, crude product, yellow oil). LC-MS (ESI) m/z: 538.9 [M+H]+.
2-(5-Bromopyridin-2-yl)-N-(2,6-dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-yl)acetamide (283 mg, 0.524 mmol), Pd2(dba)3 (48.0 mg, 0.0524 mmol), xantphos (30.3 mg, 0.0524 mmol), DIEA (0.173 mL, 1.05 mmol) and ethanethiol (0.0784 ML, 1.05 mmol) were added to 1,4-dioxane (2 mL). Under nitrogen protection, the reaction mixture was stirred in a sealed tube at 100° C. overnight, and monitored by LC-MS for completion. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (PE:EA=4:1-2:1) to obtain the target compound (170 mg, yield 74.6% over two steps, yellow oil). LC-MS (ESI) m/z: 520.9 [M+H]+.
N-(2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(5-(ethylthio)pyridin-2-yl)acetamide (170 mg, 0.326 mmol) was dissolved in DCM (5 mL), and m-chloroperoxybenzoic acid (132 mg, 0.652 mmol) was slowly added to the reaction solution under ice bath. Upon the addition was complete, the ice bath was removed, and the reaction was continued under stirring at room temperature for 1.5 hours, and monitored by LC-MS for completion. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (20 mL), extracted with DCM (10 mL×2), the organic phases were combined, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (PE:EA=1:9) to obtain crude product (120 mg). Further separation and purification by reverse phase column (water:acetonitrile=3:7) gave the target compound (76.0 mg, yield 42.1%, white solid). LC-MS (ESI) m/z: 552.9 [M+H]+.
1H NMR (500 MHz, MeOD) δ 8.90 (d, J=2.4 Hz, 1H), 8.20 (dd, J=8.2, 2.4 Hz, 1H), 7.91 (d, J=8.5 Hz, 2H), 7.73 (s, 2H), 7.62 (d, J=8.2 Hz, 1H), 7.42 (d, J=8.5 Hz, 2H), 3.25-3.20 (m, 2H), 2.71 (s, 2H), 2.68-2.63 (m, 1H), 1.21-1.19 (m, 2H), 1.17 (t, J=7.4 Hz 3H), 1.03-1.01 (m, 2H).
2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-amine (30 mg, 0.0877 mmol) and 4-(ethylsulfonyl)phenyl)-3-hydroxypropionic acid (27.1 mg, 0.105 mmol) were added to DCM (3 mL), and HOBt (17.8 mg, 0.132 mmol), EDC (25.3 mg, 0.132 mmol) and DIEA (0.043 mL, 0.263 mmol) were added successively. The reaction mixture was stirred at room temperature for 3 days. To the reaction mixture was added saturated aqueous sodium bicarbonate solution (10 mL) and DCM (10 mL). The aqueous phase was separated and extracted with DCM (10 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by preparative plate (silica gel, dichloromethane:methanol=15:1), to obtain the target compound (6 mg, yield 11.7%, white solid). LC-MS (ESI) m/z 582.0 [M+H]+.
1H NMR (400 MHz, MeOH-d4) δ 7.95 (d, J=8.4 Hz, 2H), 7.91 (d, J=8.4 Hz, 2H), 7.68 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.4 Hz, 2H), 6.70 (s, 2H), 4.09-4.04 (m, 1H), 3.90-3.84 (m, 1H), 3.60-3.54 (m, 1H), 3.20 (q, J=7.6 Hz, 2H), 2.78-2.72 (m, 1H), 1.31-1.27 (m, 2H), 1.23 (t, J=7.4 Hz, 3H), 1.15-1.08 (m, 2H).
2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-amine (100 mg, 0.292 mmol) and 2-(4-(ethylsulfonyl)phenyl) acetic acid (79.9 mg, 0.350 mmol) were added to DCM (5 mL). To the reaction mixture was added successively HATU (133 mg, 0.350 mmol) and DIEA (0.145 mL, 0.876 mmol). The reaction mixture was stirred at room temperature for 2 hours. Saturated aqueous sodium bicarbonate solution (10 mL) and DCM (10 mL) were added. The organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by column chromatography (silica gel, EA:PE=0˜40%), to obtain the target compound (150 mg, yield 93.2%, colorless oil). LC-MS (ESI) m/z: 569.0 [M+NH4]+.
N-(2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(4-(ethylsulfonyl)phenyl) acetamide (60 mg, 0.109 mmol) and paraformaldehyde (2.94 mg, 0.098 mmol) were added to anhydrous dimethylsulfoxide (2 mL). To the reaction mixture was added sodium methoxide (0.24 mg, 0.004 mmol). The reaction mixture was stirred at room temperature for 2 hours. EA (10 mL) and water (10 mL) were added. The organic phase was separated, the aqueous phase was extracted with EA (10 mL×2), the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by column chromatography (silica gel, EA/(DCM/PE=1/2)=0˜100%), to obtain the target compound (30 mg, yield 47.6%, white solid). LC-MS (ESI) m/z: 599.0 [M+NH4]+.
1H NMR (500 MHz, DMSO-d6) δ 10.68 (s, 1H), 7.99 (d, J=8.4 Hz, 2H), 7.91-7.84 (m, 4H), 7.66 (d, J=8.4 Hz, 2H), 7.55 (d, J=8.4 Hz, 2H), 5.14 (t, J=5.2 Hz, 1H), 4.11-4.06 (m, 1H), 4.00-3.95 (m, 1H), 3.70-3.65 (m, 1H), 3.28 (q, J=7.3 Hz, 2H), 2.99-2.94 (m, 1H), 1.20-1.15 (m, 2H), 1.13-1.06 (m, 5H).
2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-amine (100 mg, 0.292 mmol) and 2-(4-(methylsulfonyl)phenyl)acetic acid (75.0 mg, 0.350 mmol) were added to DCM (5 mL). To the reaction mixture was successively added HATU (133 mg, 0.350 mmol) and DIEA (0.145 mL, 0.876 mmol). The reaction mixture was stirred at room temperature for 2 hours. Saturated aqueous sodium bicarbonate solution (10 mL) and DCM (10 mL) were added. The organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by column chromatography (silica gel, EA:PE=0˜40%), to obtain the target compound (145 mg, yield 92.4%, yellow solid). LC-MS (ESI) m/z: 555.0 [M+NH4]+.
N-(2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(4-(methylsulfonyl)phenyl) acetamide (60 mg, 0.111 mmol) and paraformaldehyde (3.0 mg, 0.100 mmol) were added to anhydrous dimethylsulfoxide (2 mL). To the reaction mixture was added sodium methoxide (0.24 mg, 0.004 mmol). The reaction mixture was stirred at room temperature for 2 hours. Ethyl acetate (10 mL) and water (10 mL) were added. The separated aqueous phase was extracted with EA (10 mL×2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by column chromatography (silica gel, EA: (DCM:PE=1:2)=0˜100%), to obtain the target compound (35 mg, yield 55.6%, white solid). LC-MS (ESI) m/z: 585.0 [M+NH4]+.
1H NMR (500 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.00 (d, J=8.4 Hz, 2H), 7.92 (d, J=8.4 Hz, 2H), 7.86 (s, 2H), 7.65 (d, J=8.4 Hz, 2H), 7.55 (d, J=8.4 Hz, 2H), 5.14 (t, J=5.2 Hz, 1H), 4.13-4.06 (m, 1H), 4.01-3.94 (m, 1H), 3.70-3.63 (m, 1H), 3.20 (s, 3H), 3.00-2.93 (m, 1H), 1.20-1.16 (m, 2H), 1.12-1.07 (m, 2H).
Racemate N-(2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-yl)-3-methoxy-2-(4-(methylsulfonyl)phenyl)propanamide (Example 20, 500 mg dissolved in about 80 mL methanol, injection volume 5.0 mL) was separated through a Waters SFC 150 (room temperature, 100 bar, 214 nm) and 250*25 mm 10 μm Dr. maish Reprosil Chiral-OM (similar to DAICELCHIRALCEL® OD) (supercritical carbon dioxide:methanol, 50:50, 3.0 min, 70 mL/min) to obtain Example 34 (212 mg, white solid, Ret.time=1.284 min, ee 99%), LC-MS (ESI) m/z: 582.1 [M+H]+/584.1 [M+2+H]+; 1H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 8.05-7.87 (m, 4H), 7.69 (s, 2H), 7.64-7.53 (m, 2H), 7.43 (d, J=6.9 Hz, 2H), 4.09-3.91 (m, 2H), 3.85-3.75 (m, 1H), 3.51 (s, 3H), 3.07 (s, 3H), 2.60-2.49 (m, 1H), 1.47-1.36 (m, 2H), 1.16-1.03 (m, 2H); and Example 35 (208 mg, white solid, Ret. time=1.711 min, ee 9.8%); LC-MS (ESI) m/z: 582.1 [M+H]+/584.1 [M+2+H]+. 1H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 8.00-7.91 (m, 4H), 7.69 (s, 2H), 7.59 (d, J=8.3 Hz, 2H), 7.43 (d, J=8.3 Hz, 2H), 4.07-4.00 (m, 1H), 3.99-3.93 (m, 1H), 3.82-3.75 (m, 1H), 3.50 (s, 3H), 3.06 (s, 3H), 2.59-2.50 (m, 1H), 1.45-1.38 (m, 2H), 1.13-1.06 (m, 2H).
Racemate N-(2,6-dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(4-(methylsulfonyl)phenyl)-3-hydroxypropionamide (Example 33, 500 mg dissolved in about 120 mL methanol, injection volume 2.5 mL) was separated through a Waters SFC 150 (room temperature, 100 bar, 214 nm) and a 250*25 mm 10% m Dr. maish Reprosil Chiral-OM (similar to DAICELCHIRALCEL® OD) (supercritical carbon dioxide:methanol, 55:45, 3.0 min, 70 mL/min) to obtain Example 36 (158 mg, white solid, Ret.time=1.373 min, ee 99%), LC-MS (ESI) m/z: 567.9 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 7.97 (d, J=8.5 Hz, 2H), 7.91 (d, J=8.4 Hz, 2H), 7.70 (s, 2H), 7.56 (d, J=8.4 Hz, 2H), 7.43 (d, J=8.5 Hz, 2H), 4.23-4.16 (m, 1H), 4.05-3.99 (m, 1H), 3.98-3.92 (m, 1H), 3.07 (s, 3H), 2.58-2.50 (m, 1H), 1.44-1.38 (m, 2H), 1.13-1.06 (m, 2H); and Example 37 (102 mg, white solid, Ret.time=1.701 min, ee 99%), LC-MS (ESI) m/z: 567.9 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H), 7.97 (d, J=8.4 Hz, 2H), 7.93 (d, J=8.2 Hz, 2H), 7.69 (s, 2H), 7.57 (d, J=8.2 Hz, 2H), 7.43 (d, J=8.3 Hz, 2H), 4.26-4.17 (m, 1H), 4.07-3.99 (m, 1H), 3.98-3.91 (m, 1H), 3.07 (s, 3H), 2.59-2.48 (m, 1H), 1.44-1.38 (m, 2H), 1.13-1.07 (m, 2H).
Racemate N-(2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(4-(ethylsulfonyl) phenyl)-3-hydroxypropionamide (Example 22, 500 mg dissolved in about 150 mL methanol, injection volume 5.0 mL) was separated through a Waters SFC 150 (room temperature, 100 bar, 214 nm) and a 250*25 mm 10 m Dr. maish Reprosil Chiral-OM (similar to DAICELCHIRALCEL®OD) (supercritical carbon dioxide:methanol, 55:45, 3.2 min, 70 mL/min) to obtain Example 38 (165 mg, white solid, Ret. time=1.370 min, ee 99%), LC-MS (ESI) m/z: 581.9 [M+H]+/583.9 [M+2+H]+; 1H NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.97 (d, J=8.4 Hz, 2H), 7.88 (d, J=8.3 Hz, 2H), 7.70 (s, 2H), 7.56 (d, J=8.3 Hz, 2H), 7.43 (d, J=8.4 Hz, 2H), 4.24-4.16 (m, 1H), 4.06-3.99 (m, 1H), 3.99-3.91 (m, 1H), 3.14 (q, J=7.4 Hz, 2H), 2.59-2.50 (m, 1H), 1.44-1.38 (m, 2H), 1.31 (t, J=7.4 Hz, 3H), 1.13-1.06 (m, 2H); and Example 39 (117 mg, white solid, Ret. time=1.759 min, ee 99%), LC-MS (ESI) m/z 581.9 [M+H]+/583.9 [M+2+H]+; 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.97 (d, J=8.4 Hz, 2H), 7.87 (d, J=8.2 Hz, 2H), 7.71 (s, 2H), 7.56 (d, J=8.3 Hz, 2H), 7.43 (d, J=8.4 Hz, 2H), 4.24-4.17 (m, 1H), 4.06-3.99 (m, 1H), 3.98-3.90 (m, 1H), 3.13 (q, J=7.4 Hz, 2H), 2.59-2.49 (m, 1H), 1.44-1.38 (m, 2H), 1.31 (t, J=7.4 Hz, 3H), 1.13-1.06 (m, 2H).
Racemate N-(2,6-Dichloro-4′-(cyclopropylsulfonyl)-[1,1′-biphenyl]-4-yl)-2-(4-(ethylsulfonyl) phenyl)-3-methoxypropionamide (Example 16, 500 mg dissolved in about 100 mL methanol, injection volume 8.0 mL) was separated through a Waters SFC 150 (room temperature, 100 bar, 214 nm) and 250*25 mm 10 m Dr. maish Reprosil Chiral-OM (similar to DAICELCHIRALCEL® OD) (supercritical carbon dioxide:methanol, 50:50, 3.5 min, 70 mL/min) to obtain Example 40 (175 mg, white solid, Ret. time=1.287 min, ee 99%), LC-MS (ESI) m/z 596.1[M+H]+/598.1 [M+2+H]+; 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 7.97 (d, J=8.3 Hz, 2H), 7.91 (d, J=8.3 Hz, 2H), 7.69 (s, 2H), 7.58 (d, J=8.3 Hz, 2H), 7.43 (d, J=8.3 Hz, 2H), 4.06-3.99 (m, 1H), 3.97-3.92 (m, 1H), 3.82-3.77 (m, 1H), 3.50 (s, 3H), 3.13 (q, J=7.4 Hz, 2H), 2.58-2.51 (m, 1H), 1.44-1.38 (m, 2H), 1.30 (t, J=7.4 Hz, 3H), 1.12-1.06 (m, 2H); and Example 41 (153 mg, white solid, Ret. time=1.797 min, ee 99%), LC-MS (ESI) m/z 596.1 [M+H]+/598.1 [M+2+H]+; 1H NMR (400 MHz, CDCl3) δ 8.52 (s, 1H), 7.97 (d, J=8.3 Hz, 2H), 7.88 (d, J=8.3 Hz, 2H), 7.71 (s, 2H), 7.58 (d, J=8.3 Hz, 2H), 7.43 (d, J=8.3 Hz, 2H), 4.07-4.00 (m, 1H), 4.00-3.94 (m, 1H), 3.80-3.73 (m, 1H), 3.48 (s, 3H), 3.13 (q, J=7.4 Hz, 2H), 2.59-2.50 (m, 1H), 1.45-1.37 (m, 2H), 1.30 (t, J=7.4 Hz, 3H), 1.15-1.06 (m, 2H).
To a solution of 4-bromo-3-(trifluoromethyl)-aniline (1.00 g, 4.17 mmol) and 2-(4-(cyclopropylsulfonyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.93 g, 6.23 mmol) in 1,4-dioxane (40.0 mL) was added Pd(dppf)Cl2 (305 mg, 0.417 mmol) and potassium carbonate (1.15 g, 8.32 mmol). Under argon protection, the reaction mixture was stirred at 100° C. for 12 hours, and then the reaction was stopped. After cooling the reaction solution to room temperature, the solvent was removed by rotary evaporation under reduced pressure, and the obtained residue was separated and purified by column chromatography (PE:EA=10:1, 5:1-3:1) to obtain the target compound (820 mg, yield 57.7%, yellow solid). LC-MS (ESI) m/z 342.1 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 7.90 (d, J=8.2 Hz, 2H), 7.48 (d, J=8.1 Hz, 2H), 7.15-7.06 (m, 2H), 6.94 (d, J=7.5 Hz, 1H), 2.63-2.39 (m, 1H), 1.45-1.34 (m, 2H), 1.10-1.05 (m, 2H).
To a solution of 4′-(cyclopropylsulfonyl)-2-(trifluoromethyl)-[1,1′-biphenyl]-4-amine (200 mg, 0.586 mmol), 2-(4-(methylsulfonyl)phenyl)-3-methoxypropionic acid (303 mg, 1.17 mmol) and N-methylimidazole (241 mg, 2.93 mmol) in acetonitrile (4.00 mL) was added N,N,N′,N′-tetra methylchloroformamidine hexafluorophosphate (493 mg, 1.76 mmol). The reaction solution was stirred at room temperature for 16 hours, and then the reaction was stopped. The reaction solution was directly preoperatively purified by high pressure liquid chromatography (acetonitrile/water containing 0.05% formic acid) to obtain the target compound (91.4 mg, yield 26.8%, white solid). LC-MS (ESI) m/z: 581.9 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H), 7.98-7.90 (m, 5H), 7.81 (dd, J=8.3, 2.1 Hz, 1H), 7.61 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.3 Hz, 2H), 7.28-7.26 (d, J=8.4, 1H), 4.09-4.02 (m, 1H), 4.01-3.95 (m, 1H), 3.83-3.78 (m, 1H), 3.51 (s, 3H), 3.06 (s, 3H), 2.56-2.48 (m, 1H), 1.43-1.37 (m, 2H), 1.12-1.05 (m, 2H).
Racemate N-(4′-(cyclopropylsulfonyl)-2-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-3-methoxy-2-(4-(methylsulfonyl)phenyl)propanamide (Example 42, 90 mg dissolved in about 30 mL methanol, injection volume 2.0 mL) was separated through a Waters SFC 150 (room temperature, 100 bar, 214 nm) and 250*25 mm 10 m Dr. maish Reprosil Chiral-OM (similar to DAICELCHIRALCEL® OD) (supercritical carbon dioxide:methanol, 55:45, 4.2 min, 70 mL/min) to obtain Example 43 (37.7 mg, white solid, Ret.time=2.080 min, ee 100%), LC-MS (ESI) m/z: 581.9 [M+H]+; 1H NMR (400 MHz, CD3OD) δ 8.15 (d, J=2.0 Hz, 1H), 7.97-7.93 (m, 4H), 7.86 (dd, J=8.4, 1.9 Hz, 1H), 7.72 (d, J=8.4 Hz, 2H), 7.55 (d, J=8.2 Hz, 2H), 7.33 (d, J=8.4 Hz, 1H), 4.15-4.07 (m, 2H), 3.69-3.65 (m, 1H), 3.39 (s, 3H), 3.11 (s, 3H), 2.77-2.70 (m, 1H), 1.30-1.24 (m, 2H), 1.13-1.06 (m, 2H); and Example 44 (32.3 mg, white solid, Ret.time=2.972 min, ee 98%), LC-MS (ESI) m/z: 581.9 [M+H]+; 1H NMR (400 MHz, CD3OD) δ 8.15 (d, J=1.9 Hz, 1H), 7.98-7.92 (m, 4H), 7.86 (dd, J=8.4, 1.9 Hz, 1H), 7.72 (d, J=8.4 Hz, 2H), 7.55 (d, J=8.1 Hz, 2H), 7.34 (d, J=8.4 Hz, 1H), 4.16-4.06 (m, 2H), 3.69-3.65 (m, 1H), 3.39 (s, 3H), 3.11 (s, 3H), 2.77-2.69 (m, 1H), 1.30-1.25 (m, 2H), 1.13-1.07 (m, 2H).
A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used for the compounds of the present disclosure, to determine the agonistic activity of the compounds on the RORγ protein receptor. The agonistic activity is expressed by the concentration for 50% of maximal effect (EC50). The experiment was performed basically with reference to the methods described in ONCOIMMUNOLOGY, 2016, VOL. 5, NO. 12, e1254854.
Experimental Methods:
1. Preparation of RORγ Basal Buffer
A 100 mL basal buffer was prepared, mixed homogeneously and stored for use;
2. Preparation of Solutions of Test Compounds
The compounds of the examples shown in the table below were prepared into solutions with DMSO as the solvent, serially diluted at 3-fold dilution to 10 concentrations starting from 10 mM, and added to the corresponding wells of a 384-well plate with each concentration of each compound in duplicate;
The solution of reference substance LYC-55716 was similarly prepared, starting from 10 mM, serially diluted at 3-fold dilution to 10 concentrations, and added to the corresponding control wells of 384-well plate;
3. Preparation of Protein Solution Mixture
a. A 75 nM solution of GST-RORγ LBD ((263-509) (produced at ChemPartner)) and a 4 nM solution of Biotin-SRC1 (Glsbiochem) were prepared with the above basal buffer. The two solutions were 1:1 mixed evenly, and 10 μL of the mixed solution was added into each well of the above 384-well plate. The plate was immediately centrifugated at 1000 rpm for 10 seconds;
b. A 1 nM solution of Anti-GST Eu (Cisbio, #61GSTKLA) and a 50 nM solution of SA-APC (Cisbio, #61GSTKLA) were prepared with the above basal buffer. The two solutions were 1:1 mixed evenly, and 10 μL of the mixed solution was added into each well of the above 384-well plate. The plate was immediately centrifugated at 1000 rpm for 10 seconds;
c. The resulting 384-well plate was incubated at room temperature for 15 minutes. Data were read on an Envision microplate reader, EC50 values and E max % were calculated.
4. Analysis of Experimental Results
a. Readings were taken at 665 nm and 615 nm wavelengths respectively on the Envision Microplate Reader, and the ratios between them were normalized.
b. Calculation was carried out with the following equation:
wherein X is the reading of compounds, Min is the reading of the blank solution, and Max is the reading of 10 μM SRC.
c. The concentration-response curve data were fitted by a four-parameter logistic nonlinear regression model, and Emax was the TOP value of the fitted curve.
The test results in the above table show that the compounds of the present disclosure have good agonistic activity on the RORγ protein receptor.
This assay was basically carried out according to the method described in the literature ONCOIMMUNOLOGY, 2016, VOL. 5, NO. 12, e1254854.
The RORγ-LBD coding sequence was inserted into pBIND plasmid (Promega, E1581). This expression vector and a reporter vector (pGL4.35 carrying a stably integrated luciferase reporter gene driven by GAL4 promoter) were co-expressed in HEK293T host cells. When the agonist binds to the corresponding chimeric receptor, the chimeric receptor binds to the GAL4 binding site on the reporter gene carrier and stimulates reporter gene expression. The agonistic activity of the compound on RORγ was determined according to the intensity of the chemiluminescence signal.
Experimental Procedure:
1. Preparation of test compounds
1.1 All test compounds were serially diluted at 3-fold dilution with DMSO, with 10 serial dilutions starting from 10 mM; LIT-00093 solution was prepared similarly.
1.2 The positive control LYC-55716 was serially diluted at 3-fold dilution with DMSO, with 10 serial dilutions starting from 5 mM.
1.3 1000× positive control (5 mM LYC-55716) and 1000× negative control (100% DMSO) were prepared.
1.4 The compound plate was sealed and shaken for 5 min.
2. Test procedure
Cells were cultured according to ATCC standard operation, and 6*106 HEK293T cells were seeded into 100 mm cell culture dishes, cultured overnight in a 37° C., 5% CO2 incubator, and then transfected. Compounds of corresponding concentrations were added for treatment, and the Steady-Glo™ kit (Lot E 2520) was used for further detection.
Chemiluminescence values were detected with Envision 2104; EC50 values were calculated.
% Activity was calculated with the following equation:
RLU: Fluorescence value
The log values of the compound concentration and % Activity were fitted using Graphad 8.0, to calculate EC50s of the compounds. The results are shown in Table 2.
The test results in the above table show that the compounds of the present disclosure have good agonistic activity on the RORγt luciferase reporter gene.
Experiment Materials:
Test procedure: the mouse spleen lymphocytes were first isolated, and the lymphocytes were subject to the conditions of stimulating factors (anti-mCD28: 5 μg/mL; rmTGF-β1: 1 ng/mL; rmIL-6: 50 ng/mL; rmIL-23: 5 ng/mL) and differentiated to Th17, while adding different concentrations of compounds. All test compounds were serially diluted at 3-fold dilution with DMSO, with 8 serial dilutions starting from the maximum concentration 3 μM. The supernatants were collected after 48 hours for IL-17 ELISA detection, and compared with the solvent group, to determine the activation rate of the compounds on Th17 cells to secrete IL-17. EC50 values were fitted with Graphad 8.0, and the TOP values fitted by the software was Emax %.
The test results show that the compounds of the present disclosure have good ability to increase the differentiation of Th17 cells to secrete IL-17 on mouse lymphocytes (as shown in Table 3)
Experiment Materials:
Test procedure: PBMC cells were first thawed and plated, and then stimulated with stimulating factors (anti-hCD28: 5 μg/mL; rhTGF-β1: 5 ng/mL; rhIL-6: 20 ng/mL; rhIL-23: 10 ng/Ml) to differentiate to Th17, while adding different concentrations of compounds. All test compounds were serially diluted at 3-fold dilution with DMSO, with 8 serial dilutions starting from the maximum concentration 3 μM. The supermatants were collected after 48 hours for IL-17 ELISA detection, and compared with the solvent group, to determine the activation rate of the compounds on Th17 cells to secrete IL-17. EC50 values were fitted with Graphad8.0, and the TOP values fitted by the software was Emax %.
The test results show that the compounds of the present disclosure have good ability to increase the differentiation of Th17 cells to secrete IL-17 on on human PBMCs (as shown in Table 4).
According to standard methods of in vitro metabolic stability studies in the art, eg., the methods described in Kerns, Edward H. and Di Li (2008). Drug-like Properties: Concepts, Structure Design and Methods: from ADME to Toxicity Optimization. San Diego: Academic Press; Di, Li et al., Optimization of a Higher Throughput Microsomal Stability Screening Assay for Profiling Drug Discovery Candidates, J. Biomol. Screen. 2003, The method described in 8(4), 453, the liver microsomal metabolic stability test of the compounds of the present disclosure was carried out analogously as follows.
The incubation system contained 0.5 mg protein/mL microsomes, cofactors, and PBS, was pre-incubated at 37° C. for 3 min, and then added the substrate (i.e., the test compounds) to initiate the reaction. Samples were taken at 0, 1, 5, 10, 15, 20, 30 and 60 min from the start of the reaction, and an appropriate terminator was added to terminate the reaction.
The terminator was an ice acetonitrile solution containing 100 ng/mL tolbutamide and 100 ng/mL labetalol.
Sample treatment (n=3): each appropriate sample was added, vortexed and centrifuged at high speed. The supernatants were collected, and the substrate was detected by HPLC-MS/MS. The peak area at the 0 min was taken as 100%. The peak areas at other time points were converted to percentage of the remaining amount. The natural logarithms of the percentage of remaining amount at each time point was plotted against the incubation time, and the slope (−k) was calculated by linear regression. Then, Clint (uL/min/mg) and half-life (T½, min) of the compounds were calculated following the equation of the inherent clearance rate (Clint)=(k* volume of incubation solution)/mass of liver microsomes. The results are shown in Table 5.
The PK of each compound was determined as follows: 6 CD-1 mice (obtained from Shanghai Lingchang Biotechnology Co., Ltd.) were divided into two groups, 3 mice in each group. One group was administered intravenously at a dose of 1 mg/kg, and the vehicle was 5% DMSO/40% PEG400/55% (20% 3-CD); one group was administered by oral gavage at a dose of 5 mg/kg, and the vehicle was DMSO/1% methylcellulose (1:99). Blood samples were collected from saphenous vein of lower leg in each group at 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after administration. Approximately 40 μL of the blood samples were collected into anticoagulant tubes containing EDTA-K2. Immediately after collection, the tubes were inverted at least 5 times to ensure uniform mixing, and then placed on ice. The blood samples collected at each time point were centrifuged at 4° C., 8000 rpm for 5 minutes to obtain plasma. Another 1.5 mL centrifuge tube was marked with the compound name, animal number, and time point, and the plasma was transferred to this tube. The plasma was stored at −80° C. until analysis.
Compound concentrations in plasma were determined by UPLC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNolin 6.4 pharmacokinetic software on the obtained data.
The specific test results are as follows, showing that the compounds have good pharmacokinetic absorption and have the PK advantages.
1 mg/kg
5 mg/kg
The structures of the control compounds used in the above experiments are as follows:
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910938067.2 | Sep 2019 | CN | national |
| 202010966741.0 | Sep 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/118905 | 9/29/2020 | WO |
