CN110709402A

CN110709402A - Heteroaryl pyrimidone derivatives, preparation method and medical application thereof

Info

Publication number: CN110709402A
Application number: CN201880030204.6A
Authority: CN
Inventors: 吕贺军; 刘俊; 郝旭辉; 关东亮; 陈磊; 白骅
Original assignee: ZHEJIANG HAIZHENG PHARMACEUTICAL CO Ltd
Current assignee: Zhejiang Hisun Pharmaceutical Co Ltd; Shanghai Aryl Pharmtech Co Ltd
Priority date: 2017-06-15
Filing date: 2018-06-12
Publication date: 2020-01-17
Anticipated expiration: 2038-06-12
Also published as: WO2018228369A1; TW201904974A; CN110709402B

Abstract

The invention relates to heteroaryl pyrimidinone derivatives represented by formula (I), a preparation method thereof and application thereof as a therapeutic agent, in particular as an acetyl-CoA carboxylase (ACC) inhibitor, wherein the definition of each substituent in the formula (I) is the same as that in the specification.

Description

Heteroaryl pyrimidone derivatives, preparation method and medical application thereof

The present application claims priority of chinese patent application entitled "derivatives of heteroarylopyrimidinones, methods of preparation thereof and pharmaceutical use thereof" filed by the chinese patent office on 6/15/2017, application No. 201710450023.6, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to a heteroaryl pyridone derivative, a preparation method thereof, a pharmaceutical composition containing the derivative and application of the derivative as a therapeutic agent, in particular as an acetyl-CoA carboxylase (ACC) inhibitor.

Background

Acetyl-CoA carboxylase (ACC), one of the important proteins involved in fatty acid metabolism, catalyzes an irreversible reaction of Acetyl-CoA (Acetyl-CoA) to malonyl-CoA (malonyl-CoA) with biotin (biotin) as a coenzyme, thereby providing a substrate for the subsequent synthesis of fatty acid or regulating a fatty acid oxidation signal, which is the first step of fatty acid metabolism and is the rate-limiting step. The catalytic reaction can be divided into two steps, which depend on the Biotin Carboxylase (BC) and Carboxytransferase (CT) activities of ACC.

ACC exists in human body in 2 subtypes, ACC1 and ACC2, which are separately encoded and expressed by two genes, ACACACA and ACACACB, respectively. The two have difference in tissue distribution and intracellular distribution, and ACC1 is a cytoplasmic enzyme and is mainly expressed at high level in fat synthesis tissues (such as fat and mammary gland tissues); ACC2 localized to the mitochondrial membrane, was mainly enriched in oxidized tissues (such as heart and skeletal muscle), and was expressed at high levels in the liver. Thus, ACC1 is primarily involved in regulating fatty acid synthesis, and ACC2 is primarily responsible for the regulation of fatty acid oxidation processes. The activity of ACC is regulated by a variety of proteins, cytokines, endocrine hormones, and receptors. Wherein AMPK is a major substance that regulates ACC activity, and can inhibit ACC activity by directly phosphorylating it; protein phosphorylase 2 dephosphorylates ACC, thereby enhancing ACC function. Under physiological conditions, free fatty acids synthesized in the cytoplasm are transported into mitochondria for oxidative energy supply by carnitine palmitoyl transferase 1(CPT1) on the mitochondrial membrane. While the cytoplasmic malonyl-coa allosterically inhibits CPTl, rendering its activity at a lower level, thereby limiting fatty acid oxidation. When the organism is under stress or the energy consumption is increased, the AMPK pathway can be immediately activated, the ACC at the downstream of the AMPK pathway is inactivated, the malonyl CoA level is rapidly reduced, the inhibition effect on CPTl is further relieved, the fatty acid oxidation energy supply is promoted, and more ATP is provided for the organism.

Dysregulation of fatty acid metabolism resulting from increased fatty acid synthesis and impaired fatty acid oxidation is a common feature of a variety of metabolic diseases involving diseases including: hepatic steatosis, dyslipidemia, obesity, metabolic syndrome, nonalcoholic steatohepatitis (NASH), type 2 diabetes (T2DM), and atherosclerosis. In addition, abnormal fatty acid metabolism is also one of the characteristics of neoplastic diseases, and is involved in regulating the abnormal cell proliferation process of malignant tumors. Because ACC is a key regulatory protein of lipid metabolism, drug inhibition of ACC can restrict fatty acid synthesis in lipid-derived tissues and stimulate and promote fatty acid oxidation in oxidized tissues, thereby providing an attractive treatment mode for treating the diseases with abnormal lipid metabolism.

A series of ACC inhibitor patents have been published including WO2014182943, WO2014182945, WO2014182950 and the like, and research and application of ACC inhibitors have been advanced, for example, firsocostat of gillidd is in clinical phase II, but the compounds and test drugs disclosed in the prior art are still unsatisfactory in terms of effectiveness, safety or applicability, and there is still a need to continue research and development of new ACC inhibitors to meet the ever-increasing medical and health needs of people.

Disclosure of Invention

The present inventors have unexpectedly found through experimental studies that a compound of the following formula (I) can effectively inhibit ACC.

Accordingly, in a first aspect, the present invention provides a class of heteroarylopyrimidinone derivatives according to formula (I):

including stereoisomers, tautomers or pharmaceutically acceptable salts thereof,

wherein:

x is selected from-NH-, -O-or-S-; preferably-S-;

ring A is selected from cycloalkyl, and R attached to ring A²And N, not attached to the same carbon atom;

R¹selected from hydrogen atoms, alkyl groups or halogens, wherein said alkyl groups are optionally further substituted by one or more groups selected from halogen, hydroxy, cyano, nitro, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Substituted with the substituent(s);

R²selected from hydrogen atom, hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Wherein said alkyl groupAlkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl optionally further substituted with one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

R³is selected from aryl or heteroaryl, wherein said aryl or heteroaryl is optionally further substituted by one or more groups selected from R⁷Substituted with the substituent(s);

R⁴and R⁵Each independently selected from hydrogen atom, alkyl group, -OR¹⁰、-SR¹⁰、-NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹；

Or, R⁴、R⁵Together with the atoms to which they are attached form a 3-to 8-membered saturated or partially unsaturated cycloalkyl group, or form a cyclic alkyl group having 1 or more members selected from N, O, S (O)_qA 4-to 8-membered saturated or partially unsaturated heterocyclic group of the heteroatom (A); wherein said cycloalkyl or heterocyclyl is optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Substituted with the substituent(s);

R⁶selected from halogen, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹(ii) a Preferably a heteroaryl group;

or, R¹、R⁶Together with the atoms to which they are attached form a 3-to 8-membered saturated or partially unsaturated cycloalkyl group, or form a cyclic alkyl group having 1 or more members selected from N, O, S (O)_qA 4-to 8-membered saturated or partially unsaturated heterocyclic group of the heteroatom(s), or form a 5-to 10-membered aryl or heteroaryl group; wherein said cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more groups selected from hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Substituted with the substituent(s);

R⁷each independently selected from hydroxy, halogen, cyano, nitro, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Substituted with the substituent(s);

R⁸、R⁹and R¹⁰Each independently selected from hydrogen atom, alkyl group, -OR¹³Cyano, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

or, R⁸、R⁹Together with the N atom to which they are attached form a 4-8 membered heterocyclic group in which the 4-8 membered heterocyclic group contains one or more of N, O or S (O)_qAnd said 4-to 8-membered heterocycle is further substituted with one or more substituents selected from the group consisting of hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, ═ O, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

R¹¹、R¹²and R¹³Each independently selected from a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group, wherein the alkyl group, cycloalkyl group, heterocyclyl group, aryl group, or heteroaryl group is optionally further substituted with one or more substituents selected from the group consisting of hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl group, aryl group, heteroaryl group, carboxylic acid, or carboxylic acid ester; and is

q is 0, 1 or 2.

Herein, the compounds of formula (I) (as well as the compounds of formulae (II) to (IV)) also include in scope stereoisomers, tautomers or pharmaceutically acceptable salts thereof.

In some preferred embodiments of the invention, the compound of formula (I) has the structure of formula (II):

wherein:

m is 1,2, 3,4 or 5; and is

Ring A, R¹、R²、R⁶、R⁷And R¹⁰As defined in formula (I).

In some preferred embodiments of the invention, the compound of formula (I) has a specific steric configuration, i.e. has the structure described by formula (III):

wherein:

m is 1,2, 3,4 or 5; and is

Ring A, R¹、R²、R⁶、R⁷And R¹⁰As defined in formula (I).

In some preferred embodiments of the invention, the compound of formula (I) has the structure of formula (IV):

wherein:

m is 1,2, 3,4 or 5; and is

Ring A, R¹、R²、R⁶、R⁷And R¹⁰As defined in formula (I).

In some preferred embodiments of the invention, there are provided compounds of formula (I), (II), (III) or (IV), wherein R¹Selected from methyl or trifluoromethyl.

In some preferred embodiments of the present invention, there are provided compounds of formula (I), (II), (III) or (IV), wherein:

R²selected from tetrazolyl, -C (O) OR¹³or-C (O) NR⁸R⁹；

R⁸Selected from a hydrogen atom or an alkyl group;

R⁹selected from cyano OR-OR¹³；

R¹³Selected from hydrogen atom, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted by one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, arylHeteroaryl, carboxylic acid or carboxylic acid ester;

R²preferably-C (O) OH.

In some preferred embodiments of the present invention, there are provided compounds of formula (I), (II), (III) or (IV), wherein ring a is selected from the group consisting of:

preferably, it is

In some preferred embodiments of the invention, there are provided compounds of formula (I), (II), (III) or (IV), wherein R⁶Selected from 5-membered heteroaryl groups, preferably thiazolyl.

In some preferred embodiments of the invention, there are provided compounds of formula (I), (II), (III) or (IV), wherein R⁷Selected from halogen or alkoxy, preferably methoxy.

In some preferred embodiments of the invention, there are provided compounds of formula (I), (II), (III) or (IV), wherein R¹⁰Is tetrahydropyran-4-yl.

Typical compounds of the invention include, but are not limited to:

the above typical compounds include stereoisomers, tautomers or pharmaceutically acceptable salts thereof.

Further, the present invention provides a process for preparing a compound of formula (I), which process comprises:

reacting a compound of formula (IA) with R⁶(ii) reacting the substituted tributylstannane, optionally further hydrolyzing the resulting compound, and optionally further resolving the optically pure isomer of the resulting compound to obtain the compound of formula (I);

wherein: x¹Selected from halogens; and X, ring A, R¹～R⁶As defined in formula (I).

The present invention provides a compound represented by formula (IA):

wherein:

X¹selected from halogens; and X, ring A, R¹～R⁵As defined in formula (I).

Typical compounds of formula (IA) include, but are not limited to:

Further, the present invention provides a process for the preparation of a compound of formula (IA), said process comprising:

reacting a compound of formula (IB) with a compound of formula (IC) in the presence of triphenylphosphine to give a compound of formula (IA);

wherein:

X¹is selected fromHalogen; and X, ring A, R¹～R⁵As defined in formula (I).

In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I), (II), (III) or (IV), optionally together with a pharmaceutically acceptable carrier, excipient or combination thereof.

In yet another aspect, the present invention provides a method of inhibiting ACC comprising contacting ACC with a compound of formula (I), (II), (III) or (IV) of the present invention or a pharmaceutical composition thereof. The present invention accordingly also provides a method of preventing or treating a disease or condition associated with ACC, comprising administering to a subject in need thereof a compound or pharmaceutical composition according to the present invention.

In another aspect, the present invention provides the use of a compound of formula (I), (II), (III) or (IV), or a pharmaceutical composition thereof, in the manufacture of a medicament for use as an ACC inhibitor.

The invention also provides the use of a compound of formula (I), (II), (III) or (IV) or a pharmaceutical composition thereof, for the manufacture of a medicament for the prevention or treatment of a disease or condition associated with ACC, wherein the disease or condition is preferably a metabolic disease, cancer, fungal, parasitic or bacterial infection, wherein the metabolic disease is preferably hepatic steatosis, non-alcoholic fatty liver disease, obesity, dyslipidemia, hyperlipidemia, type II diabetes or metabolic syndrome, wherein the obesity is preferably Prader-Willi syndrome, bardt-Biedl syndrome or Cohen syndrome or MOMO syndrome, wherein the cancer is preferably hepatocellular carcinoma, non-small cell lung carcinoma, gastric carcinoma, colorectal carcinoma, head and neck tumors, melanoma, Ovarian or cervical cancer, more preferably hepatocellular carcinoma and non-small cell lung cancer.

Detailed description of the invention

Unless stated to the contrary, some of the terms used in the specification and claims of the present invention are defined as follows:

"alkyl" as a group or as a member of a groupWhen partial, is meant to include C₁-C₂₀Straight-chain or branched C₁-C₂₀Aliphatic hydrocarbon group, preferably C₁-C₁₀Alkyl, more preferably C₁-C₆Alkyl, particularly preferably C₁-C₄An alkane. Examples of alkyl groups include, but are not limited to, 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. Alkyl groups may be substituted or unsubstituted.

An "alkylene" is a divalent alkyl group. Preferably C₁-C₁₀Alkylene, more preferably C₁-C₆Alkylene, particularly preferably C₁-C₄An alkylene group. Examples of alkylene groups include, but are not limited to, methylene, ethylene, and,

N-propylene, and the like. The alkylene group may be substituted or unsubstituted.

"alkenyl" means an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, representative examples include but are not limited to ethenyl, 1-propenyl, 2-propenyl, 1-, 2-or 3-butenyl, and the like. Preferably C₂-C₄An alkylene group. The alkenyl group may be optionally substituted or unsubstituted.

"alkynyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing one carbon-carbon triple bond, which may be straight or branched. Preferably selected is C₂-C₁₀Alkynyl, more preferably C₂-C₆Alkynyl, most preferably C₂-C₄Alkynyl. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynylA 2-propynyl group, a 1-, 2-or 3-butynyl group, and the like. Alkynyl groups may be substituted or unsubstituted.

"cycloalkyl" refers to a saturated or partially saturated monocyclic, fused, bridged, or spiro carbocyclic ring. Preferably C₃-C₁₂Cycloalkyl, more preferably C₃-C₈Cycloalkyl, most preferably C₃-C₆A cycloalkyl group. Examples of monocyclic cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, with cyclopropyl, cyclohexenyl being preferred.

"cycloalkylene" is a divalent cycloalkyl group. Preferably C₃-C₁₂Cycloalkylene, more preferably C₃-C₈Cycloalkylene radical, most preferably C₃-C₆Cycloalkylene radicals. Examples of alkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, and the like. Cycloalkylene groups may be substituted or unsubstituted.

"spirocycloalkyl" refers to a 5 to 18 membered polycyclic group containing two or more cyclic structures wherein the individual rings share a common carbon atom (called the spiro atom) with each other, and which may contain 1 or more double bonds within the ring, but none of the rings have a completely conjugated pi-electron aromatic system. Preferably 6 to 14, more preferably 7 to 10. Spirocycloalkyl groups are classified according to the number of spiro atoms shared between rings into mono-spiro, di-spiro, or multi-spiro cycloalkyl groups, preferably mono-spiro and di-spiro cycloalkyl groups, preferably 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered. Non-limiting examples of "spirocycloalkyl" include, but are not limited to: spiro [4.5] decyl, spiro [4.4] nonyl, spiro [3.5] nonyl, spiro [2.4] heptyl.

"fused cycloalkyl" refers to a 5 to 18 membered all carbon polycyclic group containing two or more cyclic structures sharing a pair of carbon atoms with each other, wherein one or more of the rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron aromatic system, preferably 6 to 12, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic fused alkyl groups according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicycloalkyl groups. Non-limiting examples of "fused ring alkyl" include, but are not limited to: bicyclo [3.1.0] hexyl, bicyclo [3.2.0] hept-1-enyl, bicyclo [3.2.0] heptyl, decalinyl or tetradecaphenanthryl.

"bridged cycloalkyl" refers to a 5 to 18 membered all carbon polycyclic group containing two or more cyclic structures sharing two non-directly attached carbon atoms with each other, wherein one or more of the rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron aromatic system. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic bridged cycloalkyl groups according to the number of constituent rings, and are preferably bicyclic, tricyclic or pyridone, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cycloalkyl" groups include, but are not limited to: (1s,4s) -bicyclo [2.2.1] heptyl, bicyclo [3.2.1] octyl, (1s,5s) -bicyclo [3.3.1] nonyl, bicyclo [2.2.2] octyl, and (1r,5r) -bicyclo [3.3.2] decyl.

The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the ring to which the parent structure is attached is cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, and the like. Cycloalkyl groups may be optionally substituted or unsubstituted.

"Heterocyclyl", "heterocycle" or "heterocyclic" are used interchangeably herein and all refer to non-aromatic heterocyclic groups in which one or more of the ring-forming atoms is a heteroatom, such as oxygen, nitrogen, sulfur, and the like, including monocyclic, fused, bridged, and spiro rings. Preferably having a 5 to 7 membered monocyclic ring or a 7 to 10 membered bi-or tricyclic ring, which may contain 1,2 or 3 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1, 1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo [3.2.1] octyl, and piperazinyl. The heterocyclic group may be substituted or unsubstituted.

"Spiroheterocyclyl" means 5 to 18 membered, containing two or twoPolycyclic radicals of the above cyclic structure in which the individual rings share one atom with one another, containing 1 or more double bonds in the ring, but no ring has a completely conjugated pi-electron aromatic system, in which one or more ring atoms are selected from nitrogen, oxygen or S (O)_q(wherein q is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. The spirocycloalkyl group is classified into a single spiroheterocyclic group, a double spiroheterocyclic group or a multiple spiroheterocyclic group, preferably a single spiroheterocyclic group and a double spiroheterocyclic group, according to the number of spiro atoms shared between rings. More preferred are 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered mono spiroheterocyclic groups. Non-limiting examples of "spiroheterocyclyl" include, but are not limited to: 1, 7-dioxaspiro [4.5]]Decyl, 2-oxa-7-azaspiro [4.4]Nonyl, 7-oxaspiro [3.5]]Nonyl and 5-oxaspiro [2.4]]A heptyl group.

"fused heterocyclyl" refers to an all-carbon polycyclic group containing two or more cyclic structures sharing a pair of atoms with each other, wherein one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system, wherein one or more ring atoms is selected from nitrogen, oxygen, or S (O)_q(wherein q is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic fused heterocyclic groups according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of "fused heterocyclic groups" include, but are not limited to: octahydropyrrolo [3,4-c]Pyrrolyl, octahydro-1H-isoindolyl, 3-azabicyclo [3.1.0]Hexyl, octahydrobenzo [ b ]][1,4]Dioxins (dioxines).

"bridged heterocyclyl" means a 5-to 18-membered, preferably 5-to 14-membered, polycyclic radical which contains two or more cyclic structures and which shares two atoms not directly attached to one another, where one or more of the rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron aromatic system, where one or more of the ring atoms is selected from nitrogen, oxygen or S (O)_q(wherein q is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably of between 6 and 14 members in length,more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic bridged heterocyclic groups according to the number of constituent rings, preferably bicyclic, tricyclic or pyridone, more preferably bicyclic or tricyclic. Non-limiting examples of "fused heterocyclic groups" include, but are not limited to: 2-azabicyclo [2.2.1]Heptyl, 2-azabicyclo [2.2.2]Octyl and 2-azabicyclo [3.3.2]A decyl group. The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is heterocyclyl. The heterocyclic group may be optionally substituted or unsubstituted.

"Heterocyclylene" refers to a divalent heterocyclic radical. Preferably with a 5 to 7 membered monocyclic heterocyclylene or a 7 to 10 membered bicyclic or tricyclic heterocyclylene, which may contain 1,2 or 3 atoms selected from nitrogen, oxygen and/or sulfur. Heterocyclylene groups may be substituted or unsubstituted.

"aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be joined together in a fused fashion. The term "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl. Preferably aryl is C₆-C₁₀Aryl, more preferably aryl is phenyl and naphthyl, most preferably phenyl. The aryl group may be substituted or unsubstituted. The "aryl" may be fused to a heteroaryl, heterocyclyl or cycloalkyl group, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples include, but are not limited to:

"heteroaryl" refers to an aromatic 5-to 6-membered monocyclic or 9-to 10-membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heteroaryl" include, but are not limited to, furyl, pyridyl, 2-oxo-1, 2-dihydropyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2, 3-thiadiazolyl, benzodioxolyl, benzimidazolyl, indolyl, isoindolyl, 1, 3-dioxo-isoindolyl, quinolinyl, indazolyl, benzisothiazolyl, benzoxazolyl, and benzisoxazolyl. Heteroaryl groups may be substituted or unsubstituted. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples include, but are not limited to:

"alkoxy" refers to a radical of (alkyl-O-). Wherein alkyl is as defined herein. C₁-C₆Alkoxy of (2) is preferred, with C being especially preferred₁-C₄An alkoxy group. Examples include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.

"hydroxy" refers to an-OH group.

"halogen" means fluorine, chlorine, bromine and iodine, preferably chlorine, bromine and iodine.

"amino" means-NH₂。

"cyano" means-CN.

"nitro" means-NO₂。

"benzyl" means-CH₂-phenyl.

"carboxy" refers to-C (O) OH.

"carboxylate" refers to-C (O) O (alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

"DMSO" refers to dimethyl sulfoxide.

"Et" refers to ethyl.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable in combination with carbon atoms having unsaturated bonds (e.g., olefinic bonds).

As used herein, "substituted" or "substituted," unless otherwise specified, means that the group may be substituted with one or more groups selected from: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, ═ O, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Substituted with the substituent(s);

"pharmaceutically acceptable salts" refers to certain salts of the above compounds which retain their biological activity and are suitable for pharmaceutical use. The pharmaceutically acceptable salts of the compounds of formula (I) may be metal salts, preferably alkali metal, alkaline earth metal salts, with suitable acids, including inorganic and organic acids, for example acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, malic acid, maleic acid, mandelic acid, methanesulfonic acid, nitric acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid and the like. Particularly preferred are hydrochloric acid, hydrobromic acid, phosphoric acid and sulfuric acid, with the hydrochloride salt being most preferred.

"pharmaceutical composition" means a mixture containing one or more compounds described herein (including pharmaceutically acceptable salts or stereoisomers, tautomers or prodrugs thereof, and the like) and optionally other pharmaceutically active ingredients, which may contain other optional ingredients such as pharmaceutically acceptable carriers and/or excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

Herein, the term "plurality" includes two or more, such as two, three, four, etc.

Synthesis of the Compounds of the invention

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

the invention relates to a preparation method of a compound shown as a formula (I), which comprises the following steps:

reacting a compound of formula (IA) with R⁶(ii) reacting the substituted tributylstannane such that the resulting compound is optionally further hydrolyzed, and the resulting compound is optionally further resolved into optically pure isomers to yield the compound of formula (I);

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

The examples show the preparation of representative compounds represented by formula (I) and the associated structural identification data. It must be noted that the following examples are intended to illustrate the invention and are not intended to limit the invention.¹The H NMR spectrum was obtained using a Bruker instrument (400MHz) and the chemical shifts were expressed in ppm. Tetramethylsilane internal standard (0.00ppm) was used.¹Method for H NMR expression: s is singlet, d is doublet, t is triplet, q is quartet, m is multiplet, br is broadened, dd is doublet of doublet, dt is doublet of triplet. If a coupling constant is provided, it is in Hz.

The mass spectrum is measured by an LC/MS instrument, and the ionization mode can be ESI or APCI.

The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

In the following examples, unless otherwise indicated, all temperatures are in degrees Celsius and unless otherwise indicated, the various starting materials and reagents are commercially available or synthesized according to known methods, and none of the commercially available materials and reagents are used without further purification, unless otherwise indicated, commercially available manufacturers include, but are not limited to, Aldrich Chemical Company, ABCR GmbH & Co. KG, Acros Organics, Sciadopsis Tech, and the like.

CD₃OD: deuterated methanol.

CDCl₃: deuterated chloroform.

DMSO-d₆: deuterated dimethyl sulfoxide.

The argon atmosphere means that the reaction flask is connected with an argon balloon having a volume of about 1L.

In the examples, the solution in the reaction is an aqueous solution unless otherwise specified.

Purifying the compound by silica gel column chromatography and silica gel thin-plate chromatography, wherein the developing agent or eluent system is selected from: a: petroleum ether and ethyl acetate systems; b: dichloromethane and methanol systems; c: dichloromethane: ethyl acetate; the volume ratio of the solvent is different according to the polarity of the compound, and a small amount of acidic or basic reagent such as acetic acid or triethylamine can be added for adjustment.

Example 1

(1R,3R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid

First step of

2- (2-methoxyphenyl) oxirane

2-methoxybenzaldehyde 1a (20.0g, 146.9mmol) was dissolved in 100mL of dimethyl sulfoxide, and tert-butylthiohypoiodate (36.0g, 173.3mmol) and sodium hydroxide (24.7g, 441.0mmol) were added in this order, and the mixture was heated to 80 ℃ for reaction for 1.5 hours. The reaction was cooled to room temperature, 200mL of water was added, extraction was performed with petroleum ether (200 mL. times.3), the organic phases were combined, washed with saturated sodium chloride solution (200mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: System A) to give 2- (2-methoxyphenyl) oxirane 1b (13.1g, colorless oil) in yield: 57 percent.

¹H NMR(400MHz,CDCl₃)δ7.27(t,J＝1.2Hz,1H),7.17(d,J＝7.6Hz,1H),6.98(t,J＝1.2Hz,1H),6.89(d,J＝7.6Hz,1H),4.22(t,J＝0.4Hz,1H),3.87(s,3H),2.71(dd, J＝5.6,2.4Hz,1H)3.14(dd,J＝5.6,2.4Hz,1H).

Second step of

2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethanol

(2-methoxyphenyl) oxirane 1b (26.0g, 173.0mmol) was added to stirred tetrahydro-2H-pyran-4-ol 1c (53.1g, 519.7mmol) and aluminum triflate (4.10g, 8.65mmol) and reacted at room temperature for 3 hours. To the reaction solution were added 200mL of dichloromethane and 200mL of water, the organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: system a) to give 2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethanol 1d (13.0g, white solid), yield: 30 percent.

¹H NMR(400MHz,CDCl₃)δ7.42(d,J＝8.0Hz,1H),7.26(t,J＝7.2Hz,1H),6.98(t,J＝7.2Hz,1H),6.87(d,J＝8.0Hz,1H),5.07(dd,J＝8.0,4.0Hz,1H),3.87-4.00(m,2H),3.83(s,3H),3.62-3.72(m,1H),3.46-3.58(m,2H),3.32-3.43(m,2H),2.35-2.37(m,1H),1.99-2.03(m,1H),1.77-1.80(m,1H),1.60-1.70(m,2H).

The third step

2- (tributylstannyl) oxazole

Oxazole 1e (500mg, 7.24mmol) was dissolved in 12mL of tetrahydrofuran. Under nitrogen, the mixture was cooled to-78 ℃ and stirred for 5 minutes, n-butyllithium (4.56mL, 7.29mmol) was slowly added thereto, and after completion of the addition, the mixture was stirred at-78 ℃ for 30 minutes. Tributyltin chloride (1.96mL, 7.24mmol) was then added, stirred at-78 ℃ for 10 minutes, and allowed to warm to room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, 15mL of n-hexane was added to the residue, filtered, and the filtrate was concentrated under reduced pressure to give 2- (tributylstannyl) oxazole 1f (1.8g, pale yellow liquid), yield: 70 percent.

¹H NMR(400MHz,CDCl3):7.84(1H,s),7.18(1H,s),1.67-1.53(6H,m)，1.42-1.29(6H,m),1.20(6H,m),0.89(9H,t,J＝7Hz).

The fourth step

2-amino-4-methylthiophene-3-carboxylic acid ethyl ester

Under the protection of argon, 1g (185.5g,1.64mol) of ethyl 2-cyanoacetate, 100g (1.72 mol) of acetone and 53g (1.64 mol) of sulfur were dissolved in 500mL of absolute ethanol, and morpholine was slowly added dropwise over 1h (149.6g,1.64mol) over 20 minutes. The reaction was carried out at 45 ℃ for 10 hours. The reaction solution was cooled to room temperature and filtered to remove the remaining sulfur powder. The filtrate was concentrated under reduced pressure, 900mL of 75% ethanol was added, and the mixture was stirred at room temperature for 30 minutes. A yellow solid precipitated, and was filtered to remove the solid, the filtrate was concentrated under reduced pressure, 600mL of 40% ethanol was added to the residue, stirred at 70 ℃ for 30 minutes, naturally cooled to gradually precipitate the solid, and filtered to obtain ethyl 2-amino-4-methylthiophene-3-carboxylate 1i (95g, green solid), yield: 31.4 percent.

MS m/z(ESI)：185.9[M+1]

The fifth step

3-Oxocyclobutane-1-carboxylic acid tert-butyl ester

3-Oxocyclobutane-1-carboxylic acid 1j (5.7g,50mmol), tert-butanol (9.25g,125mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (11.5g,60mmol) and 4-dimethylaminopyridine (3.1g,25mmol) were dissolved in 130mL of dichloromethane and stirred at room temperature for 12 hours. The reaction solution was diluted with 100mL of dichloromethane, washed with water (100mL), 1N aqueous hydrochloric acid (50mL) and saturated aqueous sodium chloride (50mL) in this order, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give tert-butyl 3-oxocyclobutane-1-carboxylate 1k (8.5g, red brown oil), yield: 100 percent.

The sixth step

(1S,3S) -3-Hydroxycyclobutane-1-carboxylic acid tert-butyl ester

Tert-butyl 3-oxocyclobutane-1-carboxylate 1k (6.5g, 38.2mmol) was dissolved in 72mL of tetrahydrofuran/methanol (V: V ═ 8:1), and sodium borohydride (0.71g,19.1mmol) was added portionwise at 0 ℃ and reacted at room temperature for one hour. 80mL of a saturated aqueous potassium carbonate solution was added, extraction was performed with ethyl acetate (100 mL. times.3), the organic phases were combined, washed with a saturated aqueous sodium chloride solution (80mL), the aqueous layer was separated, the organic phase was dried over anhydrous sodium sulfate, and concentration was performed under reduced pressure to obtain 1l (6.58g, viscous solid) of tert-butyl (1S,3S) -3-hydroxycyclobutane-1-carboxylate, yield: 100 percent;

seventh step

(1R,3R) -3- (1, 3-dioxoisoindolin-2-yl) cyclobutane-1-carboxylic acid tert-butyl ester

1l (4.9g,28.45mmol) of tert-butyl (1S,3S) -3-hydroxycyclobutane-1-carboxylate, 1m (4.6g,31.3mmol) of isoindoline-1, 3-dione, and triphenylphosphine (11.2g,42.1mmol) were dissolved in 80mL of tetrahydrofuran, and azodiisopropyldicarboxylic acid (8.6g,42.7mmol) was added with stirring and reacted at room temperature for 2 hours. Concentration under reduced pressure, dissolution of the residue in 20mL of ethyl acetate, addition of 100mL of petroleum ether, precipitation of a large amount of triphenoxyphos, filtration to remove the triphenoxyphos, concentration of the filtrate under reduced pressure, purification of the residue by silica gel column chromatography (eluent: System A) to give (1R,3R) -3- (1, 3-dioxoisoindolin-2-yl) cyclobutane-1-carboxylic acid tert-butyl ester 1n (7.5g, white solid), yield: 87.5 percent.

Eighth step

(1R,3R) -3-Aminocyclobutane-1-carboxylic acid tert-butyl ester

Tert-butyl (1R,3R) -3- (1, 3-dioxoisoindolin-2-yl) cyclobutane-1-carboxylate 1n (4.5g, 14.9mmol) was dissolved in 45mL of ethanol, hydrazine hydrate (2.2g,44.8mmol) was added, and the reaction was carried out at room temperature for 12 hours. Filtration, washing of the filter cake with ethanol (10 mL. times.2), concentration of the filtrate under reduced pressure, and purification of the residue by silica gel column chromatography (eluent: System A) gave (1R,3R) -3-aminocyclobutane-1-carboxylic acid tert-butyl ester 1o (1.2g, colorless oil), yield: 66.4 percent.

The ninth step

2- (3- (1R,3R) -3- (tert-butoxycarbonyl) cyclobutyl) ureido) -4-methylthiophene-3-carboxylic acid ethyl ester

Ethyl 2-amino-4-methylthiophene-3-carboxylate 1i (1.44g, 7.8mmol) was dissolved in 60mL of methylene chloride, and a solution of bis (trichloromethyl) carbonate (810mg,2.7mmol) in methylene chloride (10mL) was added dropwise at 0 ℃ and stirred at room temperature for 30 minutes. Triethylamine (2.4g,23.4mmol) was added at 0 ℃ and stirred at room temperature for 30 minutes. (1R,3R) -3-Aminocyclobutane-1-carboxylic acid tert-butyl ester 1o (1.3g, 7.8mmol) was added at 0 ℃ and reacted at room temperature for 30 minutes. The reaction solution was diluted with 100mL of dichloromethane, washed with saturated aqueous sodium chloride solution (50mL), the aqueous layer was separated, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: System A) to give ethyl 2- (3- (1R,3R) -3- (tert-butoxycarbonyl) cyclobutyl) ureido) -4-methylthiophene-3-carboxylate 1p (2.5g, white solid) in yield: 83 percent.

MS m/z(ESI)：383.0[M+1]

The tenth step

(1R,3R) -3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid tert-butyl ester

Ethyl 2- (3- (1R,3R) -3- (tert-butoxycarbonyl) cyclobutyl) ureido) -4-methylthiophene-3-carboxylate 1p (250mg,0.65mmol) and cesium carbonate (426mg,1.31mmol) were dissolved in 3ml N, N-dimethylformamide and reacted at 100 ℃ for 2 hours. The reaction solution was diluted with 50mL of ethyl acetate, the organic phase was washed with water (10 mL. times.3) and saturated aqueous sodium chloride (10mL) successively, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: System A) to give (1R,3R) -3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid tert-butyl ester 1q (200mg, white solid), yield: 91.3 percent.

MS m/z(ESI)：280.9[M-55]

The eleventh step

(1R,3R) -3- (6-bromo-5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid tert-butyl ester

Tert-butyl (1R,3R) -3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylate 1q (2.2g,6.54mmol) was dissolved in 50mL of dichloromethane, N-bromosuccinimide (1.4g,7.85mmol) was added at 0 ℃ and reacted at 0 ℃ for 30 minutes. The reaction solution was diluted with 200mL of dichloromethane, washed with saturated aqueous sodium chloride solution (50mL), the aqueous layer was separated, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: System A) to give (1R,3R) -3- (6-bromo-5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid tert-butyl ester 1R (2.5g, white solid), yield: 92.0 percent.

MS m/z(ESI)：358.8[M-55]

The twelfth step

(1R,3R) -3- (6-bromo-1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid tert-butyl ester

Under nitrogen protection, (1R,3R) -3- (6-bromo-5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid tert-butyl ester 1R (723mg,1.74mmol), 2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethanol 1d (834mg,3.30mmol) and triphenylphosphine (912mg,3.48mmol) were dissolved in 20mL anhydrous tetrahydrofuran. After cooling to 0 ℃ and stirring for 3 minutes, a solution of diisopropyl azodicarboxylate (0.69mL,3.48mmol) in 4mL of tetrahydrofuran was added, and the mixture was allowed to warm to room temperature and reacted for 18 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: system a) to give tert-butyl (1R,3R) -3- (6-bromo-1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylate 1s (986mg, white solid), yield: 87 percent.

MS m/z(ESI)：649.8[M+1]

Thirteenth step

(1R,3R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid tert-butyl ester

Under nitrogen protection, (1R,3R) -3- (6-bromo-1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid tert-butyl ester 1s (986mg, 1.52mmol), 2- (tributylstannyl) oxazole 1f (817mg, 2.28mmol) and tetratriphenylphosphine palladium (245mg, 0.21mmol) were dissolved in 12mL toluene and heated to 110 ℃ for 7 hours. The reaction was cooled to room temperature, 40mL of water was added, extraction was performed with ethyl acetate (15mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: system a) to give tert-butyl (1R,3R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylate 1t (756mg, light yellow solid), yield: 78 percent.

MS m/z(ESI)：638.8[M+1]

Fourteenth step

Tert-butyl (1R,3R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylate 1t (756mg, 1.18mmol) was dissolved in 10mL dichloromethane. After 2mL of trifluoroacetic acid was added dropwise at 0 ℃ and the reaction was allowed to warm to room temperature for 3 hours. To the reaction solution was added 25mL of water, extracted with ethyl acetate (10mL × 3), washed with water (30mL × 4), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel thin-plate chromatography (developing agent: system B) to give (1R,3R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid 1(185mg, white solid), yield: 27 percent. MS m/z (ESI): 582.2[ M +1]

¹H NMR(400MHz,DMSO)δ12.31(s,1H),8.23(s,1H),7.49(d,J＝7.4Hz,1H),7.39(s,1H),7.29(t,J＝7.4Hz,1H),7.13-6.89(m,2H),5.53(m,1H),5.31(m,1H),4.02(m,2H),3.78(s,3H),3.55(m,2H),3.36(m,1H),3.24(m,2H),3.07(m,3H),2.78(s,3H),2.41(s,2H),1.63(s,2H),1.42-1.12(m,2H).

Example 2 and example 3

(1R,3R) -3- (1- ((S) -2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid

(1R,3R) -3- (1- ((R) -2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid

First step of

Mixing (1R,3R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazole-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2, 3-d)]Pyrimidin-3 (4H) -yl) cyclobutane1-Carboxylic acid 1(120mg,0.60mmol) the chiral isomers were resolved by preparative chromatography and a chiral column using Supercritical Fluid Chromatography (SFC) (chiral column ChiralPak AS, 250X 30mm I.D.,5 μm; 70 mL/min; mobile phase A (for CO)₂) And B (for methanol) (0.1% NH)₃.H₂O)) to give (1R,3R) -3- (1- ((S) -2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2, 3-d)]Pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid 2(56.3mg, white solid), yield: 46.9 percent, the retention time of 5.289min and the ee value of 99.7 percent; (1R,3R) -3- (1- ((R) -2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2, 3-d)]Pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid 3(63.3mg, white solid), yield: 52.28%, retention time 4.505min, ee value 99.8%.

Compound 2

MS m/z(ESI)：582.2[M+1]

Compound 3

MS m/z(ESI)：582.2[M+1]

Example 4

(1S,3S) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid

First step of

3-Oxocyclobutane-1-carboxylic acid ethyl ester

3-Oxocyclobutane-1-carboxylic acid 1j (4.56g,40mmol), ethanol (2.3g,50mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (9.2g,48mmol) and 4-dimethylaminopyridine (2.4g,20 mmol) were dissolved in 100mL of dichloromethane and stirred at room temperature for 12 hours. The reaction mixture was diluted with 100mL of dichloromethane, washed with water (150mL), 1N aqueous hydrochloric acid (50mL) and saturated aqueous sodium chloride (100mL) in this order, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give ethyl 3-oxocyclobutane-1-carboxylate 4a (5.5g, colorless oil) in yield: 96.7 percent.

Second step of

(1S,3S) -3- (dibenzylamino) cyclobutane-1-carboxylic acid ethyl ester

Ethyl 3-oxocyclobutane-1-carboxylate 4a (5.5g,40mmol) and dibenzylamine (8.58g,44mmol) were dissolved in 180mL of tetrahydrofuran and stirred at room temperature for 1 hour. Sodium triacetoxyborohydride (17.1g,80mmol) and 20mL of acetic acid were added and reacted at room temperature for 12 hours. The tetrahydrofuran was removed under reduced pressure, a saturated aqueous sodium bicarbonate solution was added, the pH was adjusted to be basic, extraction was performed with ethyl acetate (50mL × 3), the organic phases were combined, washed with 50mL of a saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the obtained residue was purified by silica gel thin-plate chromatography (developer: system a) to give (1S,3S) -ethyl 3- (dibenzylamino) cyclobutane-1-carboxylate 4b (6.0g, colorless oil), yield: 46.5 percent.

The third step

(1S,3S) -3-Aminocyclobutane-1-carboxylic acid ethyl ester

Ethyl (1S,3S) -3- (dibenzylamino) cyclobutane-1-carboxylate 4b (5.0g,15.5mmol) was dissolved in 150mL of ethanol, and 10% Pd-C (200mg, 4%) was added to replace hydrogen gas 3 times, followed by reaction at room temperature for 12 hours. Filtration to remove the remaining Pd-C and concentration of the filtrate under reduced pressure gave (1S,3S) -3-aminocyclobutane-1-carboxylic acid ethyl ester 4C (2.1g, colorless oil), yield: 95.5 percent.

The fourth step

2- (3- ((1S,3S) -3- (ethoxycarbonyl) cyclobutyl) ureido) -4-methylthiophene-3-carboxylic acid ethyl ester

Ethyl 2-amino-4-methylthiophene-3-carboxylate 1i (2.78g, 15.0mmol) was dissolved in 100mL of methylene chloride, and a solution of bis (trichloromethyl) carbonate (1.56g,5.25mmol) in methylene chloride (20mL) was added dropwise at 0 ℃ and stirred at room temperature for 30 minutes. Triethylamine (4.5g,45mmol) was added at 0 ℃ and stirred at room temperature for 30 minutes. Ethyl (1S,3S) -3-aminocyclobutane-1-carboxylate 4c (2.2g, 15.5mmol) was added at 0 ℃ and reacted at room temperature for 30 minutes. The reaction solution was diluted with 100mL of dichloromethane, washed with saturated aqueous sodium chloride solution (50mL), the aqueous layer was separated, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: System A) to give ethyl 2- (3- ((1S,3S) -3- (ethoxycarbonyl) cyclobutyl) ureido) -4-methylthiophene-3-carboxylate 4d (4.7g, pale yellow solid), yield: 89 percent.

MS m/z(ESI)：354.9[M+1]

The fifth step

(1S,3S) -3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid ethyl ester

Ethyl 2- (3- ((1S,3S) -3- (ethoxycarbonyl) cyclobutyl) ureido) -4-methylthiophene-3-carboxylate 4d (4.7g,13.3mmol) and cesium carbonate (8.67g,26.6mmol) were dissolved in 30mLN, N-dimethylformamide and reacted at 100 ℃ for 1 hour. The reaction solution was diluted with 150mL of ethyl acetate, washed successively with water (50 mL. times.3) and saturated aqueous sodium chloride (50 mL. times.1), the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: System A) to give (1S,3S) -3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid ethyl ester 4e (2.0g, pale yellow solid), yield: 49.0 percent.

MS m/z(ESI)：308.9[M+1]

The sixth step

(1S,3S) -3- (6-bromo-5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid ethyl ester

Ethyl (1S,3S) -3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylate 4e (2.0g,6.5mmol) was dissolved in 65mL of dichloromethane, N-bromosuccinimide (1.2g,6.5mmol) was added at 0 ℃ and reacted at 0 ℃ for 30 minutes. The reaction solution was diluted with 200mL of dichloromethane, washed with saturated aqueous sodium chloride solution (50mL), the aqueous layer was separated, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: System A) to give (1S,3S) -3- (6-bromo-5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid ethyl ester 4f (2.0g, white solid), yield: 80.0 percent.

MS m/z(ESI)：386.8[M+1]

Seventh step

(1S,3S) -3- (6-bromo-1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid ethyl ester

Under nitrogen, (1S,3S) -ethyl 3- (6-bromo-5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylate 4f (2.0mg, 5.2mmol), 2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethanol 1d (1.52g, 6.0mmol) and triphenylphosphine (1.58g, 6.0mmol) were dissolved in 25mL anhydrous tetrahydrofuran. After cooling to 0 ℃ and stirring for 3 minutes, a solution of diisopropyl azodicarboxylate (1.2g, 6.0mmol) in 4mL of tetrahydrofuran was added, and the mixture was allowed to warm to room temperature for reaction for 18 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: system a) to give 4g (2.9g, white solid) of ethyl (1S,3S) -3- (6-bromo-1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylate, yield: 90 percent.

MS m/z(ESI)：621.2[M+1]

Eighth step

(1S,3S) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid ethyl ester

Under nitrogen protection, (1S,3S) -3- (6-bromo-1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid ethyl ester 4g (1.24g, 2.0mmol), 2- (tributylstannyl) oxazole 1f (1.4g, 3mmol) and tetratriphenylphosphine palladium (323.5mg, 0.28mmol) were dissolved in 20mL toluene. The reaction was heated to 110 ℃ for 7 hours. The reaction was cooled to room temperature, 60mL of water was added, extraction was performed with ethyl acetate (30mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: system a) to give ethyl (1S,3S) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylate 4H (936mg, light yellow solid), yield: 78 percent.

MS m/z(ESI)：609.9[M+1]

The ninth step

Ethyl (1S,3S) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylate 4H (936mg, 1.5mmol) and lithium hydroxide (108mg,4.5mmol) were dissolved in 10mL tetrahydrofuran/methanol (V: V ═ 1: 1). The reaction was carried out at room temperature for 12 hours. Concentrated under reduced pressure and the resulting residue was purified by silica gel thin-plate chromatography (developer: system B) to give (1S,3S) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid 4(760mg, white solid), yield: 87.3 percent.

MS m/z(ESI)：582.2[M+1]

¹H NMR(400MHz,DMSO)δ12.17(s,1H),8.22(s,1H),7.49(d,J＝6.7Hz,1H),7.39(s,1H),7.30(t,J＝7.2Hz,1H),7.10-6.92(m,2H),5.33-5.26(m,1H),5.04(m, 1H),4.16-3.89(m,2H),3.78(s,3H),3.56(m,2H),3.43-3.35(m,1H),3.24(m,2H),3.00-2.81(m,3H),2.77(s,3H),2.46(m,2H),1.63(s,2H),1.34-1.13(m,2H).

Examples 5 and 6

(1S,3S) -3- (1- ((S) -2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid

(1S,3S) -3- (1- ((R) -2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid

First step of

Mixing (1S,3S) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazole-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2, 3-d)]Pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid 4(130mg,0.23mmol) was prepared by resolving chiral isomers using preparative chromatography and a chiral column using Supercritical Fluid Chromatography (SFC) (chiral column ChiralCel OJ,250 x 30mm i.d.,5 μm; 70 mL/min; mobile phase A (for CO)₂) And B (for methanol) (0.1% NH)₃.H₂O)) to give (1S,3S) -3- (1- ((S) -2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2, 3-d)]Pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid 5(59.12mg, white solid), yield: 45.48%, retention time 3.736min, ee value 100%; (1S,3S) -3- (1- ((R) -2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran)-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d]Pyrimidin-3 (4H) -yl) cyclobutane-1-carboxylic acid 6(68.73mg, white solid), yield: 52.87 percent, the retention time is 4.066min, and the ee value is 99.8 percent.

Compound 5

MS m/z(ESI)：582.2[M+1]

¹H NMR(400MHz,DMSO)δ12.17(s,1H),8.22(s,1H),7.49(d,J＝6.7Hz,1H),7.39(s,1H),7.30(t,J＝7.2Hz,1H),7.10-6.92(m,2H),5.33-5.26(m,1H),5.04(m,1H),4.16-3.89(m,2H),3.78(s,3H),3.56(m,2H),3.43-3.35(m,1H),3.24(m,2H),3.00-2.81(m,3H),2.77(s,3H),2.46(m,2H),1.63(s,2H),1.34-1.13(m,2H).

Compound 6

MS m/z(ESI)：582.2[M+1]

Biological evaluation

Test example 1 inhibition of enzymatic Activity IC of Compounds of the present invention against ACC1 and ACC2₅₀Measurement of (2)

The following methods were used to determine the extent of inhibition of the enzymatic activity of recombinant human ACC1, ACC2 protein by preferred compounds of the invention under in vitro conditions.

The principle of the method is based on the reaction of catalyzing acetyl-CoA to generate malonyl-CoA by ACC protein. ATP is consumed and ADP is produced during the reaction. By using ADP-Glo from Promega (Promega)^TMThe kinase kit can convert ADP generated by the reaction into ATP again, and the ATP can react with luciferase-luciferin in the kit to generate a chemiluminescent signal. Thus, by measuring the intensity of the chemiluminescent signal, the amount of ADP produced in the catalyzed reaction can be reflected, thereby indirectly determining the enzymatic activity of the ACC protein and the effect of the test compound on enzymatic activity. The main reagents used included: ACC1, ACC2 protein (purchased from BPS)bioscience, ACC1 cat # 50200, ACC2 cat # 50201), acetyl-CoA (acetyl-CoA, available from Sigma, cat # A2056), NaHCO3 (available from Sigma, cat # S6014), ADP-Glo^TMKinase assay kit (available from Promega, cat # V9102).

The test procedure is briefly described as follows: first, 1 × buffer solution required for reaction is prepared, and the composition of the buffer solution is as follows: 50mM HEPES (pH7.4 from Invitrogen, cat. No. 15630), 2mM magnesium chloride (MgCl)₂Purchased from Sigma, cat # M1028), 2mM Potassium citrate (Potassium citrate, purchased from Sigma, cat # 89306), 0.01% Brij-35 detergent (purchased from Merck, cat # 203728), 2mM DTT (purchased from Sigma, cat # D0632). The test compound powders were dissolved in DMSO to make up a 10mM stock solution, and then sequentially diluted 3-fold to make up the concentration required for the test, with 10 concentration points for each compound, in the range of 10. mu.M-0.5 nM. Firstly, a proper amount of ACC protein (2nM) is added into a 384-well microplate, and then diluted test compound solutions with different concentrations are added into each well, wherein each concentration is provided with a multi-well control, and simultaneously, a solvent control (blank group) and a negative control group (DMSO group) are arranged. The 384-well plate was then shaken well on a microplate shaker and incubated at room temperature for 15 minutes. Then, a substrate mixture containing ATP, acetyl-CoA and NaHCO3 diluted with the aforementioned buffer was added to each well to start the reaction, and the final concentrations of the three components were ATP 20. mu.M, acetyl-CoA 10. mu.M and NaHCO 330 mM, respectively. After 30 minutes reaction at room temperature, according to ADP-Glo^TMThe method in the kit instruction book of Kinase assay kit is to add the corresponding reaction solution and detection solution to each well (the specific operation method can refer to the kit instruction book), and finally, the Relative Light Unit (RLU) value of each well is measured on an Envision 2104 multifunctional microplate reader (Perkin Elmer). The percent inhibition of accase activity by a compound at a certain concentration is calculated according to the following formula:

percent inhibition [% mean negative control wells RLU-mean blank wells RLU- ] -mean test wells RLU-mean blank wells RLU) ]/(mean negative control wells RLU-mean blank wells RLU) × 100

Finally, the concentration log and phase of the compound were measured in GraphPad Prism5 softwareNon-linear regression analysis of percent inhibition in concentration to obtain the half inhibitory concentration value (IC) of the compound₅₀)。

TABLE 1 IC inhibition of ACC1 and ACC2 enzymatic activities by compounds of the invention₅₀Data of

Example numbering	IC ₅₀(nM)/ACC1	IC ₅₀(nM)/ACC2
1	1.3	ND
3	1.5	4.0
4	0.9	ND
5	ND	2.0
6	0.5	ND

Remarking: ND means not measured.

And (4) conclusion: the compound has better inhibition effect on ACC1 enzyme and ACC2 enzyme.

Test example 2, Compound of the present invention [ p ], [¹⁴C]Acetate incorporation into HepG2 cell inhibitory activity assay

The following method is used to determine the presence of the compound of the present invention under in vitro conditions¹⁴C]Degree of cellular inhibition of acetate incorporation into HepG 2.

1. Reagent and apparatus

1.1. Reagent and consumable

TABLE 2 reagents and consumables

TABLE 3 reagents and consumables

Reagent	Manufacturer(s)	Reagent
Sodium hydroxide	Tianjin chemical reagent factory	Sodium hydroxide
Potassium hydroxide	Beijing Ministry of precision chemical products (Limited liability Co., Ltd.)	Potassium hydroxide
Acetic acid	Tianjin City Guang Compound technology development Co Ltd	Acetic acid
Trichloromethane	Beijing chemical plant	Trichloromethane
Ether (A)	Fine chemical reagent factory of Tianjin City Jindongtianzheng	Ether (A)
Petroleum ether	Fine chemical reagent factory of Tianjin City Jindongtianzheng	Petroleum ether
Hydrochloric acid	Beijing Xingqinghong fine chemicals science and technology limited	Hydrochloric acid
N-hexane	Beijing chemical plant	N-hexane

N-heptane	Beijing chemical plant	N-heptane
Anhydrous ethanol	Beijing chemical plant	Anhydrous ethanol

1.2. Instrument for measuring the position of a moving object

TABLE 4 Instrument

Instrument for measuring the position of a moving object	Manufacturer(s)	Goods number
Biological safety cabinet	Thermo Scientific	1300 Series A2
Centrifugal machine	Eppendorf	5702
Carbon dioxide incubator	Thermo Scientific	1300 SERIES A2
Cell counter	Invitrogen	C10281
Liquid-transfering gun	BIOHIT	Easypet
Microscope	Olympus	CKX41
Liquid transfer device	BIOHIT	Proline Plus
Vortex oscillation instrument	IKA	MS3 basic
MicroBeta	PerkinElmer	2450

1.3 preparation of the Compounds of the invention

The compounds of the invention to be tested were dissolved in DMSO at 10mM and stored at 4 ℃ before use.

2. Experimental procedure

2.1. Cell culture

HepG2 cells were purchased from American Type Culture Collection (ATCC) resource stocks. The cells were cultured in DMEM containing 10% fetal bovine serum, penicillin (100units/mL) and streptomycin (100. mu.g/mL) and incubated in an incubator containing 5% carbon dioxide at 37 ℃ for passage every 2 to 3 days.

2.2.[2-¹⁴C]Assay for Acetate uptake

(1) On the first day, HepG2 cells were seeded at 2X 105 cells per well in a 24-well plate and incubated in an incubator at 37 ℃ containing 5% carbon dioxide.

(2) On day four, the medium containing the compound was changed. The compounds of the invention were initially at 3. mu.M, diluted 4-fold, and incubated in 5 concentration gradients with a final DMSO concentration of 0.5% (v/v) for 1 hour at 37 ℃ in an incubator containing 5% carbon dioxide.

(3) 2 μ Ci 2-¹⁴C]Acetate, incubated for a further 5 hours in an incubator at 37 ℃ containing 5% carbon dioxide.

(4) The medium was transferred to a 15mL centrifuge tube, 0.5mL of 0.1M NaOH was added to each well, and the cell lysate was transferred to the corresponding 15mL centrifuge tube.

(5) 1mL of ethanol and 0.17mL of 50% KOH were added to each tube, and the tube was incubated in a water bath at 90 ℃ for 1 hour.

(6) The sample was removed, after cooling to room temperature 5mL of petroleum ether was added to each tube, inverted several times and centrifuged at 1000rpm for 5 minutes. The upper organic phase was discarded and the aqueous phase was retained for fatty acid extraction.

(7) 1mL of concentrated HCl was added to each tube (to ensure that the pH was below 1).

(8) 5mL of petroleum ether was added to each tube, inverted several times, 1000rpm, centrifuged for 5 minutes, and a 4mL petroleum ether layer was transferred to a new glass tube (18X 180 mm).

(9) And (5) repeating the step (8).

10) The pooled extracts were evaporated overnight in a 64 ℃ water bath.

(11) On the fifth day, the fatty acid was dissolved in 240. mu.L of chloroform/n-hexane (1:1) containing 200. mu.g of linoleic acid.

(12) 10 μ L of the solution was spotted in a silica gel plate and washed in n-heptane: diethyl ether: carrying out chromatography for 10 minutes in a mixed solution of acetic acid (the volume ratio is 90:30: 1).

(13) The fatty acid bands were visualized with iodine vapor and cut into scintillation vials, and 2mL of ULTIMA GOLD was added and incubated for 10 minutes at room temperature.

14) Scintillation signals were recorded with MicroBeta.

Nonlinear regression analysis of the concentration of the compound and scintillation signal at the corresponding concentration in GraphPad Prism5 software to obtain the half maximal inhibitory concentration value (IC) of the compound₅₀)。

TABLE 5 Compound of the present invention pairs¹⁴C]IC of acetate incorporation into HepG2 cell inhibition₅₀Value of

Example numbering	IC ₅₀(nM)
Firsocostat (control compound)	50
3	6.25
6	19.8

And (4) conclusion: the compound of the present invention to [ alpha ]¹⁴C]The incorporation of acetate into HepG2 cells had a clear inhibitory effect, superior to Firsocostat as a control compound.

Test example 3 study of oral pharmacokinetics of ICR mouse, a Compound of the present invention

1. Abstract

ICR mice are used as test animals, and LC-MS/MS method is adopted to measure the drug concentration in plasma and liver at different time after the mice are gavaged with the compound of example 3 and the control firsocostat, so as to study the pharmacokinetic characteristics of the compound in the mice.

2. Experimental protocol

2.1 Experimental drugs and animals

The compound of example 3 and Firsocostat;

healthy adult ICR male mice 18 purchased from beijing vindolizhihua experimental animal technology ltd, animal weights: 29.3-35.4 g.

2.2 drug formulation and administration

Weighing a proper amount of experimental medicine, adding 0.5% sodium carboxymethylcellulose (CMC-Na), and grinding to prepare 1mg/mL suspension;

18 healthy adult ICR male mice were divided into 2 groups and were individually gavaged after overnight fasting at a dose of 10mg/kg and a volume of 10mL/kg and were fed 4 hours after administration.

Group AThe mice are administered with 10mg/kg by gavage^-1Example 3 administration of the Compound preparation 10mg/kg was administered by gavage to group B mice^-1formulations for administration of firsocostat.

2.3 sample Collection

Blood was collected via the orbit for 80uL before administration, and CO was administered at each set time point after administration₂After deep anesthesia, 0.20mL of blood is collected through the heart, and a whole blood sample is placed in an anticoagulation tube containing EDTA-K2; and liver tissue was immediately harvested. The sampling time points are as follows:

plasma: 0 hour before administration, 0.5 hour, 1 hour, 4 hours after administration.

Liver: 1 hour after administration.

Collecting blood sample, and placing in EDTA-K-containing container₂In the anticoagulation tube, plasma was centrifuged (centrifugation conditions: 1500g, 10 minutes), and the upper plasma sample was collected into a sample tube. A portion of the liver tissue sample was weighed and homogenized by adding 20% methanol water in a ratio (tissue: homogenate: 1:5, w/v). The collected biological samples are stored in a refrigerator at-40 to-20 ℃ before analysis.

And analyzing the content of the compound to be detected in the plasma and the liver of the mouse after the compound is administrated by gavage by adopting LC-MS/MS.

3. Pharmacokinetic parameter results

The pharmacokinetic parameters for the compound of example 3 of the invention and Firsocostat are as follows:

and (4) conclusion: compared with Firsocostat, the compound of the embodiment 3 has good pharmacokinetic absorption and better pharmacokinetic property; after 1 hour of concurrent dosing, the drug concentrations in the liver were: 10740ng/g, the compound of example 3 was better enriched in liver.

Remarking: the structure of Firsocostat is as follows, prepared according to WO2013071169

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

A compound of formula (I):

including stereoisomers, tautomers or pharmaceutically acceptable salts thereof,

wherein:

x is selected from-NH-, -O-or-S-; preferably-S-;

ring A is selected from cycloalkyl, and R attached to ring A²And N is not attached to the same carbon atom;

R¹selected from hydrogen atoms, alkyl groups or halogens, wherein said alkyl groups are optionally further substituted by one or more groups selected from halogen, hydroxy, cyano, nitro, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Substituted with the substituent(s);

R²selected from hydrogen atom, hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Wherein said alkyl, alkylOxy, cycloalkyl, heterocyclyl, aryl or heteroaryl optionally further substituted with one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

R³is selected from aryl or heteroaryl, wherein said aryl or heteroaryl is optionally further substituted by one or more groups selected from R⁷Substituted with the substituent(s);

R⁴and R⁵Each independently selected from hydrogen atom, alkyl group, -OR¹⁰、-SR¹⁰、-NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹；

Or, R⁴、R⁵Together with the atoms to which they are attached form a 3-to 8-membered saturated or partially unsaturated cycloalkyl group, or form a cyclic alkyl group having 1 or more members selected from N, O, S (O)_qWherein said cycloalkyl or heterocyclyl is optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Substituted with the substituent(s);

R⁶selected from halogen, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Preferably, it is heteroaryl;

or, R¹、R⁶Together with the atoms to which they are attached form a 3-to 8-membered saturated or partially unsaturated cycloalkyl group, or form a cyclic alkyl group having 1 or more members selected from N, O, S (O)_qOr form a 5-10 membered aryl or heteroaryl group, wherein the cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Substituted with the substituent(s);

R⁷each independently selected from hydroxy, halogen, cyano, nitro, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-OC(O)R¹⁰、-S(O)_qNR⁸R⁹、-NR⁸S(O)₂R⁹or-NR⁸C(O)R⁹Substituted with the substituent(s);

R⁸、R⁹and R¹⁰Each independently selected from hydrogen atom, alkyl group, -OR¹³Cyano, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

or, R⁸、R⁹Together with the N atom to which they are attached form a 4-to 8-membered heterocyclic group, wherein the 4-to 8-membered heterocyclic group contains one or more N, O, S (O)_qAnd 4-to 8-membered heterocycle is further substituted with one or more substituents selected from the group consisting of hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, ═ O, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

R¹¹、R¹²and R¹³Each independently selected from a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group, wherein the alkyl group, cycloalkyl group, heterocyclyl group, aryl group, or heteroaryl group is optionally further substituted with one or more substituents selected from the group consisting of hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl group, aryl group, heteroaryl group, carboxylic acid, or carboxylic acid ester; and is

q is 0, 1 or 2.
The compound of claim 1, having the structure of formula (II):

wherein:

m is 1,2, 3,4 or 5; and is

Ring A, R¹、R²、R⁶、R⁷And R¹⁰Is as defined in claim 1.
The compound of claim 2, having the structure of formula (III):

wherein:

m is 1,2, 3,4 or 5; and is

Ring A, R¹、R²、R⁶、R⁷And R¹⁰Is as defined in claim 1.
The compound of claim 2, having the structure of formula (IV):

wherein:

m is 1,2, 3,4 or 5; and is

Ring A, R¹、R²、R⁶、R⁷And R¹⁰Is as defined in claim 1.
A compound according to any one of claims 1 to 4, wherein ring A is selected from the group consisting of:

preferably, it is
A compound according to any one of claims 1 to 5, wherein R¹Selected from methyl or trifluoromethyl.
A compound according to any one of claims 1 to 6, wherein:

R²selected from tetrazolyl, -C (O) OR¹³or-C (O) NR⁸R⁹；

R⁸Selected from a hydrogen atom or an alkyl group;

R⁹selected from cyano OR-OR¹³；

R¹³Selected from a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally further substituted by one or more substituents selected from the group consisting of hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid or carboxylic acid ester;

R²is-C (O) OH.
A compound according to any one of claims 1 to 7, wherein R⁶Selected from 5-membered heteroaryl groups, preferably thiazolyl.
A compound according to any one of claims 1 to 8, wherein R⁷Selected from halogen or alkoxy, preferably methoxy.
A compound according to any one of claims 1 to 9, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein R¹⁰Is tetrahydropyran-4-yl.
The compound according to claim 1, selected from:
a process for preparing a compound of formula (I) according to claim 1, said process comprising:

reacting a compound of formula (IA) with R⁶Substituted tributylstannanesOptionally further hydrolyzing the resulting compound and optionally further resolving the optically pure isomer of the hydrolyzed compound to obtain a compound of formula (I);

wherein: x¹Selected from halogens; and X, ring A, R¹～R⁶Is as defined in claim 1.
A compound of formula (IA):

including stereoisomers, tautomers or pharmaceutically acceptable salts thereof,

wherein:

X¹selected from halogens; and X, ring A, R¹～R⁵Is as defined in claim 1.
The compound according to claim 13, selected from:
a process for the preparation of a compound of formula (IA) according to claim 13, which process comprises:

reacting a compound of formula (IB) with a compound of formula (IC) in the presence of triphenylphosphine to give a compound of formula (IA);

wherein:

X¹selected from halogens; and X, ring A, R¹～R⁵Is as defined in claim 1.
A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1-11, and optionally a pharmaceutically acceptable carrier, excipient, or combination thereof.
Use of a compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 16 in the manufacture of a medicament for use as an ACC inhibitor.
Use of a compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 16 for the manufacture of a medicament for the prevention or treatment of a disease or condition associated with ACC, wherein the disease or condition is preferably a metabolic disease, cancer, fungal, parasitic or bacterial infection; wherein the metabolic disease is preferably hepatic steatosis, non-alcoholic fatty liver disease, obesity, dyslipidemia, hyperlipidemia, type II diabetes mellitus or metabolic syndrome, wherein the obesity is preferably Prader-Willi syndrome, Bardet-Biedl syndrome or Cohen syndrome or MOMO syndrome, wherein the cancer is preferably hepatocellular carcinoma, non-small cell lung cancer, gastric cancer, colorectal cancer, head and neck tumor, melanoma, ovarian cancer or cervical cancer, more preferably hepatocellular carcinoma and non-small cell lung cancer.
A method of preventing or treating a disease or condition associated with ACC, comprising administering to a subject in need thereof a compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 16.