CN116457348A - Novel EZH2 inhibitors and uses thereof - Google Patents

Novel EZH2 inhibitors and uses thereof Download PDF

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Publication number
CN116457348A
CN116457348A CN202280007243.0A CN202280007243A CN116457348A CN 116457348 A CN116457348 A CN 116457348A CN 202280007243 A CN202280007243 A CN 202280007243A CN 116457348 A CN116457348 A CN 116457348A
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Prior art keywords
alkyl
cancer
compound
halogen
hydrogen
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Inventor
雷永华
蒋茂
王心悟
李进
陈弘道
郑杨
刘浏
杨民民
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Nanjing Maisheng Technology Co.,Ltd.
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Pharmablock Sciences Nanjing Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings

Abstract

Disclosed are a compound shown as a formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, and application thereof in preparing medicines for treating EZH2 mediated diseases.

Description

Novel EZH2 inhibitors and uses thereof Technical Field
The application belongs to the field of chemical medicine, and in particular relates to a novel EZH2 inhibitor and application thereof.
Background
Abnormalities in the activity of the core components (EZH 2, EED and SUZ 12) of the multi-comb inhibitory complex 2 (PRC 2) are closely related to the occurrence, development and invasion of a variety of diseases, especially malignant tumors. The core component EZH2 of PRC2 is obviously increased in various malignant tumor tissues such as breast cancer, prostatic cancer, colorectal cancer, soft tissue sarcoma, follicular lymphoma and the like, the over-expression level of the core component EZH2 is positively correlated with the degree of tumor deterioration and bad prognosis, and the expression of EZH2 is down-regulated by a genetic method, so that the proliferation and invasion of tumor cells can be obviously inhibited. Meanwhile, recurrent somatic mutations of EZH2 have been identified in tumor cells such as diffuse large B-cell lymphoma (DLBCL) and Follicular Lymphoma (FL).
Tazemetostat is the only EZH2 inhibitor currently approved by the FDA for the treatment of soft tissue sarcomas and follicular lymphomas (800 mg/kg BID). Meanwhile, other redevelopment EZH2 inhibitors are still in clinical stages, such as CPI-1205 (clinical I/II stage), CPI-0209 (clinical I stage), DS-3201 (clinical II stage), SHR-2554 (clinical II stage), etc. The current research on Tazemetostat shows that the Tazemetostat has the problems of insufficient stability, high administration dosage, easy generation of drug resistance and the like, so that development of EZH2 inhibitors with novel compound structures and good activity and stability is still needed to be carried out.
Disclosure of Invention
The application discloses a compound which can be used as an EZH2 inhibitor and application thereof in preparing medicines for treating EZH2 mediated diseases.
In one aspect, the present application provides a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
wherein R is 1 、R 2 、R 3 Each independently selected from hydrogen, halogen, C 1 -C 6 Alkyl, hydroxy, cyano or amino;
l, U, V, T are each independently selected from C or N, and at least one is N;
y is selected from hydrogen, halogen or C 1 -C 6 An alkyl group;
x is selected from hydrogen, halogen, C 1 -C 6 Alkyl, alkoxy or-S-R 4 ，R 4 Is C 1 -C 6 An alkyl group;
z is C 3 -C 7 Cycloalkyl or 4-to 7-membered heterocyclyl, each of which is optionally substituted with 1 to 3 substituents selected from halogen, C 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, halo C 1 -C 6 Substitution of an alkoxy group or a group of-NRaRb;
ra is hydrogen, C 1 -C 6 Alkyl or halo C 1 -C 6 An alkyl group;
rb is C 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl or 4 to 7 membered heterocyclyl, wherein said heterocyclyl is optionally substituted with 1 to 3 substituents selected from halogen, C 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy or halo C 1 -C 6 Substitution of the alkoxy group;
or Ra and Rb, together with the nitrogen atom to which they are attached, form a group optionally selected from halogen, C, 1 to 3 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl or-ORc groups substituted with 4 to 7 membered heterocyclyl;
Rc is C 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl or C 3 -C 7 Cycloalkyl;
m, n' are each independently 0, 1 or 2;
n is 0, 1, 2, 3 or 4; and
represents a single bond or a double bond.
In certain embodiments, formula (I) is formula (Ia):
wherein R is 1 、R 2 、R 3 Each independently selected from hydrogen, halogen, C 1 -C 6 Alkyl, hydroxy, cyano or amino;
l, U, V, T are each independently selected from C or N, and at least one is N;
y is selected from hydrogen, halogen or C 1 -C 6 An alkyl group;
x is selected from hydrogen, halogen, C 1 -C 6 Alkyl, alkoxy or-S-R 4 ，R 4 Is C 1 -C 6 An alkyl group;
m, n' are each independently 0, 1 or 2;
n is 0, 1, 2, 3 or 4; and
represents a single bond or a double bond.
In certain embodiments, formula (I) is formula (Ib):
wherein R is 1 、R 2 、R 3 Each independently selected from hydrogen, halogen, C 1 -C 6 Alkyl, hydroxy, cyano or amino;
u, V are each independently selected from C or N;
x is selected from hydrogen, halogen, C 1 -C 6 Alkyl, alkoxy or-S-R 4 ，R 4 Is C 1 -C 6 An alkyl group;
m, n' are each independently 0, 1 or 2;
n is 0, 1, 2, 3 or 4; and
represents a single bond or a double bond.
In certain embodiments, formula (I) is formula (Ic):
wherein R is 1 、R 2 、R 3 Each independently selected from hydrogen, halogen, C 1 -C 6 Alkyl, hydroxy, cyano or amino; u, V are each independently selected from C or N;
X is selected from hydrogen, halogen, C 1 -C 6 Alkyl, alkoxy or-S-R 4 ，R 4 Is C 1 -C 6 An alkyl group;
m, n' are each independently 0, 1 or 2;
n is 0, 1, 2, 3 or 4; and
represents a single bond or a double bond.
In certain embodiments, the compound of formula (I) is selected from:
in another aspect, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the foregoing compound, or a pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate, or isomer thereof; and a pharmaceutically acceptable carrier or excipient.
In another aspect, the present application provides the use of a compound of the foregoing, or a pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate or isomer thereof, for the manufacture of a medicament for the treatment of an EZH 2-mediated disease.
In certain embodiments, the disease is cancer.
In certain embodiments, the cancer is lung cancer, gastric cancer, liver cancer, breast cancer, nasopharyngeal cancer, pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, glioma, melanoma, prostate cancer, renal cancer, esophageal cancer, mesothelioma, head and neck cancer, bladder cancer, salivary gland cancer, leukemia, or lymphoma.
In another aspect, the present application provides a compound represented by formula (II):
Wherein R is 1 、R 2 、R 3 、U、V、m、n、n’、 As defined above, which can be used to prepare the aforementioned compounds.
Terminology:
unless stated to the contrary, the terms used in the specification and claims have the following meanings.
The term "isomer" includes enantiomeric, diastereomeric, and geometric (or conformational) isomeric forms of a given structure. For example, the present application includes R and S configuration, Z and E double bond isomers, Z and E conformational isomers, single stereochemical isomers and mixtures of enantiomers, diastereomers and geometric (or conformational) isomers for each asymmetric center.
The term "pharmaceutically acceptable salt" refers to, for example, an acid addition salt and/or a base salt thereof. Suitable acid addition salts are formed from acids which form non-toxic salts, such as hydrochloride/chloride salts. Suitable base salts are formed from bases which form non-toxic salts such as calcium and sodium salts. Semi-salts of acids and bases, such as hemisulfate and hemicalcium salts, may also be formed.
The term "therapeutically effective amount" refers to an amount of a compound of the present application that (i) treats a particular disease, condition, or disorder; (ii) Alleviating, or eliminating one or more symptoms of a particular disease, condition, or disorder; or (iii) preventing or delaying the onset of one or more symptoms of a particular disease, condition, or disorder described herein.
The term "pharmaceutically acceptable carrier or excipient" refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.
The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 12 carbon atoms, more preferably an alkyl group containing from 1 to 6 carbon atoms. Non-limiting examples of lower alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl and the like.
The term "alkenyl" refers to aliphatic hydrocarbons having at least one carbon-carbon double bond, including straight and branched chains having at least one carbon-carbon double bond. In some embodiments, the alkenyl group has 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, or 2 to 4 carbon atoms. For example, the term "C 2-6 Alkenyl "includes straight or branched chain unsaturated groups of 2 to 6 carbon atoms (having at least one carbon-carbon double bond) including, but not limited to, vinyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like.
The term "alkynyl" refers to aliphatic hydrocarbons having at least one carbon-carbon triple bond, including straight and branched chains having at least one carbon-carbon triple bond. In some embodiments, alkynyl groups have 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 3 to 6 carbonsAn atom. For example, "C 2-6 Alkynyl "includes straight or branched chain unsaturated groups of 2 to 6 carbon atoms (having at least one carbon-carbon triple bond).
The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy.
The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 6 carbon atoms (e.g., 3, 4, 5, or 6 carbon atoms), and most preferably from 5 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.
The term "spirocycloalkyl" refers to a polycyclic group sharing one carbon atom (referred to as a spiro atom) between 5-to 20-membered monocyclic rings, which may contain one or more double bonds, but each ring does not have a fully conjugated pi-electron system. Preferably 6 to 14 membered, more preferably 7 to 10 membered (e.g. 7, 8, 9 or 10 membered). The spirocycloalkyl group is classified into a single spirocycloalkyl group, a double spirocycloalkyl group or a multiple spirocycloalkyl group according to the number of common spiro atoms between rings, preferably a single spirocycloalkyl group and a double spirocycloalkyl group, more preferably a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered single spirocycloalkyl group.
The term "fused ring alkyl" refers to a 5 to 20 membered, all carbon polycyclic group wherein each ring in the system shares an adjacent pair of carbon atoms with the other rings in the system, wherein one or more of the rings may contain one or more double bonds, but each ring does not have a fully conjugated pi electron system. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The number of constituent rings may be classified as a bicyclic, tricyclic, tetracyclic or polycyclic fused ring alkyl group, preferably a bicyclic or tricyclic, more preferably a 5-membered/5-membered or 5-membered/6-membered bicycloalkyl group.
The term "bridged cycloalkyl" refers to an all-carbon polycyclic group of 5 to 20 members, any two rings sharing two carbon atoms not directly attached, which may contain one or more double bonds, but each ring does not have a fully conjugated pi-electron system. Preferably 6 to 14 membered, more preferably 7 to 10 membered. Cycloalkyl groups which may be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged according to the number of constituent rings are preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic.
The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S (O) m (where m is an integer from 0 to 2), but excluding the ring portion of-O-, -O-S-or-S-, the remaining ring atoms being carbon. Preferably containing 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; most preferably from 5 to 6 ring atoms, of which 1 to 2 or 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like, with tetrahydropyranyl, piperidinyl, pyrrolidinyl being preferred. Polycyclic heterocyclic groups include spiro heterocyclic groups, fused heterocyclic groups, and bridged heterocyclic groups.
The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group having one atom (referred to as a spiro atom) shared between 5-to 20-membered monocyclic rings, wherein one or more of the ring atoms is a heteroatom selected from nitrogen, oxygen or S (O) m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. Which may contain one or more double bonds, but each ring does not have a fully conjugated pi-electron system. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The spiroheterocyclyl group is classified into a single spiroheterocyclyl group, a double spiroheterocyclyl group or a multiple spiroheterocyclyl group according to the number of common spiro atoms between rings, preferably a single spiroheterocyclyl group and a double spiroheterocyclyl group, more preferably a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered single spiroheterocyclyl group.
The term "fused heterocyclyl" refers to a 5 to 20 membered, polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system, one or more of which may contain one or more double bonds, but each ring does not have a fully conjugated pi electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S (O) m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The number of constituent rings may be classified as a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, preferably a bicyclic or tricyclic, more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group.
The term "bridged heterocyclyl" refers to a 5 to 14 membered, polycyclic heterocyclic group in which any two rings share two atoms which are not directly attached, which may contain one or more double bonds, but each ring does not have a fully conjugated pi electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S (O) m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably 6 to 14 membered, more preferably 7 to 10 membered. Heterocyclic groups which may be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged according to the number of constituent rings are preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic.
Such heterocyclyl groups include those described above (including monocyclic, spiro, fused and bridged) fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl group, non-limiting examples of which include:
the term "aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6 to 10 membered, such as phenyl and naphthyl.
The term "heteroaryl" refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl groups are preferably 5 to 10 membered, containing 1 to 3 heteroatoms; more preferably 5 or 6 membered, containing 1 to 2 heteroatoms; preferred are, for example, imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, pyridazinyl and the like.
The heteroaryl groups include those wherein the heteroaryl groups as described above are fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:
the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.
The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.
The term "haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein the alkoxy group is as defined above.
The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.
The term "deuteroalkoxy" refers to an alkoxy group substituted with one or more deuterium atoms, wherein alkoxy is as defined above.
The term "cycloalkylalkyl" refers to an alkyl group substituted with one or more cycloalkyl groups, wherein cycloalkyl and alkyl are as defined above.
The term "cycloalkyloxy" refers to an-O-cycloalkyl group, wherein cycloalkyl is as defined above.
The term "heterocyclylalkyl" refers to an alkyl group substituted with one or more heterocyclyl groups, where heterocyclyl and alkyl are as defined above.
The term "arylalkyl" refers to an alkyl group substituted with one or more aryl groups, wherein aryl and alkyl are as defined above.
The term "hydroxy" refers to an-OH group.
The term "halogen" refers to fluorine, chlorine, bromine or iodine.
The term'Amino "means-NH 2 。
The term "cyano" refers to-CN.
The term "nitro" refers to-NO 2 。
The term "carboxy" refers to-C (O) OH.
Detailed Description
Example 1: synthesis of 10- ((1 r,4 r) -4- (dimethylamino) cyclohexyl) -8, 10-dimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxazole [4,5-e ] indol-7 (4H) -one
To the reaction flask was added methyl 3, 4-dihydroxy-2-methylbenzoate (20.00 g,0.11mol,1.0 eq.) in an ice-water bath, dichloromethane (DCM, 100 mL) and acetic acid (10 mL), followed by dropwise addition of bromine (17.57 g,0.11mol,1.0 eq.) and natural warming to room temperature for reaction over night for 12h. The reaction solution was dried by spinning, and was slurried with water (100 mL) and filtered. The filter cake was slurried with methylene chloride (20 mL) and filtered again. The filter cake was pumped down with an oil pump to afford intermediate 1.1 (24.60 g,85.9% yield). MS [ M+H ]] + ＝261。
Intermediate 1.1 (24.60 g,0.094mol,1.0 eq.) was dissolved in toluene (250 mL). Tert-butyl trans-N- (4-ethynylcyclohexyl) carbamate (25.00 g,0.11mol,1.2 eq.) and Ru were added with stirring 3 (CO) 12 (1.81 g,0.09 mol,0.03 eq.) after 3 nitrogen substitutions, heating to 105℃and overnight reaction for 10h, the tert-butyl trans-N- (4-ethynylcyclohexyl) carbamate (25.00 g,0.11mol,1.2 eq.) was supplemented with Ru 3 (CO) 12 (1.81 g,0.003mol,0.03 eq.) nitrogen was purged 3 times and then heated to 105℃for a further 10h. After the reaction solution was cooled to room temperature, it was filtered through celite. The filtrate was concentrated and purified by column chromatography (PE: EA) =10:1 to give intermediate 1.2 (36.70 g,80.4% yield). MS [ M+H ]] + ＝485。
To the reaction flask was added intermediate 1.2 (36.70 g,0.076mol,1.0 eq.) and acetic anhydride (120 mL) was added to dissolve, and 1 drop of concentrated sulfuric acid was added dropwise. Dilute nitric acid (110 mL) was added dropwise under ice salt bath conditions. The internal temperature is controlled to be not higher than 45 ℃, and the reaction is carried out for 1h at room temperature after the dripping is finished. The reaction solution was poured into ice 30% aqueous sodium hydroxide (500 mL), extracted twice with ethyl acetate, and the organic phase was washed with saturated aqueous sodium chloride solution and with MgSO 4 Drying, spin drying gave intermediate 1.3 (32.60 g,100% yield) which was directly put into the next reaction.
Intermediate 1.3 (32.60 g,0.076mol,1.0 eq.) was dissolved in DCM, and triethylamine (23.00 g,0.23mol,3.0 eq.) and DMAP (0.93 g,0.0076mol,0.1 eq.) were added. Di-tert-butyl dicarbonate (24.80 g,0.11mol,1.5 eq.) was added under ice-bath conditions. The reaction was transferred to room temperature and reacted for 2h. The reaction was concentrated and purified by column chromatography (PE: ea=4:1) to give intermediate 1.4 (22.89 g,56.9% yield). MS [ M+H ]] + ＝530。
To a reaction flask containing intermediate 1.4 (2.20 g,0.0043mol,1.0 eq.) was added diisopropylamine (200 mL) and tetrahydrofuran (20 mL). After dissolution with stirring, cuI (7.74 mg,0.043mmol,0.01 eq.) TMSA (1.57 g,0.0086mol,2 eq.) and Pd (PPh) were added 3 ) 2 Cl 2 (0.090 g,0.13mmol,0.03 eq.). After three times of nitrogen substitution, the temperature is raised to 70 ℃ and the reaction is carried out for 6 hours. The reaction solution was filtered through celite. After spin-drying the reaction, it was purified by column chromatography (PE: ea=4:1) to give intermediate 1.5 (1.89 g,80.3% yield) as a dark brown oil. MS [ M+H ]] + ＝547。
To a reaction flask containing intermediate 1.5 (1.89 g,0.035mol,1.0 eq.) was added a tetrahydrofuran solution of TBAF (1M, 10 mL) and stirred at room temperature for 1h. After the reaction solution was dried by spin drying, water was added, extraction was performed twice with ethyl acetate, and the mixture was washed with a saturated aqueous sodium chloride solution and dried over anhydrous magnesium sulfate. Purification by column chromatography (PE: ea=4:1) afforded intermediate 1.6 (1.49 g,90.9% yield). MS [ M+H ] ] + ＝475。
To a reaction flask containing intermediate 1.6 (1.49 g, 0.09 mol,1.0 eq.) was added DCM (15 mL) and DMF (15 mL). Stirring and dissolving, and adding dihydrateStannous chloride (3.56 g,0.150mol,5.0 eq.). Stirring for 2h at room temperature. To the reaction solution was added saturated aqueous sodium hydrogencarbonate solution (2L). Ethyl acetate extraction was twice, washed with saturated aqueous sodium chloride, and MgSO 4 And (5) drying. After spin-drying, the intermediate 1.7 (0.89 g,63.9% yield) was isolated and purified by column chromatography (PE: ea=4:1). MS [ M+H ]] + ＝445。
To a reaction flask containing intermediate 1.7 (0.89 g,0.002mol,1.0 eq.) was added pyridine (10 mL), cp (PPh) 3 ) 2 RuCl (0.15 g,0.003mol,0.1 eq.). After three times of nitrogen substitution, the temperature is raised to 90 ℃ for reaction for 2 hours. The reaction solution was filtered through celite. Purification by column chromatography (PE: ea=4:1) afforded intermediate 1.8 (0.81 g,90.6% yield). MS [ M+H ]] + ＝445。 1 H NMR(400MHz,DMSO-d 6 )δ10.91(s,1H),7.30-7.24(m,1H),6.75(d,J＝7.9Hz,1H),6.37-6.34(m,1H),3.87(s,3H),3.25-3.10(m,1H),2.48(s,3H),1.93-1.74(m,5H),1.59(s,3H),1.36(s,9H),1.30-1.06(m,4H).
To a reaction flask containing intermediate 1.8 (0.81 g,0.0018mol,1.0 eq.) was added DMF (50 mL), stirred, cooled to 0deg.C, naH (0.087 g,0.0022mol,1.2 eq.) was slowly added, and after 0.5h the reaction was followed by the addition of tert-butyl 1,2, 3-oxazolidine-3-carboxylate 2, 2-dioxide (0.81 g,0.0036mol,2.0 eq.). Naturally heating to room temperature, and reacting for 6h. Adding water to the reaction solution, extracting twice with ethyl acetate, washing the organic phase with saturated aqueous sodium chloride solution, and MgSO 4 And (5) drying. Spin-drying afforded intermediate 1.9 (0.62 g,58.1% yield). MS [ M+H ]] + ＝588.
To a reaction flask containing intermediate 1.9 (0.62 g,0.001mol,1.0 eq.) was added methanol (12 mL), followed by 1, 4-dioxane hydrochloride solution (4 m,3 mL), and the temperature was raised to 70 ℃ and the reaction was carried out for 0.5h. The intermediate 1.10 after the reaction liquid is dried by spin is directly put into the next reaction.
Methanol (10 mL) was added to a reaction flask containing intermediate 1.10, cesium carbonate (0.98 g,0.003mol,3.0 eq.) was added after stirring and dissolution, and the temperature was raised to 70℃for 2h. The intermediate 1.11 after the reaction liquid is dried by spin is directly put into the next reaction.
Directional dressDCM (10 mL) was added to a reaction flask containing intermediate 1.11, and triethylamine (0.30 g,0.003mol,3.0 eq.) was added after stirring and dissolution, and di-tert-butyl dicarbonate (0.33 g,0.0015mol,1.5 eq.) was added under an ice-water bath. Transfer to room temperature and react for 1h. After spin-drying the reaction solution, it was purified by column chromatography (DCM: meoh=20:1) to give intermediate 1.12 (0.29 g,61.5% yield). MS [ M+H ]] + ＝456。
To a reaction flask containing intermediate 1.12 (0.29 g,0.60mmol,1.0 eq.) was added DMF (5 mL), stirred, ice-water bath added t-BuOK (0.14 g,12.0mol,2.0 eq.) and after stirring for 20min 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine (0.2 g,0.72mmol,1.2 eq.). After 3h of reaction, water (50 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (50 mL. Times.3). Saturated aqueous sodium chloride solution washing, mgSO 4 And (5) drying. After spin-drying, the intermediate 1.13 (0.39 g,90.4% yield) was isolated and purified by column chromatography (PE: ea=4:1). MS [ M+H ]] + ＝713。
Intermediate 1.13 (0.39 g,5.4mmol,1.0 eq.) was dissolved in 5mL of methanol, 1, 4-dioxane hydrochloride (1 mL, 7M) was added, and the temperature was raised to 70℃and the reaction was carried out for 0.5h. The reaction mixture was dried to give intermediate 1.14 (0.28 g,100% yield).
Intermediate 1.14 (0.28 g,5.4mmol,1.0 eq.) was dissolved in 5mL methanol, aqueous formaldehyde (0.5 mL,37%,10 eq.) was added, and after stirring for 15min, STAB (0.57 g,2.7mmol,5 eq.) was added and stirring was continued for a further 15min. The reaction was then dried by spin-drying and purified by column chromatography (DCM: meoh=8:1) to give the title compound of example 1 (0.28 g,94.3% yield). MS [ M+H ]] + ＝551。 1 H NMR(400MHz,DMSO-d 6 )11.70(s,1H),7.21(s,1H),6.30(s,1H),6.14(s,1H),4.73(s,2H),4.07(s,2H),3.51(s,2H),2.65(s,3H),2.41(d,J＝7.7Hz,6H),2.32-2.36(m,1H),2.30(s,3H),2.21(s,3H),1.89(s,5H),1.59(s,3H),1.19-1.28(m,4H)。
Example 2: 8-chloro-10- ((1 r,4 r) -4- (dimethylamino) cyclohexyl) -10-methyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
To the reaction flask was added methyl 3, 4-dihydroxy-2-chlorobenzoate (20 g,0.10mol,1.0 eq.) in an ice-water bath, dichloromethane (100 mL) and acetic acid (10 mL), followed by dropwise addition of bromine (17.50 g,0.1mol,1.1 eq.) and natural warming to room temperature for reaction over night for 12h. The reaction solution was dried by spinning, and was slurried with water (100 mL) and filtered. The filter cake was slurried with methylene chloride (20 mL) and filtered again. The filter cake was pumped down with an oil pump to afford intermediate 2.1 (24.00 g,85.7% yield). MS [ M+H ] ] + ＝280。 1 H NMR(400MHz,DMSO),7.35(d,J＝3.3Hz,1H),3.70(s,3H)。
Intermediate 2.1 (24.00 g,0.085mol,1.0 eq.) was dissolved in toluene (200 mL). Tert-butyl trans-N- (4-ethynylcyclohexyl) carbamate (20.00 g,0.88mol,0.88.00 eq.) and Ru were added with stirring 3 (CO) 12 (1.60 g,0.00255mol,0.03 eq.) after 3 nitrogen substitutions, heating to 105 ℃, overnight reaction for 10h, make up tert-butyl trans-N- (4-ethynylcyclohexyl) carbamate (20.00 g,0.88mol,0.88 eq.) after 3 nitrogen purges, heating to 105 ℃, and further reaction for 10h. After the reaction solution was cooled to room temperature, it was filtered through celite. After concentration, purification by column chromatography (PE: EA) =10:1 gave intermediate 2.2 (24.00 g,56.6% yield). MS [ M+H ]] + ＝504。
To the reaction flask was added intermediate 2.2 (24.00 g,0.047mol,1.0 eq.) and acetic anhydride (120 mL) was added to dissolve and 1 drop of concentrated sulfuric acid was added dropwise. Dilute nitric acid (60 mL) was added dropwise under ice-salt bath. The internal temperature is controlled to be not higher than 45 ℃, and the reaction is carried out for 1h at room temperature after the dripping is finished. The reaction solution was poured into ice 30% aqueous sodium hydroxide (300 mL), extracted twice with ethyl acetate, and the organic phase was washed with saturated aqueous sodium chloride solution and MgSO 4 Drying, spin drying gave intermediate 2.3 (21.30 g,100% yield) which was directly put into the next reaction.
DCM dissolved intermediate 2.3 (21.30 g,0.047mol,1.0 eq.) and triethylamine (15.00 g,0.15mol,3.0 eq.) was added, DMAP (0.57 g,0.0047mol,0.1 eq.). Di-tert-butyl dicarbonate (17.50 g,0.64mol,1.5 eq.) was added under ice-bath conditions. Transfer to room temperature and react for 2h. The reaction was concentrated and purified by column chromatography (PE: ea=4:1) to give intermediate 2.4 (20.80 g,75.0% yield). MS [ M+H ]] + ＝549。 1 H NMR(400MHz,DMSO),7.51(s,1H),6.75(d,J＝3.3Hz,1H),3.80(s,3H),3.21-3.05(m,1H),1.95-1.65(m,5H),1.70(s,3H),1.45(s,9H),1.30-1.15(m,4H)。
To a reaction flask containing intermediate 2.4 (2.00 g,3.80mmol,1.0 eq.) was added diisopropylamine (20 mL) and tetrahydrofuran (5 mL). After dissolution with stirring, cuI (7.10 mg,0.038mmol,0.01 eq.) TMSA (1.38 g,7.6mol,2 eq.) Pd (PPh) was added 3 ) 2 Cl 2 (83.60 mg,0.11mmol,0.03 eq.). After three times of nitrogen substitution, the temperature is raised to 70 ℃ and the reaction is carried out for 6 hours. The reaction solution was filtered through celite. After spin-drying the reaction mixture was isolated and purified by column chromatography (PE: ea=4:1) to afford intermediate 2.5 (1.89 g,80.3% yield) as a dark brown oil. MS [ M+H ]] + ＝567。
To a reaction flask containing intermediate 2.5 (1.89 g,3.30mmol,1.0 eq.) was added a tetrahydrofuran solution of TBAF (1M, 10 mL) and stirred at room temperature for 1h. After the reaction solution was dried by spin drying, water was added, extraction was performed twice with ethyl acetate, and the mixture was washed with a saturated aqueous sodium chloride solution and dried over anhydrous magnesium sulfate. Purification by column chromatography (PE: ea=4:1) afforded intermediate 2.6 (1.49 g,90.3% yield) as a dark brown oil. MS [ M+H ] ] + ＝495。
To a reaction flask containing intermediate 2.6 (1.49 g,0.0030mmol,1.0 eq.) was added DCM (15 mL) and DMF (15 mL). Stannous chloride dihydrate (3.38 g,0.0150mmol,5.0 eq.) was added after stirring to dissolve and stirred at room temperature for 2h. To the reaction solution was added saturated aqueous sodium hydrogencarbonate solution (2L). Ethyl acetate extraction was twice, washed with saturated aqueous sodium chloride, and MgSO 4 And (5) drying. After spin-drying, the intermediate 2.7 (1.10 g,78.6% yield) was isolated and purified by column chromatography (PE: ea=4:1). MS [ M+H ]] + ＝465。
To a reaction flask containing intermediate 2.7 (1.10 g,0.0023mol,1.0 eq.) was added pyridine (50 mL), cp (PPh) 3 ) 2 RuCl (0.15 g,0.00023mol,0.1 eq.). After three times of nitrogen substitution, the temperature is raised to 90 ℃ for reaction for 2 hours. The reaction solution was filtered through celite. Purification by column chromatography (PE: ea=4:1) afforded intermediate 1.8 (1.00 g,90.9% yield). MS [ M+H ]] + ＝465。
To a reaction flask containing intermediate 2.8 (1.00 g,0.0022mol,1.0 eq.) was added DMF (20 mL). Stirring and reducing the temperature to 0 ℃. NaH (0.10 g,0.0026mol,1.2 eq.) was slowly added. After 0.5h, tert-butyl 1,2, 3-oxathiolane-3-carboxylate 2, 2-dioxide (1.46 g,0.0044mol,2.0 eq.) was added. Naturally heating to room temperature, and reacting for 6h. Adding water to the reaction solution, extracting twice with ethyl acetate, washing the organic phase with saturated aqueous sodium chloride solution, and MgSO 4 And (5) drying. Spin-drying afforded intermediate 2.9 (0.95 g,73.0% yield). MS [ M+H ]] + ＝608。
To a reaction flask containing intermediate 2.9 (0.95 g,0.0015mol,1.0 eq.) was added methanol (12 mL), followed by 1, 4-dioxane hydrochloride solution (4M, 3 mL), and the mixture was warmed to 70℃and reacted for 0.5h. The intermediate 2.10 after the reaction liquid is dried by spin is directly put into the next reaction.
To a reaction flask containing intermediate 2.10 was added methanol (10 mL), and cesium carbonate (1.38 g,0.0045mol,3.0 eq.) was added after stirring and dissolution, and the temperature was raised to 70℃for 2h. The intermediate 2.11 after the reaction liquid is dried by spin is directly put into the next reaction.
To a reaction flask containing intermediate 2.11 was added DCM (60 mL), and after stirring to dissolve, triethylamine (0.58g,0.0045 mol,3.0eq.) was added, and di-tert-butyl dicarbonate (0.49 g,0.0225mol,1.5 eq.) was added under ice-water. Transfer to room temperature and react for 1h. The reaction was dried by spin-drying and purified by column chromatography (DCM: meoh=20:1) to afford intermediate 2.12 (0.51 g,71.4% yield). MS [ M+H ]] + ＝476。
To a reaction flask containing intermediate 2.12 (0.51 g,0.001mol,1.0 eq.) was added DMF (30 mL), stirred, ice-water bath added t-BuOK (0.22 g,0.002mol,2.0 eq.) and stirred for 20min followed by 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine (0.32 g,0.0012mol,1.2 eq.). After 3 hours of reaction, add to the reaction solution Water (200 mL) followed by extraction with ethyl acetate (80 mL. Times.3). Saturated aqueous sodium chloride solution washing, mgSO 4 And (5) drying. After spin-drying, the intermediate 2.13 (0.65 g,91.5% yield) was isolated as a yellow solid by column chromatography (PE: ea=4:1). MS [ M+H ]] + ＝733.
Intermediate 2.13 (0.65 g,0.89mmol,1.0 eq.) was dissolved in 5mL methanol and 1, 4-dioxane hydrochloride (0.5 mL,7 m) was added and the temperature was raised to 70 ℃ and reacted for 0.5h. The reaction mixture was dried to give intermediate 2.14 (0.56 g,100% yield).
Intermediate 2.14 (0.56 g,0.89mmol,1.0 eq.) was dissolved in 50mL methanol, aqueous formaldehyde (1.0 mL,37%,10 eq.) was added, and after stirring for 15min, STAB (0.95 g,4.45mmol,5 eq.) was added and stirring was continued for another 15min. The reaction was then dried by spin-drying and purified by column chromatography (DCM: meoh=8:1) to give the title compound of example 2 (0.28 g,48.3% yield). MS [ M+H ]] + ＝571。 1 H NMR(400MHz,DMSO)δ11.73(s,1H),7.35(d,J＝3.3Hz,1H),6.38(d,J＝3.3Hz,1H),6.15(s,1H),4.73(d,J＝17.8Hz,2H),4.23(d,J＝6.5Hz,2H),3.55(s,2H),2.63(s,3H),2.43(s,3H),2.49(s,3H),2.30-2.36(m,1H),2.22(s,3H),1.95-2.05(m,4H),1.65(d,J＝6.4Hz,3H),1.48-1.55(m,1H),1.25-1.40(m,4H)。
EXAMPLE 3 Synthesis of 10- ((1 r,4 r) -4- (dimethylamino) cyclohexyl) -8, 10-dimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-2, 3-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indazol-7 (4H) -one
Methyl 3, 4-dihydroxy-2, 5-dimethylbenzoate (10.0 g,51.48mmol,1.0 eq.) was dissolved in toluene (150 mL). Tert-butyl trans-N- (4-ethynylcyclohexyl) carbamate (11.50 g,51.47mmol,1.0 eq.) and Ru were added with stirring 3 (CO) 12 (0.64 g,1.54mmol,0.03 eq.) after 3 nitrogen substitutions, the mixture was heated to 105℃and reacted overnight for 10h, after which the trans-N- (4-ethynylcyclohexyl) amino group was addedTert-butyl formate (11.50 g,51.48mmol,1.2 eq.) Ru 3 (CO) 12 (0.639 g,1.54mmol,0.03 eq.) nitrogen is pumped 3 times and heated to 105℃and reacted for a further 10h. After the reaction solution was cooled to room temperature, it was filtered through celite. After concentration, isolation and purification by column chromatography (PE: EA) =10:1 gave intermediate 3.1 (12.86 g,59.53% yield). MS [ M+H ]] + ＝420。
To the reaction flask was added 3.1 (12.00 g,0.028mol,1.0 eq.) of intermediate, acetic anhydride (60 mL) was added to dissolve, and 1 drop of concentrated sulfuric acid was added dropwise. Dilute nitric acid (55 mL) was added dropwise under ice-salt bath. The internal temperature is controlled to be not higher than 45 ℃, and the reaction is carried out for 1h at room temperature after the dripping is finished. The reaction solution was poured into ice 30% aqueous sodium hydroxide (250 mL), extracted twice with ethyl acetate, and the organic phase was washed with saturated aqueous sodium chloride solution and MgSO 4 Drying, spin drying to give intermediate 3.2 (10.42 g,100% yield) which is directly fed to the next reaction.
Intermediate 3.2 (10.42 g,0.028mol,1.0 eq.) was dissolved in DCM and triethylamine (8.67 g,0.085mol,3.0 eq.) and DMAP (0.37 g,0.0028mol,0.1 eq.) were added. Di-tert-butyl dicarbonate (9.36 g,0.043mol,1.5 eq.) was added under ice-bath conditions. Transfer to room temperature and react for 2h. The reaction was concentrated and purified by column chromatography (PE: ea=4:1) to afford intermediate 3.3 (6.75 g,50.8% yield). MS [ M+H ] ] + ＝465。
To a reaction flask containing intermediate 3.3 (6.75 g,0.0145mol,1.0 eq.) was added 80mL of DCM and 80mL of DMF. Stannous chloride dihydrate (16.4 g,0.073mol,5.0 eq.) was added after stirring to dissolve. Stirring for 2h at room temperature. To the reaction solution was added saturated aqueous sodium hydrogencarbonate solution (1L). Ethyl acetate extraction was twice, washed with saturated aqueous sodium chloride, and MgSO 4 And (5) drying. After spin-drying, the intermediate 3.4 (5.10 g,80.9% yield) was isolated and purified by column chromatography (PE: ea=4:1). MS [ M+H ]] + ＝435。
To a reaction flask containing intermediate 3.4 (5.10 g,0.012mol,1.0 eq.) was added chloroform (50 mL) acetic anhydride (8.17 g,0.029mol,2.5 eq.) potassium acetate (0.58 g,0.006mol,0.5 eq.). After heating to 70 ℃, isoamyl nitrite (3.46 g,0.029mol,2.5 eq.) was added dropwise. Reacting for 1 hour, and reversingWater (50 mL) was added to the reaction solution. Ethyl acetate 100ml x 2 was extracted twice, washed with saturated aqueous sodium chloride, mgSO 4 And (5) drying. After spin-drying, the intermediate 3.5 (4.20 g,80.7% yield) was isolated and purified by column chromatography (PE: ea=4:1). MS [ M+H ]] + ＝446。
To a reaction flask containing intermediate 3.5 (1.00 g,0.0022mol,1.0 eq.) was added DMF (10 mL). Stirring and reducing the temperature to 0 ℃. NaH (0.108 g,0.0027mol,1.2 eq.) was added slowly. After 0.5h, tert-butyl 1,2, 3-oxathiolane-3-carboxylate 2, 2-dioxide (1.02 g,0.0045mol,2.0 eq.) was added. Naturally heating to room temperature, and reacting for 6h. Adding water to the reaction solution, extracting twice with ethyl acetate, washing the organic phase with saturated aqueous sodium chloride solution, and MgSO 4 And (5) drying. Spin-drying afforded intermediate 3.6 (0.850 g,64.4% yield). MS [ M+H ]] + ＝589。
To a reaction flask containing 3.6 (0.850 g,0.0015mol,1.0 eq.) of intermediate was added methanol (10 mL), followed by 1, 4-dioxane hydrochloride solution (4M, 3 mL), and the mixture was warmed to 70℃and reacted for 0.5h. The intermediate 3.7 after the reaction liquid is dried by spin is directly put into the next reaction.
To a reaction flask containing intermediate 3.7 was added methanol (10 mL), and cesium carbonate (1.41 g,0.0045mol,3.0 eq.) was added after stirring and dissolution, and the temperature was raised to 70℃for 2h. The intermediate 3.8 after the reaction liquid is dried by spin is directly put into the next reaction.
To a reaction flask containing intermediate 3.8 was added DCM (10 mL), and after stirring to dissolve, triethylamine (0.438 g,0.0045mol,3.0 eq.) was added, followed by di-tert-butyl dicarbonate (0.470 g,0.0022mol,1.5 eq.) in an ice-water bath. Transfer to room temperature and react for 1h. The reaction was dried by spin-drying and purified by column chromatography (DCM: meoh=20:1) to afford intermediate 3.9 (0.35 g,53.1% yield). MS [ M+H ]] + ＝457。
To a reaction flask containing intermediate 3.9 (0.35 g,0.765mmol,1.0 eq.) was added DMF (10 mL), stirred, ice-water bath added t-BuOK (0.174 g,1.53mmol,2.0 eq.) and stirred for 20min then 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine (0.256 g,0.92mmol,1.2 eq.). After 3 hours of reaction, water (50 mL) was added to the reaction mixture After extraction with ethyl acetate (50 mL. Times.3). Saturated aqueous sodium chloride solution washing, mgSO 4 And (5) drying. After spin-drying, the intermediate 3.10 (0.24 g,43.8% yield) was isolated as a yellow solid by column chromatography (PE: ea=4:1). MS [ M+H ]] + ＝714。
Intermediate 3.10 (0.24 g,0.336mmol,1.0 eq.) was dissolved in 10mL methanol and 1, 4-dioxane hydrochloride (3 mL,7 m) was added and the temperature was raised to 70 ℃ and reacted for 0.5h. The reaction mixture was dried to give intermediate 2.11 (0.51 g,100% yield).
Intermediate 3.10 (0.51 g,0.336mmol,1.0 eq.) was dissolved in 10mL methanol, aqueous formaldehyde (1.10 mL,37%,10 eq.) was added, and after stirring for 15min, STAB (0.35 g,1.68mmol,5 eq.) was added and stirring was continued for a further 15min. The reaction was then dried by spin-drying and purified by column chromatography (DCM: meoh=8:1) to give the title compound of example 3 (0.11 g,59.5% yield). MS [ M+H ]] + ＝552。 1 H NMR(400MHz,DMSO-d 6 )11.71(s,1H),8.04(s,1H),6.15(s,1H),4.73(s,2H),4.07(s,2H),3.51(s,2H),2.65(s,3H),2.41(d,J＝7.7Hz,6H), 2.32-2.36(m,1H),2.30(s,3H),2.21(s,3H),1.89(s,5H),1.59(s,3H),1.19-1.28(m,4H)。
Intermediate 1.4.1 methyl (R) -7-bromo-2- ((1R, 4S) -4- ((tert-butoxycarbonyl) amino) cyclohexyl) -2, 4-dimethyl-6-nitrobenzo [ d ] [1,3] dioxin-5-carboxylate and intermediate 1.4.2 methyl (S) -7-bromo-2- ((1R, 4R) -4- ((tert-butoxycarbonyl) amino) cyclohexyl) -2, 4-dimethyl-6-nitrobenzo [ d ] [1,3] dioxin-5-carboxylate
Compound 1.4.1 and compound 1.4.2 were prepared by chiral separation of intermediate 1.4 as follows:
Separation apparatus Waters UPC2 analytical SFC
Chromatographic column: chiralCel OJ, 150X 4.6mm I.D.,3 μm
Mobile phase A for CO 2 and B for Methanol(0.1％NH 3 H 2 O)
Gradient B10%
Flow rate 2.0mL/min
Wavelength of 220nm
Compound 1.4.1 retention time 4.22 min, [ α ] = -30.3 ° (MeOH, c=1.0 g/100 mL)
Compound 1.4.2 retention time 3.70 min, [ α ] = 25.4 ° (MeOH, c=1.0 g/100 mL)
HPLC apparatus: shimadzu LC-20AD
MS:[M+H] + ＝529。 1 H NMR(400MHz,DMSO)δ6.76(d,J＝7.8Hz,1H),3.79(s,3H),3.18(s,1H),2.18(s,3H),1.90-1.75(m,5H),1.70(s,3H),1.37(s,9H),1.27-1.09(m,4H)。
EXAMPLE 4 Synthesis of (R) -10- ((1R, 4R) -4- (dimethylamino) cyclohexyl) -8, 10-dimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 4 was synthesized using a similar procedure as in example 1, intermediate 1.4 was resolved and replaced with 1.4.1 (2.20 g,0.0043mol,1.0 eq.). MS [ M+H ]] + ＝551。 1 H NMR(400MHz,DMSO-d 6 )δ11.71(s,1H),7.21(d,J＝3.1Hz,1H),6.30(d,J＝3.0Hz,1H),6.14(s,1H),4.72(s,2H),4.06(s,2H),3.51(s,2H),2.68(s,1H),2.54(s,6H),2.42(s,3H),2.40(s,3H),2.21(s,3H),2.08-1.76(m,5H),1.59(s,3H),1.36-1.20(m,4H)。
EXAMPLE 5 Synthesis of (S) -10- ((1 r, 4S) -4- (dimethylamino) cyclohexyl) -8, 10-dimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 5 was synthesized using a similar procedure as in example 1, intermediate 1.4 was resolved and replaced with 1.4.2 (2.20 g,0.0043mol,1.0 eq.). MS [ M+H ] ] + ＝551。 1 H NMR(400MHz,DMSO-d 6 )11.70(s,1H),7.21(s,1H),6.30(s,1H),6.14(s,1H),4.73(s,2H),4.07(s,2H),3.51(s,2H),2.65(s,3H),2.41(d,J＝7.7Hz,6H),2.32-2.36(m,1H),2.30(s,3H),2.21(s,3H),1.89(s,5H),1.59(s,3H),1.29-1.19(m,4H)。
Example 6 Synthesis of (4R, 10R) -10- ((1 r, 4R) -4- (dimethylamino) cyclohexyl) -4,8, 10-trimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 6 was synthesized using a procedure similar to that described in example 4 substituting tert-butyl 1,2, 3-oxathiazolidine-3-carboxylate 2, 2-dioxide with (S) -5-methyl-1, 2, 3-oxathiazolidine-3-carboxylate-2, 2-dioxide. MS [ M+H ]] + ＝565。 1 H NMR(400MHz,DMSO-d 6 )δ11.65(s,1H),7.36-7.26(m,1H),6.29(s,1H),6.13(s,1H),5.12(s,2H),4.53(s,2H),4.23(s,1H),3.46(s,3H),2.68(s,1H),2.51(s,3H),2.44(s,3H),2.39(s,3H),2.21(s,3H),1.95(m,5H),1.60(s,3H),1.43(s,3H),1.32(m,4H)。
EXAMPLE 7 Synthesis of (4S, 10R) -10- ((1 r, 4R) -4- (dimethylamino) cyclohexyl) -4,8, 10-trimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 7 was synthesized using a procedure similar to that described in example 6 substituting (R) -4-methyl-1, 2, 3-oxazolidine-3-carboxylic acid tert-butyl 2, 2-dioxide with (R) -5-methyl-1, 2, 3-oxazolidine-3-carboxylic acid tert-butyl 2, 2-dioxide. MS [ M+H ]] + ＝565。 1 H NMR(400MHz,DMSO-d 6 )δ11.67(s,1H),7.31(s,1H),6.30(s,1H),6.13(s,1H),5.11(s,1H),4.51(d,J＝15.8Hz,2H),3.50-3.43(m,2H),2.68(s,1H),2.63(s,6H),2.44(s,3H),2.39(s,3H),2.21(s,3H),2.09-1.87(m,5H),1.61(s,3H),1.44(s,3H),1.39-1.20(m,4H)。
EXAMPLE 8 Synthesis of (10R) -10- ((1R, 4R) -4- (dimethylamino) cyclohexyl) -5,8, 10-trimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 8 was synthesized using a similar procedure as in example 6 substituting (R) -4-methyl-1, 2, 3-oxazolidine-3-carboxylic acid tert-butyl ester 2, 2-dioxide with 4-methyl-1, 2, 3-oxazolidine-3-carboxylic acid tert-butyl ester 2, 2-dioxide. MS [ M+H ]] + ＝565。 1 H NMR(400MHz,DMSO)δ11.69(s,1H),7.18(d,J＝2.9Hz,1H),6.30(d,J＝3.0Hz,1H),6.13(s,1H),4.93(d,J＝13.8Hz,1H),4.54(d,J＝13.7Hz,1H),4.31(dd,J＝13.5,4.5Hz,1H),4.15(d,J＝13.5Hz,1H),3.90-3.80(m,1H),2.52(s,3H),2.47(s,3H),2.42(s,6H),2.38-2.25(m, 1H),2.20(s,3H),2.06-1.83(m,5H),1.60(s,3H),1.42-1.20(m,4H),0.59(d,J＝7.0Hz,3H)。
EXAMPLE 9 Synthesis of (R) -10- ((1R, 4R) -4- (dimethylamino) cyclohexyl) -6- ((4-methoxy-6-methyl-2-oxo-1, 2-dihydropyridin-3-yl) methyl) -8, 10-dimethyl-5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 9 was synthesized using a procedure similar to that described in example 4 substituting 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine with 2- (benzyloxy) -3- (chloromethyl) -4-methoxy-6-methylpyridine. MS [ M+H ]] + ＝535。 1 H NMR(400MHz,DMSO-d 6 )11.58(s,1H),7.21(s,1H),6.29(s,1H),6.14(s,1H),5.15(s,2H),4.33(s,2H),3.80(s,3H),3.51(s,2H),2.40(d,J＝8.0Hz,6H),2.32-2.36(m,1H),2.30(s,3H),2.21(s,3H),1.95-1.79(m,5H),1.59(s,3H),1.29-1.19(m,4H)。
EXAMPLE 10 Synthesis of (S) -10- ((1 r, 4S) -4- (dimethylamino) cyclohexyl) -6- ((4-methoxy-6-methyl-2-oxo-1, 2-dihydropyridin-3-yl) methyl) -8, 10-dimethyl-5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 10 was synthesized using a procedure similar to that described in example 5 substituting 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine with 2- (benzyloxy) -3- (chloromethyl) -4-methoxy-6-methylpyridine. MS [ M+H ] ] + ＝535。 1 H NMR(400MHz,DMSO-d 6 )11.58(s,1H),7.21(s,1H),6.29(s,1H),6.14(s,1H),4.56(s,2H),4.33(s,2H),3.79(s,3H),3.51(s,2H),2.40(s,6H),2.32-2.36(m,1H),2.30(s,3H),2.21(s,3H),1.95-1.79(m,5H),1.59(s,3H),1.29-1.19(m,4H)。
EXAMPLE 11 Synthesis of (R) -6- ((4, 6-dimethyl-2-oxo-1, 2-dihydropyridin-3-yl) methyl) -10- ((1R, 4R) -4- (dimethylamino) cyclohexyl) -8, 10-dimethyl-5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compounds of formula (I)11 was synthesized using a procedure similar to that described in example 4 substituting 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine with 2- (benzyloxy) -3- (chloromethyl) -4, 6-dimethylpyridine. MS [ M+H ]] + ＝519。 1 H NMR(400MHz, DMSO-d 6 )11.66(s,1H),7.23(s,1H),6.31(s,1H),5.94(s,1H),4.74(s,2H),4.13(s,2H),3.63(s,2H),2.80(s,1H),2.38(s,3H),2.19(s,3H),2.15(s,3H),1.95-1.80(m,5H),1.60(s,3H),1.41-1.24(m,4H),1.11(s,6H)。
Synthesis of intermediate 3.5.1 methyl (R) -2- ((1R, 4R) -4- ((tert-butoxycarbonyl) amino) cyclohexyl) -2, 4-dimethyl-6H- [1,3] dioxa [4,5-e ] indazole-5-carboxylate and intermediate 3.5.2 methyl (S) -2- ((1R, 4S) -4- ((tert-butoxycarbonyl) amino) cyclohexyl) -2, 4-dimethyl-6H- [1,3] dioxa [4,5-e ] indazole-5-carboxylate
Compound 3.5.1 and compound 3.5.2 were prepared by chiral separation of intermediate 3.5 as follows:
separation apparatus Waters UPC2 analytical SFC
Chromatographic column: chiralCel OJ, 150X 4.6mm I.D.,3 μm
Mobile phase A for CO 2 and B for Methanol(0.1％NH 3 H 2 O)
Gradient of B40%
Flow rate 2.0mL/min
Wavelength of 220nm
Compound 3.5.1 retention time 2.72 min, [ α ] = 38.0 ° (MeOH, c=1.0 g/100 mL)
Compound 3.5.2 retention time 4.39 min, [ α ] = -29.1 ° (MeOH, c=1.0 g/100 mL)
MS:[M+H] + ＝446。 1 H NMR(400MHz,DMSO)δ12.91(s,1H),8.11(d,J＝1.3Hz,1H),6.75(d,J＝7.9Hz,1H),3.90(s,3H),3.23-3.11(m,1H),2.54(s,3H),1.94-1.77(m,5H),1.64(s,3H),1.36(s,9H),1.24-1.05(m,4H)。
Example 12: synthesis of (R) -10- ((1R, 4R) -4- (dimethylamino) cyclohexyl) -8, 10-dimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indazol-7 (4H) -one
Compound 12 was synthesized using a similar procedure as in example 3, intermediate 3.5 was resolved and replaced with 3.5.1 (2.20 g,0.0043mol,1.0 eq.). MS [ M+H ]] + ＝552。 1 H NMR(400MHz,DMSO)δ11.69(s,1H),8.03(s,1H),6.15(s,1H),4.75(s,2H),4.26(s,2H),3.60(s,2H),2.49(s,3H),2.42(s,3H),2.23(s,3H),2.17(s,6H),2.15-2.08(m,1H),1.89(m,5H),1.64(s,3H),1.21(m,4H)。
Example 13: synthesis of (S) -10- ((1 r, 4S) -4- (dimethylamino) cyclohexyl) -8, 10-dimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indazol-7 (4H) -one
Compound 13 was synthesized using a similar procedure as in example 12 substituting 3.5.1 for 3.5.2 (2.20 g,0.0043mol,1.0 eq.) to yield intermediate. MS [ M+H ]] + ＝552。 1 H NMR(400MHz,DMSO)δ11.71(s,1H),8.04(s,1H),6.15(s,1H),4.75(s,2H),4.26(s,2H),3.59(s,2H),2.52(s,3H),2.49(s,6H),2.42(s,3H),2.34(s,1H),2.23(s,3H),1.98m,5H),1.65(s,3H),1.33-1.14(m,4H)。
Example 14: synthesis of (4R, 10R) -10- ((1 r, 4R) -4- (dimethylamino) cyclohexyl) -4,8, 10-trimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indazol-7 (4H) -one
Example 14 was synthesized using a procedure similar to that used in example 12 substituting tert-butyl 1,2, 3-oxazolidine-3-carboxylate 2, 2-dioxide with (S) -5-methyl-1, 2, 3-oxazolidine-3-carboxylate 2, 2-dioxide. MS [ M+H ] ] + ＝566。 1 H NMR(400MHz,DMSO)δ11.70(s,1H),8.03(s,1H),6.13(s,1H),5.01(s,1H),4.60(d,J＝13.9Hz,2H),3.68-3.45(m,2H),2.46(s,3H),2.43(s,3H),2.23(s,3H),2.17(s,6H),2.14-2.08(m,1H),2.02-1.75(m,5H),1.63(s,3H),1.42(s,3H),1.25-1.14(m,4H)。
Example 15: synthesis of (4S, 10R) -10- ((1 r, 4R) -4- (dimethylamino) cyclohexyl) -4,8, 10-trimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indazol-7 (4H) -one
Example 15 was synthesized using a procedure similar to that used in example 12 substituting tert-butyl 1,2, 3-oxazolidine-3-carboxylate 2, 2-dioxide with (R) -5-methyl-1, 2, 3-oxazolidine-3-carboxylate 2, 2-dioxide. MS [ M+H ]] + ＝566。 1 H NMR(400MHz,DMSO)δ11.70(s,1H),8.03(s,1H),6.14(s,1H),5.02(s,1H),4.59(m,2H),3.55(s,2H),2.46(s,3H),2.43(s,3H),2.33(s,1H),2.27(s,6H),2.22(s,3H),1.91(d,J＝22.4Hz,5H),1.64(s,3H),1.41(d,J＝14.4Hz,3H),1.23(s,4H)。
Example 16: synthesis of (R) -1-chloro-10- ((1R, 4R) -4- (dimethylamino) cyclohexyl) -8, 10-dimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Intermediate 4.4 (0.81 g,1.8mmol,1.0 eq.) was dissolved in dichloromethane, N-chlorosuccinimide (0.36 g,2.7mmol,1.5 eq.) was added and stirred overnight at ambient temperature. The reaction mixture was then dried by spin-drying, and isolated and purified by column chromatography to give intermediate 16.1 (0.81 g,92.0% yield). MS [ M+H ]] + ＝479。
Compound 16 was prepared using a similar synthetic procedure to compound 4, substituting intermediate 16.1 for intermediate 4.4. MS [ M+H ]] + ＝586。 1 H NMR(400MHz,DMSO-d 6 )δ11.72(s,1H),7.39(s,1H),6.14(s,1H),4.71(s,3H),4.03(s,2H),3.50(s,2H),2.68(s,1H),2.54(s,2H),2.50(s,3H),2.43(s,3H),2.39(s,3H),2.21(s,3H),2.05-1.87(m,5H),1.63(s,3H),1.34-1.19(m,4H)。
EXAMPLE 17 Synthesis of (4R, 10R) -10- ((1 r, 4R) -4- (dimethylamino) cyclohexyl) -6- ((4-methoxy-6-methyl-2-oxo-1, 2-dihydropyridin-3-yl) methyl) -4,8, 10-trimethyl-5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 17 was synthesized using a similar procedure as in example 6 substituting 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine with 2- (benzyloxy) -3- (chloromethyl) -4-methoxy-6-methylpyridine. MS [ M+H ]] + ＝549.24。 1 H NMR(400MHz,DMSO-d 6 )δ:11.53(s,1H),7.30(s,1H),6.29(d,1H),6.14(s,1H),5.15(s,2H),4.33(s,2H),3.80(s,3H),3.51-3.79(m,2H),2.35(m,7H),2.21(s,3H),1.58(m,3H),1.90(m,4H),1.85(s,3H),1.39-1.23(m,7H)。
EXAMPLE 18 Synthesis of (4S, 10R) -10- ((1 r, 4R) -4- (dimethylamino) cyclohexyl) -6- ((4-methoxy-6-methyl-2-oxo-1, 2-dihydropyridin-3-yl) methyl) -4,8, 10-trimethyl-5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 18 was synthesized using a procedure similar to that described in example 7 substituting 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine with 2- (benzyloxy) -3- (chloromethyl) -4-methoxy-6-methylpyridine. MS [ M+H ]] + ＝549.27。 1 H NMR(400MHz,DMSO-d 6 )δ:11.53(s,1H),7.30(s,1H),6.29(d,1H),6.13(s,1H),5.09(brs,2H),4.23(m,2H),3.80(s,3H),2.60(m,2H),2.37(m,6H),2.21(s,3H),1.91-1.86(m,8H),1.58(s,3H),1.38-1.25(m,7H)。
EXAMPLE 19 Synthesis of (4R, 10R) -6- ((4, 6-dimethyl-2-oxo-1, 2-dihydropyridin-3-yl) methyl) -10- ((1 r, 4R) -4- (dimethylamino) cyclohexyl) -4,8, 10-trimethyl-5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 19 was synthesized using a procedure similar to that described in example 6 substituting 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine with 2- (benzyloxy) -3- (chloromethyl) -4, 6-dimethylpyridine. MS [ M+H ]] + ＝533.36。 1 H NMR(400MHz,DMSO-d 6 )δ:11.60(s,1H),7.31(s,1H),6.30(s,1H),5.92(s,1H),5.06(s,1H),4.47(s,2H),3.69(s,1H),3.59(s,1H),2.51(s,2H),2.35(s,3H),2.39(s,2H),2.22(s,6H),2.15(s,3H),1.92-1.87(m,5H),1.58(s,3H),1.44-1.43(m,3H),1.20-1.18(m,4H)。
EXAMPLE 20 Synthesis of((5R, 10R) -10- ((1 r, 4R) -4- (dimethylamino) cyclohexyl) -5,8, 10-trimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 20 was synthesized using a procedure similar to that described in example 4 substituting tert-butyl 1,2, 3-oxazolidine-3-carboxylate 2, 2-dioxide with (R) -4-methyl-1, 2, 3-oxazolidine-3-carboxylate-2, 2-dioxide. MS [ M+H ]] + ＝565.46。 1 H NMR(400MHz,DMSO-d 6 )δ:11.69(s,1H),7.18(d,J＝2.9Hz,1H),6.30(d,J＝3.0Hz,1H),6.13(s,1H),4.93(d,J＝13.8Hz,1H),4.54(d,J＝13.7Hz,1H),4.31(dd,J＝13.5,4.5Hz,1H),4.15(d,J＝13.5Hz,1H),3.90-3.80(m,1H),2.52(s,3H),2.47(s,3H),2.42(s,6H),2.38-2.25(m,1H),2.20(s,3H),2.06-1.83(m,5H),1.60(s,3H),1.42-1.20(m,4H),0.59(d,J＝7.0Hz,3H)。
EXAMPLE 21 Synthesis of (S) -8-chloro-10- ((1 r, 4S) -4- (dimethylamino) cyclohexyl) -10-methyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 21 was prepared by following a similar procedure to that used in example 2, resolution of intermediate 1.4 followed by replacement with 1.4.1. MS [ M+H ]] + ＝571.14。 1 H NMR(400MHz,DMSO-d 6 )δ:11.72(s,1H),7.33(s,1H),6.38(d,1H),6.15(s,1H),4.70(s,2H),4.08(s,2H),3.55(s,2H),2.51(s,3H),2.43(s,3H),2.22(m,8H),1.92(m,4H),1.64(m,3H),1.23(m,4H)。
EXAMPLE 22 Synthesis of (4R, 10S) -8-chloro-10- ((1 r, 4S) -4- (dimethylamino) cyclohexyl) -4, 10-dimethyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 22 was synthesized using a procedure similar to that described in example 21 substituting tert-butyl 1,2, 3-oxazolidine-3-carboxylate 2, 2-dioxide with (S) -5-methyl-1, 2, 3-oxazolidine-3-carboxylate-2, 2-dioxide. MS [ M+H ]] + ＝585.16。 1 H NMR(400MHz,DMSO-d 6 )δ:11.68(s,1H),7.43(d,1H),6.28(d,1H),6.13(s,1H),5.05(s,2H),4.52(s,2H),3.50(s,2H),2.51-2.50(m,3H),2.49(s,3H),2.20(m,7H),1.91(m,4H),1.62(m,3H),1.32(m,7H)。
EXAMPLE 23 Synthesis of (R) -8-chloro-10- ((1R, 4R) -4- (dimethylamino) cyclohexyl) -10-methyl-6- ((6-methyl-4- (methylsulfanyl) -2-oxo-1, 2-dihydropyridin-3-yl) methyl) -5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxa [4,5-e ] indol-7 (4H) -one
Compound 23 was prepared by a similar procedure as in example 2, resolution of intermediate 1.4 and replacement with 1.4.2. MS [ M+H ]] + ＝572.23。 1 H NMR(400MHz,DMSO-d 6 )δ:11.73(s,1H),7.35(d,J＝3.3Hz,1H),6.38(d,J＝3.3Hz,1H),6.15(s,1H),4.73(d,J＝17.8Hz,2H),4.56(s,2H),3.55(s,2H),2.63(s,3H),2.43(s,3H),2.34(s,1H),2.22(s,7H),2.01(m,4H),1.62(m,3H),1.38(m,7.5Hz,4H)。
EXAMPLE 24 Synthesis of (4R, 10S) -8-chloro-6- ((4, 6-dimethyl-2-oxo-1, 2-dihydropyridin-3-yl) methyl) -10- ((1 r, 4S) -4- (dimethylamino) cyclohexyl) -4, 10-dimethyl-5, 6-dihydro- [1,4] diazepin [6,7,1-hi ] [1,3] dioxin [4,5-e ] indol-7 (4H) -one
Compound 24 was synthesized using a procedure similar to that described in example 22 substituting 2- (benzyloxy) -3- (chloromethyl) -6-methyl-4- (methylthio) pyridine with 2- (benzyloxy) -3- (chloromethyl) -4, 6-dimethylpyridine. MS [ M+H ]] + ＝572.42。 1 H NMR(400MHz,DMSO-d 6 )δ:11.65(s,1H),7.54(s,1H),6.38(d,1H),5.93(s,1H),5.08(s,1H),4.52(m,2H),3.74(s,1H),3.35(s,2H),2.51-2.50(m,3H),2.21(s,2H),2.06(m,6H),1.85(m,5H),1.63(m,3H),1.47(m,3H),1.14(m,5H)。
Experimental example 1
PRC2/EZH 2HTRF enzyme inhibition assay
PRC2 Complex was purchased from Carna Biosciences (Japan), histone H3 (21-44) -GK (biotin), strepitavidin-XL 665 and Mab Anti Histone H K27Me1-Eu (K) from Cisbio (France).
SAM (30 mM), DTT (1M), 10% BAS, tris-HCl (50 mM), naCl (50 mM), 10% Tween and dimethyl sulfoxide (DMSO) were purchased from Sigma.384 Kong Baiban is purchased from CORNING (united states).
The assay buffer used in the experiments consisted of 50mM Tris-HCl,50mM NaCl,1mM DTT,0.01%Tween,0.1%BSA. A mixed solution of 5% DMSO compound, PRC2 enzyme and Histone H3 (21-44) -GK is prepared by using an assay buffer, after the preparation is completed, 2 mu L of 5% DMSO compound and 4 mu L of PRC2 mixed solution are respectively added into an Opti Plate-384White pore Plate to cover the membrane 800 for 1min, incubated for 0.5H at room temperature, and 4 mu L of Histone H3 (21-44) -GK (bio)/SAM cover membrane 800 for 1min and incubated for 2H at room temperature. PRC2, histone H3 (21-44) -GK (biotin), SAM and DMSO final concentrations were 1 ng/. Mu.L, 5. Mu.M, 15. Mu.M and 1%, respectively. Then 5. Mu.L of detection antibody Mab Anti histone H K27Me1-Eu (K) and 5. Mu.L of strepitavidin-XL 665 were added and incubated for 2h at room temperature. The plates were read on a SPARK multifunctional microplate reader of TECAN (Switzerland) with excitation light at 320nm and emission light at 665nm. Compound IC was determined in Prism7 (LaJolla, CA) using an S-shaped dose response model (variable slope, four parameters) 50 Values.
Cell proliferation inhibition assay
Karpas422 cells were purchased from the cell bank of China academy of sciences (Shanghai); RPMI1640 medium, penicillin-streptomycin diabody and 0.5% pancreatin (10X) were purchased from ThermoFisher (Waltham, mass., USA). Certified Fetal Bovine Serum (FBS) was purchased from Biological Industries (Israel). CORNING 96 well cell culture plates were purchased from CORNING (USA). The Counting Kit-8 was purchased from Biyun (China).
To evaluate the proliferation inhibition level of synthetic compounds on human large cell lymphoma SU-DHL-10, human B cell non-Hodgkin lymphoma cell Karpas422, exponentially growing SU-DHL-10, karpas422 cells were inoculated in RPMI1640 medium containing 20% bovine serum and 1% penicillin-streptomycin diabody, respectively, at a density of 1000 cells/mL, 96 well plates, 100 μl per well, and placed at 37℃at 5% CO 2 Overnight in the incubator of (a). Compounds were diluted in DMSO at initial concentration of 2mm, 3-fold dilution, 12 concentrations. 1 μl of DMSO solution of the compound stock plate was added to 99 μl of cell culture medium, the final maximum concentration of compound in the assay was 10 μΜ, and the final concentration of DMSO was (0.5%). mu.L of compound solution in the medium was added to each well of SU-DHL-10 and Karpas422 cell plates. After addition of the compound solution, the 96-well plate was placed at 37℃with 5% CO 2 Incubate in incubator for 8 days. Cell viability was determined by quantifying the amount of formazan produced in cell culture in direct proportion to the number of living cells using the Counting Kit-8 detection Kit. After 2 hours incubation, readings were made using a SPARK multifunctional microplate reader from TECAN under a chemiluminescent program. Concentration of compounds to inhibit cell viability by 50% (IC) was determined in Prism 7 (LaJolla, calif.) using an S-shaped dose response model (variable slope, four parameters) 50 Values).
The results of the EZH2 enzyme inhibition experiments and cell proliferation inhibition experiments for representative compounds described herein are shown in table 1.
TABLE 1
From the test results in table 1, most of the compounds have excellent EZH2 inhibitory activity, and at the same time, part of the compounds have better results than the prior positive references, especially compound 4, compound 6, compound 7, compound 12 and compound 16. Meanwhile, the compound also shows obvious proliferation and transplantation activity and has obvious advantages compared with the drug Tazemetostat on the market.
Experimental example 2 in vitro liver microsome stability test
Human liver microsomes were derived from Corning, ICR/CD-1 rat liver microsomes (androstane) were also derived from Corning and stored in-80℃refrigerator, respectively. Sodium dihydrogen phosphate, potassium dihydrogen phosphate, NADPH were all purchased from Sigma.
The test substance was prepared as a high-concentration stock solution with DMSO, and diluted to 200. Mu.M working solution with DMSO before use, and the final concentration of the test substance was 1. Mu.M. 7.098g of disodium hydrogen phosphate is weighed first, and 500mL of pure water is added for ultrasonic dissolution to obtain solution A. 3.400g of potassium dihydrogen phosphate was weighed, and 250mL of pure water was added for ultrasonic dissolution to obtain solution B. Solution B was added to solution a until pH 7.4. The right amount of NADPH is weighed before the experiment, and a working solution with the concentration of 10mM is prepared by using phosphate solution. Transfer 25. Mu.L of NADPH or phosphate buffer to the incubation system described above, add 2. Mu.L of 200. Mu.M of the test compound. Double parallel preparation was performed on NADPH added samples; single parallel preparations were performed for NADPH negative samples. 30. Mu.L of suspension were taken at 0.5, 5, 15, 30 and 60 minutes, respectively. The reaction was quenched by the addition of 180 μl of acetonitrile containing internal standard, and vortexed for 10 minutes. Protein precipitation was then performed by centrifugation at 3220g for 20 minutes. After placing the plates in a refrigerator at 4℃for 30 minutes, they were centrifuged again at 3220g for 20 minutes. Transfer 100 μl of supernatant to sample plate, add 100 μl of purified water and mix well for UPLC-MS/MS analysis. All data calculations were performed by Microsoft Excel software. The peak area was detected by extracting the ion spectrum. Detection of in vitro half-life of the parent drug by linear fitting of the natural logarithm of the percentage of parent drug elimination to time (t 1/2 ) The clearance is calculated.
The results of the liver microsomal stability experiments for the representative compounds described herein are shown in table 2.
TABLE 2
From the test results of table 2, most of these compounds have excellent in vitro liver microsomal stability, while in vitro liver microsomal stability of some of them has better results than the existing positive references, especially compound 3, compound 4, compound 6, compound 7, compound 9, compound 11, compound 12 and compound 19.

Claims (11)

  1. A compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
    wherein R is 1 、R 2 、R 3 Each independently selected from hydrogen, halogen, C 1 -C 6 Alkyl, hydroxy, cyano or amino;
    l, U, V, T are each independently selected from C or N, and at least one is N;
    y is selected from hydrogen, halogen or C 1 -C 6 An alkyl group;
    x is selected from hydrogen, halogen, C 1 -C 6 Alkyl, alkoxy or-S-R 4 ，R 4 Is C 1 -C 6 An alkyl group;
    z is C 3 -C 7 Cycloalkyl or 4-to 7-membered heterocyclyl, each of which is optionally substituted with 1 to 3 substituents selected from halogen, C 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, halo C 1 -C 6 Substitution of an alkoxy group or a group of-NRaRb;
    ra is hydrogen, C 1 -C 6 Alkyl or halo C 1 -C 6 An alkyl group;
    rb is C 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl or 4 to 7 membered heterocyclyl, wherein said heterocyclyl is optionally substituted with 1 to 3 substituents selected from halogen, C 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy or halo C 1 -C 6 Substitution of the alkoxy group;
    or Ra and Rb, together with the nitrogen atom to which they are attached, form a group optionally selected from halogen, C, 1 to 3 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl or-ORc groups substituted with 4 to 7 membered heterocyclyl;
    rc is C 1 -C 6 Alkyl, halogenated C 1 -C 6 Alkyl or C 3 -C 7 Cycloalkyl;
    m, n' are each independently 0, 1 or 2;
    n is 0, 1, 2, 3 or 4; and
    represents a single bond or a double bond.
  2. The compound of claim 1, wherein formula (I) is formula (Ia):
    wherein R is 1 、R 2 、R 3 Each independently selected from hydrogen, halogen, C 1 -C 6 Alkyl, hydroxy, cyano or amino;
    l, U, V, T are each independently selected from C or N, and at least one is N;
    y is selected from hydrogen, halogen or C 1 -C 6 An alkyl group;
    x is selected from hydrogen, halogen, C 1 -C 6 Alkyl, alkoxy or-S-R 4 ，R 4 Is C 1 -C 6 An alkyl group;
    m, n' are each independently 0, 1 or 2;
    n is 0, 1, 2, 3 or 4; and
    represents a single bond or a double bond.
  3. The compound of claim 1, wherein formula (I) is formula (Ib):
    wherein R is 1 、R 2 、R 3 Each independently selected from hydrogen, halogen, C 1 -C 6 Alkyl, hydroxy, cyano or amino;
    u, V are each independently selected from C or N;
    x is selected from hydrogen, halogen, C 1 -C 6 Alkyl, alkoxy or-S-R 4 ，R 4 Is C 1 -C 6 An alkyl group;
    m, n' are each independently 0, 1 or 2;
    n is 0, 1, 2, 3 or 4; and
    represents a single bond or a double bond.
  4. The compound of claim 1, wherein formula (I) is formula (Ic):
    wherein R is 1 、R 2 、R 3 Each independently selected from hydrogen, halogen, C 1 -C 6 Alkyl, hydroxy, cyano or amino;
    u, V are each independently selected from C or N;
    x is selected from hydrogen, halogen, C 1 -C 6 Alkyl, alkoxy or-S-R 4 ，R 4 Is C 1 -C 6 An alkyl group;
    m, n' are each independently 0, 1 or 2;
    n is 0, 1, 2, 3 or 4; and
    represents a single bond or a double bond.
  5. A compound of formula (I) according to claim 1, selected from:
  6. a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate, or isomer thereof; and a pharmaceutically acceptable carrier or excipient.
  7. Use of a compound of claim 1, or a pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate, or isomer thereof, for the manufacture of a medicament for the treatment of an EZH 2-mediated disease.
  8. The use of claim 7, wherein the disease is cancer.
  9. The use of claim 8, wherein the cancer is lung cancer, gastric cancer, liver cancer, breast cancer, nasopharyngeal cancer, pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, glioma, melanoma, prostate cancer, renal cancer, esophageal cancer, mesothelioma, head and neck cancer, bladder cancer, salivary gland cancer, leukemia or lymphoma.
  10. A compound represented by the following formula (II):
    wherein R is 1 、R 2 、R 3 、U、V、m、n、n’、 As defined in claim 1.
  11. Use of a compound according to claim 10 for the preparation of a compound according to claim 1.
CN202280007243.0A 2021-02-26 2022-02-25 Novel EZH2 inhibitors and uses thereof Pending CN116457348A (en)

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