Indoline aromatic acid ether derivative as well as preparation method and application thereof

Abstract

The invention discloses indoline aromatic acid ether derivatives as well as a preparation method and application thereof. Specifically, the invention discloses an indoline aromatic acid ether derivative as shown in a formula (I), or a tautomer, a cis-trans-isomer, an isotope label and pharmaceutically acceptable salts thereof. The derivative shows anti-platelet aggregation activity in an AA and ADP induced platelet aggregation experiment. The compound disclosed by the invention can be used for preventing and / or treating diseases such as cerebral arterial thrombosis, Alzheimer's disease, vascular dementia and / or Parkinson's disease. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry, and specifically relates to a class of indoline aromatic acid ether derivatives and their use in drugs for treating or preventing diseases related to nerve damage. Background Art

[0002] Ischemic stroke is the most common cerebrovascular disease, accounting for about 80% of all cerebrovascular diseases, and is one of the leading causes of morbidity and mortality worldwide. Studies have shown that ischemic stroke has three main stages: thrombosis, usually caused by vascular damage, triggers platelet aggregation and adhesion to the vessel wall; then the continued growth of arterial thrombus leads to vascular occlusion; and finally, due to insufficient blood supply to the brain, inflammation and oxidative stress reactions occur in brain tissue, leading to apoptosis and necrosis of nerve cells. Therefore, the development of new drugs with dual effects of antiplatelet aggregation and neuroprotection is of great significance for the treatment of ischemic stroke.

[0003] Ferulic acid is widely found in plants such as Angelica sinensis and Ligusticum chuanxiong. It has pharmacological effects such as improving cerebral circulation, anti-inflammation, anti-oxidative stress, and anti-platelet aggregation. However, due to its weak activity, its structure can be optimized to enhance its pharmacological activity, which has certain prospects for the development of new drugs. Du Junrong et al. synthesized a series of ferulic acid derivatives (1) and found that their derivatives can not only reduce lipopolysaccharide (LPS)-induced cell inflammatory damage, but also reduce H 2 O 2 Induced cell free radical damage, improved cell viability, and had significant neuroprotective effects [Chinese invention patent CN104958287A]. Lei Haimin et al. synthesized a series of ligustrazine-substituted cinnamic acid derivatives. The results showed that most of the compounds had certain neuroprotective activity, among which compound 2 had the strongest neuroprotective activity (EC 50 =3.68μM) [Chinese invention patent CN106977464A].

[0004] Indole is an active fragment with multiple pharmacological effects such as anti-inflammatory, antioxidant, and anti-tumor, and is widely used in drug design. Indole-3-propionic acid is a naturally occurring compound that has been shown to have the property of scavenging free radicals and protecting neuronal cells from oxidative damage caused by beta-amyloid protein. Zaikin et al. surprisingly found that the reduction products of indole (such as general formula 4 and general formula 5) have stronger anti-inflammatory and antioxidant activities by structural modification, showing significant protective activity against RAW264.7 macrophages [Shani Z, Tehilla W, Corina B, et al. Synthesis and Biological Evaluation of Derivatives of Indoline as Highly Potent Antioxidant and Anti-inflammatory Agents [J], J Med Chem, 2018, 61, 4004-4009].

[0005] Summary of the invention

[0006] The purpose of the present invention is to try to find a drug with both anti-platelet aggregation activity and neuroprotective activity by combining and modifying the structures of dihydroindoline with anti-inflammatory and antioxidant effects and ferulic acid and its derivatives.

[0007] In one aspect, the present invention provides a dihydroindole aromatic acid ether derivative, which is a compound represented by the general formula (I):

[0008]

[0009] or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts,

[0010] Among them, R 1 , R 2 , X, m, and n are as defined herein.

[0011] On the other hand, the present invention provides a pharmaceutical composition comprising a compound represented by formula (I) or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier.

[0012] In another aspect, the present invention provides the use of the compound represented by formula (I) or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts and / or pharmaceutical compositions containing the same in the preparation of drugs for preventing and / or treating diseases related to nerve damage.

[0013] Beneficial effects of the present invention: The compounds of the present invention exhibit good antiplatelet aggregation activity in platelet aggregation tests induced by arachidonic acid (AA) and / or adenosine diphosphate (ADP). Moreover, the compounds of the present invention also exhibit significant neuroprotective activity, significantly improving animal neurological scores and reducing cerebral infarction rates. DETAILED DESCRIPTION

[0014] definition

[0015] As used in this specification, the following words and phrases are generally intended to have the meanings set forth below, unless the context in which they are used indicates otherwise.

[0016] As used herein, the term "alkyl" refers to a monovalent group of a straight or branched saturated hydrocarbon chain having 1 to 20 carbon atoms, more typically 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, 1-propyl (n-propyl), 2-propyl (isopropyl), 1-butyl (n-butyl), 2-methyl-1-propyl (isobutyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (tert-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, 1-nonyl, 1-decyl, and the like.

[0017] As used herein, the term "alkenyl" refers to a linear or branched unsaturated hydrocarbon chain monovalent group having 2 to 20 carbon atoms (more typically 2 to 10 carbon atoms, 2 to 8 carbon atoms, or 2 to 6 carbon atoms) and having carbon-carbon double bonds (e.g., 1, 2 or 3 carbon-carbon double bonds). The term is exemplified by groups such as vinyl (i.e., -CH=CH 2 ), propen-1-yl (i.e. -CH=CHCH 3 ), propen-3-yl (or allyl, i.e. -CH 2 CH=CH 2 ), propene-2-yl (i.e. -C(CH 3 )=CH 2 ), butadienyl (including 1,2-butadienyl and 1,3-butadienyl), etc.

[0018] As used herein, the term "alkynyl" refers to a linear or branched unsaturated hydrocarbon chain monovalent group having 2 to 20 carbon atoms (more typically 2 to 10 carbon atoms, 2 to 8 carbon atoms, or 2 to 6 carbon atoms) and having carbon-carbon triple bonds (e.g., 1, 2 or 3 carbon-carbon triple bonds). The term is exemplified by groups such as ethynyl (i.e., -C≡CH), propargyl (i.e., -CH 2 C≡CH), propynyl (i.e. -C≡CCH 3 )wait.

[0019] As used herein, the term "aryl" refers to an aromatic carbocyclic group of 6 to 14 carbon atoms (more typically 6 to 10 carbon atoms, or 6 carbon atoms) having a single ring (e.g., phenyl) or multiple rings (e.g., biphenyl) or multiple condensed (fused) rings (e.g., naphthyl, fluorenyl, and anthracenyl). The term is exemplified by groups such as phenyl, fluorenyl, naphthyl, anthracenyl, 1,2,3,4-tetrahydronaphthalene (if the point of attachment is through the aryl group), and the like.

[0020] As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0021] As used herein, the term "alkoxy" refers to an "alkyl-O-" group, wherein alkyl is as defined herein. This term is exemplified by groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.

[0022] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a halogen, wherein alkyl is as defined herein. The term is exemplified by the groups trifluoromethyl, difluoromethyl, monofluoromethyl, 2,2,2-trifluoroethyl, 1,1,-difluoroethyl, etc.

[0023] As used herein, the term "carbocycle", "carbocyclic radical" refers to a monocyclic saturated or partially unsaturated group of 3 to 8 members of a monocyclic ring or a plurality of thick (fused) rings or bridged rings or spirocycles having 3 to 14 carbon atoms as ring atoms.Carbocycle or carbocyclic radical can be saturated or partially unsaturated, and can be fused with another saturated, partially unsaturated or aromatic ring, provided that the ring atom connected to the target molecule is not an aromatic carbon.Examples of carbocycle or carbocyclic radical include, but are not limited to cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclopentadiene, etc.

[0024] As used herein, the term "heteroaromatic ring", "heteroaryl" refers to an aromatic ring group containing a monocyclic or multiple condensed (fused) ring (e.g., containing 2 or 3 rings) of 5 to 14 ring atoms in the ring, wherein in addition to carbon atoms, the ring atoms also contain at least one heteroatom selected from oxygen, nitrogen and / or sulfur. If the ring is aromatic, the sulfur and nitrogen atoms can also exist in oxidized form. Multiple condensed (fused) ring heteroaryl is a monocyclic heteroaryl as defined above fused with one or more rings selected from the following to form a multiple condensed ring system: heteroaryl (to form, for example, naphthyridinyl, such as 1,8-naphthyridinyl), heterocyclic (for example, 1,2,3,4-tetrahydronaphthyridinyl, such as 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocyclic (to form, for example, 5,6,7,8-tetrahydroquinolinyl) and aryl (to form, for example, indazolyl). Such multiple fused ring systems can be optionally substituted with one or more (e.g., 1, 2, 3, or 4) oxo groups on the carbocyclic or heterocyclic portions of the fused rings. When valence requirements permit, the rings of the multiple fused ring systems can be interconnected by fusion, spirocyclic, and bridging bonds. It should be understood that the individual rings of the multiple fused ring systems can be connected to each other in any order. It should also be understood that the attachment point of the multiple fused ring system can be at any position of the multiple fused ring system, including heteroaryl, heterocyclic, aryl, or carbocyclic portions of the multiple fused system. It should also be understood that the attachment point of the heteroaryl group can be on any suitable atom of the heteroaryl group, including carbon atoms and heteroatoms (e.g., nitrogen). Exemplary heteroaryl groups include, but are not limited to, pyridinyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, oxadiazolyl, thiadiazolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, quinazolinyl, 5,6,7,8-tetrahydroisoquinolinyl, benzofuranyl, benzo[d][1,3]dioxolyl, benzimidazolyl, thiaindenyl, pyrrolo[2,3-b]pyridinyl, quinazolinyl-4(3H)-one, triazolyl, 4,5,6,7-tetrahydro-1H-indazolyl, and 3b,4,4a,5-tetrahydro-1H-cyclopropane[3,4]cyclopenta[1,2-c]pyrazolyl.

[0025] As used herein, the terms "heterocycle", "heterocyclyl" and "heterocyclic group" refer to a monoradical saturated or partially unsaturated group having a 3- to 8-membered monocyclic ring or multiple condensed (fused) rings or bridged rings having 3 to 14 ring atoms in the ring, wherein in addition to carbon atoms, the ring atoms also contain at least one heteroatom selected from oxygen, nitrogen and / or sulfur. Examples of heterocyclyl groups include, but are not limited to, aziridine, azetidine, tetrahydropyrrole, piperidine, azepane, aziroxane, oxetane, tetrahydrofuran, tetrahydropyran, oxepane, oxocane, thiirane, thietane, tetrahydrothiophene, tetrahydrothiopyran, thiepane, thiocane, tetrahydroimidazole, tetrahydropyrazole, tetrahydrooxazole, tetrahydroisoxazole, tetrahydrothiazole, tetrahydroisothiazole, piperazine, morpholine, dioxane, thioxane, dithiane, dihydropyridyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazole, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine, and the like.

[0026] As used herein, the term "cis-trans isomers" refers to isomers that appear in a compound molecule due to restrictions on free rotation, such as C=C double bonds, C=N double bonds, C=S double bonds, N=N double bonds or alicyclic rings, which cause different arrangements of various groups in space. Organic molecules containing such isomers, such as olefins, azo compounds, alicyclic hydrocarbons, etc., are regarded as cis-trans isomers and are named according to the "cis-trans isomer nomenclature" or "ZE nomenclature".

[0027] As used herein, the term "tautomer" refers to the coexistence of two (or more) compounds that differ only in the position and electron distribution of one (or more) mobile atoms, such as keto-enol tautomers.

[0028] As used herein, the term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of a given compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts can be acid addition salts and / or base addition salts. Acid addition salts can be prepared from inorganic acids and organic acids. Salts derived from inorganic acids include hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, carbonates, bisulfates, hydrogenphosphates, dihydrogenphosphates, bicarbonates, etc.; salts derived from organic acids include formates, acetates, propionates, glycolates, pyruvates, oxalates, malates, malonates, succinates, maleates, fumarates, tartrates, citrates, benzoates, cinnamates, mandelates, methanesulfonates, ethanesulfonates, p-toluenesulfonates, salicylates, lactates, nicotinates, lauryl sulfates, naphthylsulfonates, camphorsulfonates, gluconates, glucuronates, oleates, palmitates, stearates, pamoates, trifluoroacetates, etc. Base addition salts can be formed with inorganic or organic bases. Salts derived from inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, lithium, barium, aluminum salts and the like; salts derived from organic bases include salts formed with various primary, secondary and tertiary amines, for example, ethylamine, diethylamine, n-propylamine, isopropylamine, diethanolamine, meglumine, lysine, piperazine, piperidine, morpholine, tromethamine, choline and the like.

[0029] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0030] Any formula or structure given herein, including Formula I or any formula disclosed herein, is also intended to represent unlabeled forms of the compounds as well as isotopically labeled forms. These isotopically labeled forms of compounds may also be referred to as "isotopically labeled" or "isotopically enriched analogs." Isotopically labeled compounds have structures depicted herein except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as, but not limited to, 2 H (deuterium, D), 3 H (tritium), 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 36Cl, 123 I and 125 I. Various isotope-labeled compounds of the present invention, for example, compounds into which a radioactive isotope (e.g. 3 H. 13 C and 14 C). Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogens have been replaced with deuterium.

[0031] Compound

[0032] In one embodiment, the present invention provides a compound represented by formula (I)

[0033]

[0034] or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts, wherein

[0035] R 1 Selected from hydrogen, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, halogen, hydroxyl, cyano, nitro, carboxyl;

[0036] R 2 Selected from hydrogen, C 1 -C 20 alkyl;

[0037] X is selected from C 6 -C 14 aryl, 5- to 14-membered heteroaryl, 3- to 14-membered heterocyclic group or C 3 -C 14 Carbocyclic group, which is optionally substituted by one or more groups selected from the group consisting of halogen, oxo, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, C 1 -C 20 Alkoxycarbonyloxy, hydroxy, cyano, nitro, carboxyl;

[0038] m is selected from 0 or 1;

[0039] n is selected from an integer between 0-4.

[0040] In one embodiment, in the compound represented by formula (I) provided by the present invention, X is selected from C 6 -C 14 aryl, 5- to 14-membered heteroaryl, 3- to 14-membered heterocyclic group or C 3 -C 14 Carbocyclic group, which is optionally substituted by one or more groups selected from the group consisting of halogen, oxo, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, C 1 -C 20 alkoxycarbonyloxy, hydroxy, cyano, nitro, carboxyl; preferably, X is selected from C 6 -C 10 aryl, 5- to 10-membered heteroaryl, 3- to 8-membered heterocyclic group or C 3 -C 8 Carbocyclic group, which is optionally substituted by one or more groups selected from the group consisting of halogen, C 1 -C 20 Alkyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1-C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, C 1 -C 20 Alkoxycarbonyloxy, hydroxy, cyano, nitro, carboxyl; More preferably, X is selected from C 6 -C 10 aryl, 5- to 10-membered heteroaryl, 3- to 8-membered heterocyclic group or C 3 -C 8 Carbocyclic group, which is optionally substituted by one or more groups selected from the group consisting of halogen, C 1 -C 6 Alkyl, C 1 -C 6 Haloalkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkanoyl, C 1 -C 6 Alkanoyloxy, C 1 -C 6 Alkoxycarbonyl, C 1 -C 6alkoxycarbonyloxy, hydroxyl, cyano, nitro, carboxyl; further preferably, X is selected from phenyl, naphthyl, pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, quinazolinyl, 5,6,7,8 -tetrahydroisoquinolyl, benzofuranyl, benzo[d][1,3]dioxolyl, benzimidazolyl, thiaindenyl, pyrrolo[2,3-b]pyridinyl, quinazolinyl-4(3H)-one, triazolyl, 4,5,6,7-tetrahydro-1H-indazolyl and 3b,4,4a,5-tetrahydro-1H-cyclopropane[3,4]cyclopenta[1,2-c]pyrazolyl, aziridine ring, azetidine ring, tetrahydropyrrole ring, piperidine ring, azepane ring, aziridine ring, oxirane ring, oxetane ring, tetrahydrofuran ring, tetrahydropyran ring, oxepane ring, oxocane ring, thiirane ring, thietane ring, tetrahydrothiophene ring, tetrahydrothiopyran ring, thiepane ring, thiocane ring, tetrahydroimidazole ring, tetrahydropyrazole ring, tetrahydrooxazole ring, tetrahydroisoxazole ring, tetrahydrothiazole ring, tetrahydroisothiazole ring, piperazine ring, morpholine ring, dioxane ring, thioxane ring, dithiane ring, dihydropyridyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazole, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclopentadiene, which are optionally substituted by one or more groups selected from the following: halogen, C 1 -C 6 Alkyl, C 1 -C 6 Haloalkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkanoyl, C 1 -C 6 Alkanoyloxy, C 1 -C 6 Alkoxycarbonyl, C 1 -C 6 Alkoxycarbonyloxy, hydroxy, cyano, nitro, carboxyl; further preferably, X is selected from phenyl, furanyl, thienyl, benzo[d][1,3]dioxole, benzofuranyl, pyridyl, which are optionally substituted by one or more groups selected from the following: halogen, methyl, trifluoromethyl, methoxy, cyano, nitro; most preferably, X is selected from phenyl, which is optionally substituted by one or more groups selected from the following: methoxy.

[0041] In one embodiment, in the compound of formula (I) provided by the present invention, n is an integer between 0 and 4; preferably, n is 0 or 1 or 2 or 3; more preferably, n is 0 or 1 or 2; most preferably, n is 0 or 1.

[0042] In one embodiment, in the compound represented by formula (I) provided by the present invention, each R 1 are independently selected from hydrogen, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, halogen, hydroxy, cyano, nitro, carboxyl; preferably, each R 1 are independently selected from hydrogen, C 1 -C 20 Alkyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, halogen, hydroxy, cyano, nitro, carboxyl; more preferably, each R 1 are independently selected from hydrogen, C 1 -C 6 Alkyl, C 1 -C 6 Haloalkyl, C 1 -C 6 Alkoxy, halogen; most preferably, each R 1 Independently selected from hydrogen, methoxy.

[0043] In one embodiment, in the compound represented by formula (I) provided by the present invention, R 2 Selected from hydrogen, C 1 -C 20 Alkyl; preferably, R 2 Selected from hydrogen, C 1 -C 6 More preferably, each R 2 Selected from hydrogen, C 1 -C 3 Alkyl; most preferably, R 2 Selected from hydrogen, methyl, ethyl.

[0044] In one embodiment, in the compounds provided herein, each R3 are independently selected from hydrogen, halogen, oxo, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, C 1 -C 20 alkoxycarbonyloxy, hydroxy, cyano, nitro, carboxyl; preferably, each R 3 are independently selected from hydrogen, C 1 -C 20 Alkyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, halogen, hydroxy, cyano, nitro, carboxyl; more preferably, each R 3 are independently selected from hydrogen, C 1 -C 6 Alkyl, C 1 -C 6 Haloalkyl, C 1 -C 6 Alkoxy; most preferably, each R 3 Independently selected from hydrogen, methyl, ethyl, methoxy, ethoxy.

[0045] In one embodiment, the present invention provides a compound represented by formula (II):

[0046]

[0047] or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts, wherein R 1 , R 2 , R 3 , m, n, and p are as defined herein.

[0048] In a specific embodiment, the compound of the present invention is a compound represented by formula (III):

[0049]

[0050] or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts, wherein R 1 , R 2 , R 3 , n, m, and p are as defined herein.

[0051] In a specific embodiment, the compounds of the present invention include but are not limited to the following compounds:

[0052] 4-(3-(2,3-dihydroindolin-1-yl)propoxy)-3-methoxybenzoic acid;

[0053] (E)-3-(4-(3-(2,3-dihydroindolin-1-yl)propoxy)-3-methoxyphenyl)acrylic acid;

[0054] (E)-3-(4-(3-(2,3-dihydroindolin-1-yl)propoxy)phenyl)acrylic acid;

[0055] (E)-3-(4-(2-(2,3-dihydroindolin-1-yl)ethoxy)-3-methoxyphenyl)acrylic acid;

[0056] 4-(3-(2,3-dihydroindolin-1-yl)propoxy)benzoic acid;

[0057] (E)-3-(3-methoxy-4-(2-(5-methoxy-2,3-dihydroindolin-1-yl)ethoxy)phenyl)acrylic acid;

[0058] 4-(3-(2,3-dihydroindolin-1-yl)propoxy)-3,5-dimethoxybenzoic acid;

[0059] (E)-3-(3-methoxy-4-(3-(5-methoxy-2,3-dihydroindolin-1-yl)propoxy)phenyl)acrylic acid;

[0060] 3-Methoxy-4-(3-(5-methoxy-2,3-dihydroindolin-1-yl)propoxy)benzoic acid;

[0061] (E)-3-(4-(2-(2,3-dihydroindolin-1-yl)ethoxy)phenyl)acrylic acid;

[0062] or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts.

[0063] Pharmaceutical compositions and administration

[0064] The pharmaceutical composition provided by the present invention comprises the compound of the present invention or its stereoisomer, tautomer, solvate, prodrug, isotope label, pharmaceutically acceptable salt, and at least one pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known to those skilled in the art, including diluents, lubricants, disintegrants, adhesives, buffers, preservatives, stabilizers, wetting agents, glidants, emulsifiers, colorants, flavoring agents, sweeteners, etc. According to the different routes of administration of the drug, such as oral administration, parenteral administration and rectal administration, the pharmaceutical composition of the present invention can be made in solid form (including but not limited to tablets, capsules, pills, granules, powders, powders, suppositories) or liquid form (including but not limited to solutions, suspensions, emulsions, tinctures, syrups). When the pharmaceutical composition of the present invention is in solid form, the pharmaceutically acceptable carrier generally includes one or more of the following: a) diluents, such as lactose, glucose, sucrose, mannitol, sorbitol, cellulose, etc.; b) lubricants, such as silicon dioxide, talc, stearic acid, polyethylene glycol, etc.; c) binders, such as magnesium aluminosilicate, gelatinized starch, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, etc.; d) disintegrants, such as starch, alginic acid, agar, corn starch; e) stabilizers, such as antioxidants such as ascorbic acid; f) glidants, such as silicon dioxide; g) flavoring agents, such as mint, methyl salicylate; sweeteners, such as sucrose, saccharin. When the pharmaceutical composition of the present invention is in liquid form, the pharmaceutically acceptable carrier generally includes one or more of the following: a) a diluent, such as water for injection, physiological saline, Ringer's solution, polyethylene glycol, glycerol, propylene glycol, etc.; b) an antioxidant, such as ascorbic acid or sodium bisulfite; c) a buffer, such as acetate, phosphate, etc.

[0065] The effective dose of the compounds of the present invention depends at least on the nature, extent, delivery method and pharmaceutical dosage form of the condition being treated, and will be ultimately determined by the clinician. It can be expected that about 0.0001 to about 100 mg per kilogram of body weight per day; generally about 0.01 to about 10 mg per kilogram of body weight per day; more typically, about 0.01 to about 5 mg per kilogram of body weight per day; most typically about 0.05 to about 0.5 mg per kilogram of body weight per day. For example, the daily candidate dose for an adult of about 70 kg body weight will be in the range of 1 mg to 1000 mg, preferably in the range of 5 mg to 500 mg, and can be administered in the form of a single dose or multiple doses.

[0066] Indications

[0067] The compounds of the present invention have significant anticoagulant and neuroprotective activities, and can be used to treat or prevent diseases related to nerve damage, including ischemic stroke, posterior circulation cerebral ischemia, amyotrophic lateral sclerosis, epilepsy, Huntington's disease, Alzheimer's disease, multiple sclerosis, multiple system atrophy, Parkinson's disease, primary lateral sclerosis peripheral neuropathy, and diabetic neuropathy.

[0068] General synthetic method

[0069] Compounds of the present invention can be prepared using methods disclosed herein and their modified approaches and methods well known in the art. Typical embodiments of compounds according to the present invention can be synthesized using the following general reaction schemes. It is apparent from the description herein that corresponding different products can be obtained by replacing reaction raw materials with other materials having similar structures. Reaction raw materials are typically obtained from commercial sources or synthesized using disclosed methods.

[0070] Reaction process

[0071]

[0072] Step 1, compound 1 undergoes reduction reaction to obtain intermediate 2;

[0073] Step 2, reacting intermediate 2 with reagent 2a to undergo nucleophilic substitution reaction to obtain intermediate 3;

[0074] Step 3, intermediate 3 undergoes substitution reaction with reagent 3a to obtain intermediate 4;

[0075] Step 4: Intermediate 4 is subjected to hydrolysis and acidification to obtain compound 5.

[0076] In the formula, R 1 , R 2 , R 3 , m, n and p are as defined herein.

[0077] In a preferred embodiment, in step 1, compound 1 is dissolved in acetic acid, sodium cyanoborohydride is slowly added in batches, and the reaction is stirred at room temperature for 1 hour to obtain intermediate 2;

[0078] In a preferred embodiment, in step 2, intermediate 2 and reagent 2a are dissolved in N,N-dimethylformamide, and then potassium carbonate is added, and the mixture is stirred at 70° C. for 4 h to obtain intermediate 3;

[0079] In a preferred embodiment, in step 3, intermediate 3 and reagent 3a are dissolved in acetonitrile, and then potassium carbonate is added, and the mixture is stirred at 50° C. for 4 h to obtain intermediate 4;

[0080] In a preferred embodiment, in step 4, intermediate 4 is dissolved in methanol, 10% NaOH solution is added, and the mixture is stirred at 60° C. for 1 h to obtain compound 5.

[0081] Example 1 Synthesis of (E)-3-(3-methoxy-4-(3-(5-methoxy-2,3-dihydroindole-1-yl)propoxy)phenyl)acrylic acid (Compound 5h)

[0082] Step 1: Synthesis of 5-methoxyindoline (Intermediate 2a)

[0083]

[0084] 5-Methoxyindole (1.5 g, 10.2 mmol) and glacial acetic acid (20 mL) were added to a 100 mL single-necked flask, and then NaBH was added in batches under stirring at 25 °C. 3 CN (0.77 g, 12.2 mmol), continue the reaction for 1 h, TLC (petroleum ether: ethyl acetate = 3: 1) detected that the reaction was basically complete. Add 20% NaOH solution to the reaction solution to adjust the pH to 9-10, extract with ethyl acetate (50 mL × 3), combine the organic phases, wash with water (50 mL × 3), and then add anhydrous Na 2 SO 4 The product was dried, filtered and the filtrate was concentrated. The residue was purified by silica gel column chromatography to obtain intermediate 2a as a light yellow oil with a yield of 91%.

[0085] Step 2: Synthesis of 1-(3-bromopropyl)-5-methoxy-2,3-dihydro-1H-indole (Intermediate 3a)

[0086]

[0087] In a 100 mL single-necked flask, intermediate 2a (0.6 g, 4.02 mmol), DMF (20 mL), 1,3-dibromopropane (4.87 g, 24.12 mmol), K 2 CO 3 (1.1 g, 8.0 mmol), stirred at 70 ° C for 4 h, and the reaction was basically complete by TLC (petroleum ether: ethyl acetate = 5:1). The reaction solution was diluted with 150 mL of water, extracted with ethyl acetate (50 mL × 3), and the organic phases were combined, washed with water (50 mL × 3), and then purified by anhydrous Na 2 SO 4 The product was dried, filtered and the filtrate was concentrated. The residue was purified by silica gel column chromatography to obtain intermediate 3a as a light yellow oil with a yield of 36%.

[0088] Step 3: Synthesis of (E)-methyl 3-(3-methoxy-4-(3-(5-methoxy-2,3-dihydroindolin-1-yl)propoxy)phenyl)acrylate (Intermediate 4h)

[0089]

[0090] In a 50 mL single-necked flask, intermediate 3a (0.39 g, 1.44 mmol), CH 3 CN 8mL, methyl ferulate (0.3g, 1.44mmoL), K 2 CO 3 (0.3 g, 2.16 mmol), stirred at 50 ° C for 4 h, TLC (petroleum ether: ethyl acetate = 3: 1) detected that the reaction was basically complete. The reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure to obtain a brown oil, which was then chromatographed on a silica gel column to obtain the intermediate 4h, which was a white solid of 0.45 g, mp 83.3-85.1 ° C, and a yield of 80.9%.

[0091] Step 4: Synthesis of (E)-3-(3-methoxy-4-(3-(5-methoxy-2,3-dihydroindolin-1-yl)propoxy)phenyl)acrylic acid (Compound 5h)

[0092]

[0093] In a 50mL single-necked flask, add intermediate 4h (0.40g, 1.01mmoL), CH 3 OH (8 mL), 10% NaOH solution (4 mL), heated to 60°C, stirred for 1 h, and the reaction was almost complete as detected by TLC (petroleum ether: ethyl acetate = 3:1). The reaction solution was concentrated under reduced pressure to remove CH 3 OH, add 5mL water to dilute the residue, and adjust the pH to 5-6 with 1mol / L dilute hydrochloric acid. A large amount of white solid precipitates, which is filtered and dried to obtain 0.30g of the target compound 5h, with a yield of 77.7%. mp122.2-123.5℃.ESI-MS[M+H] + :m / z=384.03, 1 H NMR (400 MHz, DMSO-d 6)δ:12.20(s,1H,OH),7.52(d,J=15.9Hz,1H,=CH),7.33(s,1H,ArH),7.18(d,J=8.4Hz,1H,ArH),6.99(d,J=8.3Hz, 1H,ArH),6.71(s,1H,ArH),6.56(d,J=8.3,1.9Hz,1H,ArH),6.44(d,J=15.9Hz,1H,=CH),4.10(t,J=6.2Hz,2H,OCH 2 ),3.84(s,3H,OCH 3 ),3.63(s,3H,OCH 3 ),3.23(t,J=8.1Hz,2H,NCH 2 ),3.11(t,J=6.9Hz,2H,NCH 2 ),2.84(t,J=8.0Hz,2H,ArCH 2 ),2.02-1.94(m,2H,CH 2 CH 2 CH 2 ).

[0094] Example 2 Synthesis of 4-(3-(2,3-dihydroindole-1-yl)propoxy)-3-methoxybenzoic acid (Compound 5a)

[0095] Referring to the synthesis method of Example 1, 5-methoxy-2,3-dihydroindoline was replaced by dihydroindoline raw material, and methyl ferulate was replaced by methyl vanillate to obtain Example 2 compound 5a, which was a white solid. mp135.6~137.1℃.ESI-MS[M+H] + :m / z=328.41. 1 H NMR (400 MHz, DMSO-d 6 )δ:12.63(s,1H,OH),7.54(d,J=8.4Hz,1H,ArH),7.46(s,1H,ArH),7.05(d,J=8.5Hz,1H,ArH),7.01(d,J=7.1Hz,1H ,ArH),6.95(t,J=7.6Hz,1H,ArH),6.55(t,J=7.3Hz,1H,ArH),6.49(d,J=7.8Hz,1H,ArH),4.13(t,J=6.3Hz,2H,OCH 2 ),3.84(s,3H,OCH 3 ),3.32(t,J=8.3Hz,2H,NCH 2 ),3.21(t,J=7.0Hz,2H,ArCH 2CH 2 ),2.88(t,J=8.3Hz,2H,ArCH 2 ),2.05-1.97(m,2H,CH 2 CH 2 CH 2 ).

[0096] Example 3 Synthesis of (E)-3-(4-(3-(2,3-dihydroindole-1-yl)propoxy)-3-methoxyphenyl)acrylic acid (Compound 5b)

[0097] Referring to the synthesis method of Example 1, 5-methoxy-2,3-dihydroindoline was replaced with 2,3-dihydroindoline as the starting material to obtain Example 3 compound 5b, which was a light yellow solid. mp 128.5~130.1℃. ESI-MS [M+H] + :m / z=354.41. 1 H NMR (400 MHz, DMSO-d 6 )δ:12.22(s,1H,OH),7.52(d,J=15.9Hz,1H,=CH),7.33(s,1H,ArH),7.18(d,J=8.3Hz,1H,ArH),7.03-6.92(m,3H, ArH),6.54(t,J=7.0Hz,1H,ArH),6.48(d,J=8.0Hz,1H,ArH),6.45(d,J=15.9Hz,1H,=CH),4.09(t,J=6.2Hz,2H,OCH 2 ),3.84(s,3H,OCH 3 ),3.31(t,J=8.3Hz,2H,NCH 2 ),3.20(t,J=7.0Hz,2H,ArCH 2 CH 2 ),2.88(t,J=8.3Hz,2H,ArCH 2 ),2.04-1.94(m,2H,CH 2 CH 2 CH 2 ).

[0098] Example 4 Synthesis of (E)-3-(4-(3-(2,3-dihydroindole-1-yl)propoxy)phenyl)acrylic acid (Compound 5c)

[0099] Referring to the synthesis method of Example 1, 5-methoxy-2,3-dihydroindoline was replaced by 2,3-dihydroindoline as the raw material, and methyl ferulate was replaced by trans-methyl coumarate as the raw material to obtain Example 4 compound 5c, which was a light yellow solid. mp 135.8~137.9℃.ESI-MS[M+H] + :m / z=324.39. 1 H NMR (400 MHz, DMSO-d 6 )δ:12.23(s,1H,OH),7.63(d,J=8.8Hz,2H,ArH),7.54(d,J=15.9Hz,1H,=CH),7.03-6.98(m,3H,ArH),6.96(t,J=7.0Hz ,1H,ArH),6.55(t,J=7.0Hz,1H,ArH),6.48(d,J=7.8Hz,1H,ArH),6.38(d,J=16.0Hz,1H,=CH),4.11(t,J=6.2Hz,2H,OCH 2 ),3.31(t,J=8.0Hz,2H,NCH 2 ),3.20(t,J=7.0Hz,2H,ArCHCH 2 ),2.88(t,J=8.3Hz,2H,ArCH 2 ),2.04-1.95(m,2H,CH 2 CH 2 CH 2 ).

[0100] Example 5 Synthesis of (E)-3-(4-(2-(2,3-dihydroindole-1-yl)ethoxy)-3-methoxyphenyl)acrylic acid (Compound 5d)

[0101] Referring to the synthesis method of Example 1, 5-methoxy-2,3-dihydroindoline was replaced by 2,3-dihydroindoline as the raw material, and 1,3-dibromopropane was replaced by 1,2-dibromoethane as the raw material to obtain Example 5 compound 5d as a white solid. mp155.4~157.8℃.ESI-MS[M+H] + :m / z=340.39. 1 H NMR (400 MHz, DMSO-d 6)δ:12.18(s,1H,OH),7.52(d,J=15.9Hz,1H,=CH),7.33(s,1H,ArH),7.19(d,J=8.3Hz,1H,ArH),7. 09-6.97(m,3H,ArH),6.60-6.55(m,2H,ArH),6.45(d,J=15.9Hz,1H,=CH),4.20(t,J=5.6Hz,2H,OCH 2 ),3.81(s,3H,OCH 3 ),3.48-3.43(m,4H,N(CH 2 ) 2 ),2.89(t,J=8.3Hz,2H,ArCH 2 ).

[0102] Example 6 Synthesis of 4-(3-(2,3-dihydroindole-1-yl)propoxy)benzoic acid (Compound 5e)

[0103] Referring to the synthesis method of Example 1, 5-methoxy-2,3-dihydroindoline was replaced by 2,3-dihydroindoline as the raw material, and methyl ferulate was replaced by methyl p-hydroxybenzoate as the raw material to obtain Example 6 compound 5e as a white solid. mp 137.2~138.5℃.ESI-MS[M+H] + :m / z=298.35. 1 H NMR (400 MHz, DMSO-d 6 )δ:12.64(s,1H,OH),7.89(d,J=8.8Hz,2H,ArH),7.04(d,J=8.8Hz,2H,ArH),7.02(d,J=4.0Hz,1H,ArH),6 .95(t,J=7.6Hz,1H,ArH),6.55(t,J=7.3Hz,1H,ArH),6.48(d,J=7.8Hz,1H,ArH),4.14(t,J=6.2Hz,2H,OCH 2 ),3.31(t,J=8.3Hz,2H,NCH 2 ),3.20(t,J=7.0Hz,2H,NCH 2 ),2.88(t,J=8.3Hz,2H,ArCH 2 ),2.05-1.97(m,2H,CH 2 CH 2 CH 2 ).

[0104] Example 7 Synthesis of (E)-3-(3-methoxy-4-(2-(5-methoxy-2,3-dihydroindole-1-yl)ethoxy)phenyl)acrylic acid (Compound 5f)

[0105] Referring to the synthesis method of Example 1, 1,3-dibromopropane was replaced with 1,2-dibromoethane as the starting material to obtain Example 7 compound 5f as a yellow solid. mp 154.3~155.8℃.ESI-MS[M+H] + :m / z=370.36. 1 H NMR (400 MHz, DMSO-d 6 )δ:12.24(s,1H,OH),7.53(d,J=15.9Hz,1H,=CH),7.33(s,1H,ArH),7.20(d,J=8.2Hz,1H,ArH),7.02(d,J=8.3Hz,1H,ArH),6 .72(s,1H,ArH),6.60(d,J=8.3Hz,1H,ArH),6.54(d,J=8.5Hz,1H,ArH),6.45(d,J=15.9Hz,1H,=CH),4.19(t,J=5.6Hz,2H,OCH 2 ),3.81(s,3H,OCH 3 ),3.65(s,3H,OCH 3 ),3.38-3.32(m,4H,NCH 2 ),2.85(t,J=8.1Hz,2H,ArCH 2 ).

[0106] Example 8 Synthesis of 4-(3-(2,3-dihydroindole-1-yl)propoxy)-3,5-dimethoxybenzoic acid (Compound 5g)

[0107] Referring to the synthesis method of Example 1, 5-methoxy-2,3-dihydroindoline was replaced by 2,3-dihydroindoline as the raw material, and methyl ferulate was replaced by methyl syringate as the raw material to obtain 5 g of the compound of Example 8 as a white solid. mp 124.4~126.9℃.ESI-MS[M+H] + :m / z=358.36. 1 H NMR (400 MHz, DMSO-d 6)δ:12.94(s,1H,OH),7.24(s,2H,ArH),7.01(d,J=4.1Hz,1H),6.97(d,J=4.2Hz,1H,A rH),6.55(t,J=6.9Hz,1H,ArH),6.48(d,J=7.8Hz,1H,ArH),4.05(t,J=6.0Hz,2H,OCH 2 ),3.83(s,6H,(OCH 3 ) 2 ),3.29(t,J=8.3Hz,2H,NCH 2 ),3.24(t,J=8.1Hz,2H,NCH 2 ),2.87(t,J=8.3Hz,2H,ArCH 2 ),1.91-1.83(m,2H,CH 2 CH 2 CH 2 ).

[0108] Example 9 Synthesis of 3-methoxy-4-(3-(5-methoxy-2,3-dihydroindole-1-yl)propoxy)benzoic acid (Compound 5i)

[0109] Referring to the synthesis method of Example 1, methyl ferulate was replaced with methyl vanillate as the raw material to obtain Example 9 compound 5i, which was an off-white solid with a yield of 72%. mp140.3~143.1℃.ESI-MS[M+H] + :m / z=358.02. 1 HNMR (400 MHz, DMSO-d 6 )δ:12.65(s,1H,OH),7.54(d,J=8.4Hz,1H,ArH),7.46(s,1H,ArH),7.06(d,J=8.4Hz,1H,ArH),6 .71(s,1H,ArH),6.56(d,J=8.4Hz,1H,ArH),6.42(d,J=8.4Hz,1H,ArH),4.13(t,J=6.2Hz,2H,OCH 2 ),3.83(s,3H,OCH 3 ),3.63(s,3H,OCH 3 ),3.24(t,J=8.1Hz,2H,NCH 2 ),3.11(t,J=6.9Hz,2H,NCH 2 ),2.84(t,J=8.1Hz,2H,ArCH 2 ),2.05-1.96(m,2H,CH 2 CH2 CH 2 ).

[0110] Example 10 Synthesis of (E)-3-(4-(2-(2,3-dihydroindole-1-yl)ethoxy)phenyl)acrylic acid (Compound 5j)

[0111] Referring to the synthesis method of Example 1, 5-methoxy-2,3-dihydroindoline was replaced by 2,3-dihydroindoline, 1,3-dibromopropane was replaced by 1,2-dibromoethane, and methyl ferulate was replaced by trans-methyl coumarate to obtain Example 10 compound 5j, which was an off-white solid. mp 177.4~179.4℃.ESI-MS[M+H] + :m / z=310.32. 1 H NMR (400 MHz, DMSO-d 6 )δ:12.22(s,1H,OH),7.63(d,J=8.6Hz,2H,ArH),7.54(d,J=15.9Hz,1H,ArCH=),7.05-6.96(m,4H,ArH),6.57(m,2H ArH), 6.38 (d, J = 16.0Hz, 1H, = CH), 4.23 (t, J = 5.5Hz, 2H, OCH 2 ),3.44(m,4H,N(CH 2 ) 2 ),2.89(t,J=8.3Hz,2H,ArCH 2 ).

[0112] Biological Activity

[0113] 1. Antiplatelet aggregation activity test of the compounds of the present invention

[0114] (1) Experimental methods

[0115] Male rabbits (weight 1.8-2.0 kg) were anesthetized with lidocaine locally, and the carotid artery was cannulated for bleeding. The rabbits were mixed with sodium citrate solution (0.38 g sodium citrate and 10 mL normal saline to prepare a 3.8% sodium citrate solution) at a ratio of 9:1 and rotated at 500-800 r·min. -1 Centrifuge for 10 min, take platelet-rich plasma (PRP), and the remaining part at 3000 r / min -1 Centrifuge and take platelet-poor plasma (PPP). Aggregation inducer was 10 μg mL -1 ADP, 200 μg mL -1AA. 10 μL of drugs of different concentrations (1 μM, 10 μM, 100 μM, 1 mM, 10 mM) were added to each tube of 180 μL PRP. 10 μL of saline was added to the PRP of the control group. The mixture was incubated for 3 min, and then the above inducers of different concentrations (10 μg mL -1 ADP, 200 μg mL -1 AA), platelet aggregation rate was detected, all experiments were repeated 3 times, and the aggregation inhibition rate was calculated according to the average aggregation rate of each group and the average aggregation rate of the blank group: inhibition rate (%) = (average maximum aggregation rate of blank control group - average maximum aggregation rate of drug-treated group) / average maximum aggregation rate of blank control group × 100%.

[0116] (2) Experimental results

[0117] Table 1: Inhibitory effect of indoline aromatic acid ether derivatives on AA-induced platelet aggregation As can be seen from Table 1, compounds 5a to 5j inhibited AA-induced platelet aggregation to varying degrees, among which compounds 5c, 5f, 5g, 5h, and 5i were more active than the control compound [3-(2,3-dihydroindole-1-yl)propanol].

[0118] Table 2. Inhibitory effect of indoline aromatic acid ether derivatives on ADP-induced platelet aggregation As can be seen from Table 2, compounds 5a to 5j can significantly inhibit ADP-induced platelet aggregation, and their activities are stronger than those of the control compound [3-(2,3-dihydroindole-1-yl)propanol] and the positive control drug aspirin.

[0119] 2. Protective effects of compounds on hypoxia / reoxygenation (OGD / R)-mediated injury of primary neurons in rat cerebral cortex

[0120] (1) Experimental animals

[0121] Clean-grade SD pregnant rats, weighing 350-400 g, female, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and housed in an SPF-grade animal room with the indoor temperature controlled at 23±2°C. They were allowed to eat and drink freely with a 12h / 12h day and night schedule.

[0122] (2) Drug grouping:

[0123] Model group (OGD / R); positive control group (butylphthalide); control group (compound ) Drug group; the compound concentrations were 0.1 μmol / L, 1 μmol / L, and 10 μmol / L, and each concentration was biologically repeated 5 times.

[0124] (3) Test methods

[0125] Take SD rats (0-1d), fix the head, disinfect the scalp, open the skull and quickly remove the whole brain tissue, put it in a glass culture dish with D-Hank's solution on ice, carefully peel off the blood vessels and meninges on the surface of the brain tissue, remove the cerebellum and brain stem, and then rinse the brain tissue repeatedly with D-Hank's solution. Take the cerebral cortex, move it into a glass culture dish, cut it into pieces, add trypsin and mix it, place it in a 37℃ constant temperature water bath for digestion, then add serum-containing culture medium to terminate digestion, filter it with a 200-mesh sieve, centrifuge the filtrate (800rpm, 10min), discard the supernatant, add an appropriate amount of culture medium (neurobasal culture medium containing penicillin (final concentration of 100U / ml), streptomycin (final concentration of 100μg / ml), 2% B27 and L-glutamine (final concentration of 0.3mg / ml)) to suspend the precipitate, inoculate it in a 6-well plastic culture plate, and place it at 37℃ and 5% CO 2 After 36 hours of incubation, cytarabine (3 μg / ml) was added, and the medium was changed after 12 hours. Thereafter, the medium was changed by half every 3 days for 7 to 14 days.

[0126] (4) Preparation of rat cortical neurons subjected to oxygen-glucose deprivation and reoxygenation (OGD / R)

[0127] The cells grown to about day 7 were selected for the preparation of in vitro OGD / R model. The cell density was adjusted to 4×10 4 The cells were inoculated in a 12-well culture plate at a density of 10 cells / well. The OGD / R model was prepared after 24 hours of conventional culture using DMEM medium containing 1% FBS and corresponding concentrations of drugs. The culture medium was first replaced with DMEM medium without glucose and serum, and the cells were placed in a 5% CO 2 , 95% N 2 The cells were cultured in a three-gas incubator for 2 h to complete the hypoxia process. The cell culture medium was then replaced with complete DMEM medium. 2 Culture in the incubator for 24 hours to complete the reoxygenation process. 4 hours before the end of incubation, add 20 μL of MTT solution (5 mg / mL) to each well. After the incubation, discard the supernatant of each well, add 150 μL of DMSO to each well, and shake on a cell shaker for 10 minutes. After the crystals are fully dissolved, use an enzyme-labeled instrument to measure the OD 570 .

[0128] (5) Experimental results

[0129] Table 3 Protective effect of compounds on OGD / R model-mediated neuronal injury in rats

[0130]

[0131]

[0132] Note: Compared with the OGD / R group, the drug treatment group * P<0.05, ** P<0.01, drug treatment group compared with the butylphthalide group with equal molar concentration, # P<0.05, ## P<0.01.

[0133] As shown in Table 3, the compounds of the present invention exhibited different degrees of activity in protecting neurons from damage. Compared with the OGD / R model group, each concentration of the positive drug butylphthalide and compounds 5c, 5d and 5j could significantly increase the cell survival rate; each concentration of compounds 5c, 5d and 5j had no significant difference compared with the same concentration of butylphthalide.

[0134] 3. Experimental study on protection against acute focal cerebral ischemia-reperfusion injury in rats

[0135] (1) Experimental groups

[0136] The rats were randomly divided into 7 groups, with 8 rats in each group, namely sham operation group, model group, low-dose group of compound 5b (4 mg / kg), high-dose group of 5b (20 mg / kg), low-dose group of 5j (4 mg / kg), high-dose group of 5j (20 mg / kg) and positive control butylphthalide group (20 mg / kg).

[0137] (2) Drug preparation

[0138] Butylphthalide and compounds 5b and 5j were dissolved in 5% DMSO + 30% PEG400 + 65% medical sodium bicarbonate (25 mL / 1.25 g) to prepare a 10 mg / mL butylphthalide solution, and 2 mg / mL and 10 mg / mL solutions of compounds 5b and 5j.

[0139] (3) Modeling method

[0140] The middle cerebral artery occlusion (MCAO) reperfusion model was used to induce cerebral ischemia injury. Rats were anesthetized with 2% chloral hydrate (0.2 mL / 10 g, ip), the neck muscle tissue was separated, the right common carotid artery (CCA) was exposed and carefully freed; the accompanying vagus nerve was stripped, the external carotid artery (ECA) branch running inward and superficial was ligated and cut; the internal carotid artery (ICA) was followed forward and the pterygopalatine artery was separated and ligated near the skull base. The proximal end of the CCA was ligated with surgical thread; the distal end of the ligature was cut with ophthalmic scissors, and the nylon thread (diameter: 0.26 mm) was inserted along the small opening until a slight resistance was felt after about 20 mm, indicating that the front end of the thread plug had reached the anterior cerebral artery. The Doppler blood flow detector was used to determine the success of the model. At this point, the blood supply of the middle cerebral artery (MCA), including the blood supply from the carotid artery and the anterior communicating branch of the cerebral Waldeyer circle, had been blocked. The depth of the thread plug insertion was determined according to the mark. The outer part of the vascular ligature was ligated and fixed to prevent the ligature from slipping. After 90 minutes of right cerebral ischemia, the nylon thread in the lumen of the internal carotid artery was carefully withdrawn and the broken end was ligated to reperfuse the blood flow of the internal carotid artery. The skin was sutured and the rats were carefully fed after returning to the cage. The same surgical procedure was performed on sham-operated rats, but the middle cerebral artery was not blocked. Neurological deficits were scored 10 minutes later, and those with obvious neurological deficits were considered successful. The animal body temperature was kept constant at 37±0.5℃ during the operation.

[0141] (4) Dosage regimen

[0142] Three days before rat modeling, the drug was administered once a day through the tail vein. The sham operation group and the model group were given an equal amount of solvent. Two hours after the third administration, the ischemic model was created by surgery. After 90 minutes of ischemia, the nylon thread in the lumen of the internal carotid artery was carefully extracted and the broken ends were ligated to reperfuse the blood flow of the internal carotid artery. 24 hours after the third administration, the compound group and the positive control (butylphthalide) group were administered once more through the tail vein, and the sham operation group and the model group were injected with an equal amount of solvent, with an administration volume of 2 mL / kg body weight, and a total of 4 administrations.

[0143] (5) Testing indicators

[0144] Neurological deficit score:

[0145] After 24 h of reperfusion, the motor function of the rats was evaluated by the Zea-Longa method. The evaluation criteria were as follows: the higher the score, the more severe the nerve damage.

[0146] Scoring criteria:

[0147] 0 points: normal, no neurological damage; 1 point: incomplete extension of the left forelimb, slight neurological damage; 2 points: the rat turns toward the paralyzed side when walking, moderate neurological damage; 3 points: the rat falls toward the paralyzed side when walking, severe neurological damage; 4 points: the rat cannot walk at all, and consciousness is impaired.

[0148] (6) Experimental methods

[0149] The volume of cerebral infarction was evaluated by TTC method. After 24h of reperfusion, rats were anesthetized and killed with 10% chloral hydrate 3.5mL / kg, and the brain was taken out and placed in a -20℃ refrigerator for 30min. Five coronal brain slices with a thickness of about 2mm were cut from the frontal pole of the forebrain to the back. The brain slices were placed in 2% TTC solution (pH=7.4), incubated at 37℃ in the dark for about 30min, the solution was discarded, and 4% formaldehyde solution was added for fixation for 30min, and then observed and photographed. Normal brain tissue is bright red, and infarcted brain tissue is pale. After rinsing with physiological saline, the brain slices were quickly arranged in a row from front to back, and the residual water marks on the surface were dried and photographed. The photos were statistically analyzed using ImageJ image analysis software, the white ischemic area and the total area were circled, and the percentage of cerebral infarction area was calculated using the following formula: infarction rate % = ischemic area / total area × 100%. The experimental results are shown in Table 4.

[0150] Table 4. Effects of MCAO-reperfusion on neurobehavior and cerebral infarction rate in rats with focal cerebral ischemia (x±s, n=8)

[0151]

[0152] * P<0.05, ** P<0.01vs model control group; # P<0.05, ## P<0.01 vs butylphthalide group

[0153] According to the results in Table 4, 24 hours after MCAO reperfusion, compared with the sham operation group, the model control group rats had significant changes in neurobehavior, and the pale infarction area on the surgical side was very obvious, and the infarction area was large, indicating that the model was successfully established. After 3 days of preventive administration and MCAO reperfusion, compared with the model control group, the 20 mg / kg butylphthalide group, the low and high dose 5b group, and the high dose 5j group could significantly improve the animal neurological scores and reduce the cerebral infarction rate (P<0.01, P<0.05). Compared with the butylphthalide group, there was no significant difference in the effects of the 5b and 5j dose groups on the neurological function scores and cerebral infarction rates of rats.

[0154] In summary, the compounds of the present invention have significant antiplatelet aggregation activity and neuroprotective activity, showing good research value and medicinal prospects.

Claims

1. Compound represented by formula (I) or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts, in, R 1 Selected from hydrogen, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, halogen, hydroxyl, cyano, nitro, carboxyl; R 2 Selected from hydrogen, C 1 -C 20 alkyl; X is selected from C 6 -C 14 aryl, 5- to 14-membered heteroaryl, 3- to 14-membered heterocyclic group or C 3 -C 14 Carbocyclic group, which is optionally substituted by one or more groups selected from the group consisting of halogen, oxo, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, C 1 -C 20 Alkoxycarbonyloxy, hydroxy, cyano, nitro, carboxyl; m is selected from 0 or 1; n is selected from an integer between 0-4.

2. The compound according to claim 1, which is a compound represented by formula (II): or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts, in, R 1 Selected from hydrogen, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, halogen, hydroxyl, cyano, nitro, carboxyl; R 2 Selected from hydrogen, C 1 -C 20 alkyl; R 3 are independently selected from hydrogen, halogen, oxo, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, C 1 -C 20 Alkoxycarbonyloxy, hydroxy, cyano, nitro, carboxyl; m is selected from 0 or 1; n and p are each independently selected from 0, 1, 2, 3 or 4.

3. The compound according to claim 2 or its tautomer, cis-trans isomer, isotope-labeled substance and pharmaceutically acceptable salt, wherein R 1 Selected from hydrogen, C 1 -C 20 Alkyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, halogen; R 2 Selected from hydrogen, C 1 -C 6 alkyl; R 3 are independently selected from hydrogen, halogen, oxo, C 1 -C 20 Alkyl, C 2 -C 20 Alkenyl, C 2 -C 20 Alkynyl, C 1 -C 20 Haloalkyl, C 1 -C 20 Alkoxy, C 1 -C 20 Alkanoyl, C 1 -C 20 Alkanoyloxy, C 1 -C 20 Alkoxycarbonyl, C 1 -C 20 Alkoxycarbonyloxy, hydroxy, cyano, nitro, carboxyl; m and n are each independently selected from 0 or 1; p is selected from 0, 1, 2, 3 or 4.

4. The compound according to claim 3 or its tautomer, cis-trans isomer, isotope-labeled substance and pharmaceutically acceptable salt, wherein R 1 is selected from hydrogen, methoxy; R 2 is selected from hydrogen, methyl, ethyl; R 3 is selected from hydrogen, methoxy; n and m are each independently selected from 0 or 1; p is selected from 0, 1 or 2.

5. The compound according to any one of claims 2 to 4, which is a compound represented by formula (III): or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts, in, R 1 , R 2 , R 3 , n, m, p are as defined in any one of claims 2-4.

6. The compound according to claim 1, selected from: 4-(3-(2,3-dihydroindolin-1-yl)propoxy)-3-methoxybenzoic acid; (E)-3-(4-(3-(2,3-dihydroindolin-1-yl)propoxy)-3-methoxyphenyl)acrylic acid; (E)-3-(4-(3-(2,3-dihydroindolin-1-yl)propoxy)phenyl)acrylic acid; (E)-3-(4-(2-(2,3-dihydroindolin-1-yl)ethoxy)-3-methoxyphenyl)acrylic acid; 4-(3-(2,3-dihydroindolin-1-yl)propoxy)benzoic acid; (E)-3-(3-methoxy-4-(2-(5-methoxy-2,3-dihydroindolin-1-yl)ethoxy)phenyl)acrylic acid; 4-(3-(2,3-dihydroindolin-1-yl)propoxy)-3,5-dimethoxybenzoic acid; (E)-3-(3-methoxy-4-(3-(5-methoxy-2,3-dihydroindolin-1-yl)propoxy)phenyl)acrylic acid; 3-Methoxy-4-(3-(5-methoxy-2,3-dihydroindolin-1-yl)propoxy)benzoic acid; (E)-3-(4-(2-(2,3-dihydroindolin-1-yl)ethoxy)phenyl)acrylic acid; or its tautomers, cis-trans isomers, isotope-labeled substances and pharmaceutically acceptable salts.

7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 6 or its tautomer, cis-trans isomer, isotope label and pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

8. Use of the compound according to any one of claims 1 to 6 or its tautomer, cis-trans isomer, isotope-labeled substance, pharmaceutically acceptable salt or the pharmaceutical composition according to claim 7 in the preparation of a medicament for antiplatelet aggregation.

9. Use of the compound according to any one of claims 1 to 6 or its tautomer, cis-trans isomer, isotope-labeled substance, pharmaceutically acceptable salt or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing and / or treating diseases related to nerve damage.

10. The use according to claim 9, wherein the nerve damage-related diseases include ischemic stroke, posterior circulation cerebral ischemia, amyotrophic lateral sclerosis, epilepsy, Huntington's disease, Alzheimer's disease, multiple sclerosis, multiple system atrophy, Parkinson's disease, primary lateral sclerosis peripheral neuropathy, and diabetic neuropathy.

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