Preparation method and application of diaza-triquinolinedione derivative

Abstract

The invention provides a preparation method and application of a diaza-triquinolinedione derivative. Provided is the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of cerebral apoplexy and / or inflammatory diseases, neuroprotection, free radical scavenging, and / or alleviation of oxidative stress. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the use of diazatriquinidinedione compounds in the preparation of drugs for treating ischemic stroke or inflammatory response. Background Art

[0002] Ischemic stroke is a serious injury caused by local brain tissue ischemia and hypoxia. It is the leading cause of disability and death among adults worldwide, and imposes a huge burden on families and society. At present, there is a lack of effective drugs for the treatment of stroke in clinical practice. The commonly used treatment strategy is to restore blood flow through thrombolysis or mechanical thrombectomy to save ischemic brain tissue, but the treatment time window is narrow, and reperfusion may cause secondary damage. Less than half of the patients actually benefit from it.

[0003] Therefore, there is an urgent need to develop compounds with neuroprotective effects in clinical practice.

[0004] Although a variety of single-target neuroprotectants have been developed based on target screening, the results in clinical trials and applications are not ideal. In addition to target screening, phenotypic screening is another effective way to discover new structures and new target drugs.

[0005] There are no reports on the neuroprotective and anti-inflammatory activities of diazatriquinidinedione compounds. Summary of the invention

[0006] Provided is a use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing stroke and / or inflammatory diseases, neuroprotection, scavenging free radicals, and / or alleviating oxidative stress,

[0007]

[0008] in,

[0009] R 1 may be an optionally substituted aryl, heteroaryl, arylvinyl, cycloalkyl, or heterocycloalkyl; and

[0010] R 2 It may be an optionally substituted aryl group, heteroaryl group, arylvinyl group, cycloalkyl group, or heterocycloalkyl group.

[0011] The compound of formula (I) of the present application or a pharmaceutically acceptable salt thereof has been shown to be able to improve ischemic damage, improve organic damage and neurobehavioral damage caused by ischemia-reperfusion, reduce neural apoptosis and ferroptosis induced by ischemia-reperfusion, reduce the accumulation of reactive oxygen species (ROS) and cell damage induced by oxygen-glucose deprivation-reperfusion, have significant neuroprotective effects, and inhibit inflammation, and can treat ischemic stroke or inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :Compound 1 (a) 1 H NMR spectrum and (b) 13 C NMR spectrum.

[0013] Figure 2 :Compound 38 (a) 1 H NMR spectrum and (b) 13 C NMR spectrum.

[0014] Figure 3 :(a) Primary neuronal oxygen glucose deprivation (OGD) model mice were treated with different concentrations of compound 1 (2.5μM, 5.0μM, 10μM, 20μM, 40μM) or 40μM positive control NA-1 (also known as nerinetide, Tat-NR2B9c), and the cell viability was measured by CCK-8. (b) OGD model mice were treated with different concentrations of compound 1, NA-1, or edaravone, or (c) different concentrations of compound 38, and the cell viability was measured by CCK-8. (a) The horizontal axis from left to right is the solvent control group, 40μM NA-1 control group, 2.5μM compound 1 group, 5.0μM compound 1 group, 10μM compound 1 group, 20μM compound 1 group, 40μM compound 1 group; (b) The horizontal axis is the logarithmic value of the concentration (μM) of compound 1, NA-1, or edaravone; (c) The horizontal axis is the concentration (μM) of compound 38. The vertical axis is the normalized cell viability value relative to the solvent control group. (b) The shape "■" represents each compound 1 group, the shape "▲" represents each NA-1 positive control group, and the shape "△" represents each edaravone positive control group. (a) ANOVA was performed first between the means of multiple groups, and then Dunnett's T test was used for post hoc analysis of the differences between groups. The displayed values ​​are mean ± standard deviation. n = 6; * p<0.05, *** p<0.001, **** p<0.0001.

[0015] Figure 4 :(a)–(b) OGD model mice were treated with different concentrations of compound 1 and edaravone, and the expression level of caspase-3 protein was detected, and vinculin was used as an internal reference. (a–b) From left to right, they are normal group, vehicle control group, 10μM edaravone group, 10μM compound 1 group, 20μM compound 1 group, and 40μM compound 1 group. (a) The upper part is caspase-3, and the lower part is vinculin. (b) The vertical axis is the normalized absorbance (OD) value relative to the internal reference of vinculin in the same group. ****p<0.0001; ns: not statistically significant.

[0016] Figure 5 :Compound 1 (10μM, 20μM, 40μM) or compound 38 (0.25, 2.5, 5μM) at different concentrations was used to inhibit the release of inflammatory factors in mouse primary microglia induced by lipopolysaccharide (LPS), and the (a) interleukin 6 (IL-6) content and (b) interleukin 1β (IL-1β) content were detected by ELISA. (a) The horizontal axis from left to right is the vehicle control group, model control group, 10μM compound 1 group, 20μM compound 1 group, 40μM compound 1 group; (b) The horizontal axis from left to right is the vehicle control group, model control group, 10μM compound 1 group, 20μM compound 1 group, 40μM compound 1 group, 0.25μM compound 38 group, 2.5μM compound 38 group, 5μM compound 38 group. The vertical axis is (a) IL-6 content (pg / mL); (b) IL-1β content (pg / mL). First, the variance analysis between the means of multiple groups was performed, and then Dunnett's T test was used for post hoc analysis of the differences between groups. n = 6; compared with the blank control group: ** p<0.01, *** p<0.001, **** p<0.0001; compared with the model control group, ###p<0.001.

[0017] Figure 6 :Compound 1 at different concentrations (10 μM, 20 μM, 40 μM) was used to inhibit the release of inflammatory factors in mouse primary neutrophils induced by lipopolysaccharide (LPS), and the IL-6 content was detected by ELISA. The horizontal axis is the vehicle control group, model control group, 10 μM compound 1 group, and 20 μM compound 1 group from left to right. The vertical axis is the normalized IL-6 content (%), with the model control group as 100%. ANOVA was performed between the means of multiple groups, and then Dunnett's T test was used for post hoc analysis of the differences between groups. n=6; compared with the blank control group: ** p<0.01, *** p<0.001, **** p<0.0001; compared with the model control group, ###p<0.001.

[0018] Figure 7: Intraluminal middle cerebral artery occlusion / reperfusion (MCAO / R) model mice were treated with different concentrations of compound 1 (20 μM, 40 μM), (a) TTC staining of mouse brain sections and (b) statistics of the percentage of infarct volume shown in the staining, as well as (c) Longa score of neural injury, (d) modified neurological score (mNSS) and (e) survival rate of mice within 24 hours. (a) and (b)-(d) horizontal axes, from left to right, are sham operation group, vehicle control group, low-dose group, and high-dose group; (e) horizontal axis, from left to right, is vehicle control group, low-dose group, and high-dose group. The vertical axis is (b) infarct volume (%); (c) Longa score of neural injury; (d) modified neurological score; and (e) survival rate (%). n=45; * p<0.05, ** p<0.01, *** p<0.001, ns: not statistically significant. DETAILED DESCRIPTION

[0019] In order to further explain the technical means and effects adopted by the present application to achieve the predetermined purpose, the specific implementation methods, structures, features and effects of the present application are described in detail below in combination with the accompanying drawings, various embodiments (including manufacturing examples and experimental examples) and preferred implementation methods.

[0020] Terms and Definitions

[0021] As used herein, singular terms refer to one or more than one. For example, "element" or "an element" refers to one element or more than one element. As used herein, the term "plurality" refers to at least two.

[0022] As used herein, all features or conditions defined in the form of numerical ranges or percentage ranges mentioned by endpoints are only for brevity and convenience. Therefore, the description of numerical ranges or percentage ranges should be deemed to have covered and specifically disclosed all possible subranges and all individual integer and fractional values ​​within the range, especially integer values. For example, the range description of "1 to 8" should be deemed to have specifically disclosed all subranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8... etc., especially subranges defined by all integer values, and should be deemed to have specifically disclosed individual values ​​such as 1, 2, 3, 4, 5, 6, 7, 8 within the range; regardless of whether these ranges or these individual values ​​are recorded literally. Similarly, the range description of "between 1 and 8" should also be deemed to have specifically disclosed all ranges such as 1 to 8, 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., and include the endpoint values ​​of these ranges, such as 1, 2, 3, 4, 5, 6, 7, 8, etc. Individual values.

[0023] As used herein, the term "about" refers to approximation, in the range of about or near. When the term "about" is used in conjunction with a numerical range, it modifies the range by expanding the limit above or below the provided numerical value. In general, the term "about" is used herein to change the numerical value up and down by 10% from the provided value. On the one hand, the term "about" refers to adding or subtracting 20% ​​of the numerical value of the number it modifies. For example, "about 50%" refers to within the range of 45%–55%. It should also be understood that all integers and fractions thereof are considered to be modified by the term "about". In this article, under the premise that the purpose of the invention can be achieved, the numerical value should be understood to have the accuracy of the significant digits of the numerical value. For example, the number 40.0 can be understood to cover the range from 39.50 to 40.49.

[0024] As used herein, the terms "comprising", "including", or "containing", as non-exclusive or open terms, are intended to indicate that a combination (e.g., a device, a composition, a method, etc.) includes the listed elements (e.g., each unit of a device, each component of a composition, a substantial step of a method, etc.), but does not exclude other elements. As used herein, the term "substantially consisting of..." when used to define compositions and methods means excluding other elements that have any substantial impact on the combination of the stated purpose, but does not exclude other elements that do not substantially affect the basic and novel features of the present invention. As used herein, the term "consisting of..." refers to a combination (units, components, substantial steps, etc.) excluding other elements, but unless otherwise specified, it is not intended to exclude trace amounts of unavoidable impurities. The embodiments defined by each of these connecting terms are within the scope of the present invention. As a specific embodiment thereof, the technical solution disclosed including the terms "comprising", "including", or "containing" should also be deemed to simultaneously disclose the corresponding technical solutions including the terms "substantially consisting of..." and "consisting of...".

[0025] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more associated listed items. When used in a list of two or more items, the term "and / or" represents that any one of the listed items can be included individually, or can include any combination of two or more listed items. For example, if a group, combination, or composition, etc., is described as including (or including) components A, B, C, and / or D, then the composition can include A alone, B alone, C alone, D alone, a combination of A and B, a combination of A and C, a combination of A and D, a combination of B and C, a combination of B and D, a combination of C and D, a combination of A and B and C, a combination of A and B and D, a combination of A and C and D, a combination of B and C and D, or a combination of A and B and C and D.

[0026] As used herein, for any use of a Markush group or option list to describe features or embodiments of the present application, a subgroup or any individual member of the member within the Markush group or option list also constitutes a part of the present application. For example, if it is stated herein that X is "selected from the group consisting of A, B, and C", it means that the technical solution in which X is A, the technical solution in which X is B, the technical solution in which X is C, the technical solution in which X is A and / or B, the technical solution in which X is A and / or C, the technical solution in which X is B and / or C, and the technical solution in which X is A and / or B and / or C are disclosed respectively where chemically permitted.

[0027] The compounds of the present application may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomers of the compounds of the present application, including but not limited to diastereomers, enantiomers, and atropisomers, and mixtures thereof such as racemic mixtures, should be considered as part of the present application.

[0028] As used herein, the term "(substituted) substituted" means that one or more hydrogens on a specified group are replaced by a specific group, radical or moiety, i.e., a substituent, under the premise that it is chemically permitted, so that the specified group has one or more of the substituents. As used herein, the term "substituent" refers to a chemical group, radical or moiety that is covalently linked or (if appropriate) fused to a parent group or moiety. As used herein, the term "optionally (substituted) substituted" means that one or more hydrogens on a specified group are optionally replaced by a substituent, which includes the parallel technical schemes of "substituted" and "unsubstituted", and can be equivalent to the term "substituted or unsubstituted". This term can further limit the substituent to a specified substituent, for example, the term "optionally methyl substituted" means that it is optionally substituted by methyl, which includes the parallel technical schemes of "substituted by methyl" and "unsubstituted", and can be equivalent to the term "substituted or unsubstituted by methyl". This term may also be further modified by quantifiers, for example, the term "optionally mono-substituted" means optionally substituted by a substituent, which includes the parallel technical solutions of "substituted by a substituent" and "not substituted", and is equivalent to the term "substituted by a substituent"; the term "optionally methyl mono-substituted" means optionally substituted by a methyl group, which includes the parallel technical solutions of "substituted by a methyl group" and "not substituted", and is equivalent to the term "substituted by a methyl group".

[0029] As used herein, the term "pharmaceutically acceptable salts" may refer to salts prepared from pharmaceutically acceptable non-toxic bases or acids.

[0030] When the compound is acidic, its corresponding salt can be prepared from pharmaceutically acceptable non-toxic bases (including inorganic bases and organic bases). Salts derived from inorganic bases include, but are not limited to, aluminum salts, ammonium salts, calcium salts, copper salts, cuprous salts, iron salts, ferrous salts, lithium salts, magnesium salts, potassium salts, sodium salts, zinc salts, etc., especially ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts. Salts derived from organic bases include salts of primary, secondary, and tertiary amines as well as cyclic amines and substituted amines (such as naturally occurring and synthetic substituted amines). Salts derived from organic bases include, but are not limited to, salts of arginine, betaine, caffeine, choline, N′,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methyl reduced glucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0031] When the compound is alkaline, its corresponding salt can be prepared from pharmaceutically acceptable non-toxic acids (including inorganic and organic acids). Salts derived from organic acids include, but are not limited to, acetate, benzenesulfonate, benzoate, camphorsulfonate, citrate, ethanesulfonate, fumarate, gluconate, glutamate, hydrobromide, hydrochloride, isethionate, lactate, maleate, malate, mandelate, methanesulfonate, mucate, nitrate, pamoate, pantothenate, phosphate, succinate, sulfate, tartrate, p-toluenesulfonate, etc.

[0032] As used herein, the term "treatment" may refer to eradicating, inhibiting, alleviating, or delaying the development of a disease, disorder, or condition, such as eradicating, inhibiting, alleviating, or delaying the development of at least one clinical symptom thereof, or alleviating or improving at least one physical parameter, including those that the patient may not recognize. "Treatment" may be physically (e.g., stabilization of discernible symptoms), physiologically) (e.g., stabilization of physical parameters), or regulating a disease, disorder, or condition in both. "Treatment" may be to slow down the onset or development rate of a condition, disorder, or disease, reduce or alleviate the symptoms associated therewith, generate complete or partial regression of a condition, or a combination thereof. The results obtained by "treatment" include, but are not limited to, reduction or alleviation of symptoms, reduction of the extent of the disease, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or slowing of the disease state, and alleviation (partial or complete slowing), whether detectable or undetectable. "Treatment" may also refer to prolonged survival relative to the expected survival time when not receiving treatment. "Treatment" may also encompass the inhibition or reduction of an increase in the severity of a pathological state or symptom relative to the absence of treatment, and is not necessarily meant to imply a complete cessation of the disease, disorder, or condition in question. An individual (e.g., a human) in need of treatment may be an individual already suffering from the disease, disorder, or condition in question.

[0033] As used herein, the term "prevention" may refer to eradication, inhibition, mitigation, or delaying the risk of developing a disease, disorder, or condition, or eradication, inhibition, mitigation, or improvement of the potential disease, disorder, or condition to be prevented. An individual (e.g., a person) in need of prevention may be an individual who has not yet suffered from the disease, disorder, or condition in question. "Prevention" includes delaying the onset of a disease relative to when treatment has not yet been given to an individual, and does not necessarily mean that the disease, disorder, or condition is permanently prevented. In order to obtain benefits in terms of prevention, a pharmaceutical composition may be administered to a patient at risk of developing a particular disease or to a patient with one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed. Unless otherwise indicated, methods for evaluating the therapeutic and / or preventive effects of a disease are generally known in the art.

[0035] Compound

[0036] In some embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided.

[0037]

[0038] R 1 It may be an optionally substituted aryl group, heteroaryl group, arylvinyl group, cycloalkyl group, or heterocycloalkyl group.

[0039] R2 It may be an optionally substituted aryl group, heteroaryl group, arylvinyl group, cycloalkyl group, or heterocycloalkyl group.

[0040] The optional substitution may refer to optional substitution by a substituent, i.e., R 1 and R 2 Each may independently be (the same or different) an aryl group, a heteroaryl group, an arylvinyl group, a cycloalkyl group, or a heterocycloalkyl group, which may be optionally substituted with one or more substituents.

[0041] In some embodiments, the substituents (when there are multiple substituents, each independently) can be: halogen (such as fluorine, chlorine, bromine, iodine); alkyl (such as C 1-6 Alkyl groups, especially C 1-4 alkyl, especially methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, etc.); haloalkyl (such as trifluoromethyl, etc.); cycloalkyl (such as C 3-8 Cycloalkyl, especially cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.); alkenyl (such as C 2-6 alkenyl, especially vinyl, prop-1-enyl, prop-2-enyl, but-1-enyl, but-2-enyl, but-3-enyl, etc.); alkynyl (such as C 2-6 The invention also includes but is not limited to alkenyl, especially ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, etc.); aryl (such as phenyl, naphthyl, anthracenyl, etc.); aralkyl (such as benzyl, phenethyl, etc.); amino; primary amino (such as methylamino, ethylamino, propylamino, butylamino, phenylamino, etc.); secondary amino (such as dimethylamino, diethylamino, diphenylamino, etc.); aminoalkyl (such as aminomethyl, aminoethyl, aminopropyl, aminobutyl, etc.); acylamino (such as formylamino, acetylamino, propionylamino, butyrylamino, benzylamino, etc.); The present invention also includes alkyl, alkyl, alkyl, alkylene ...

[0042] In some embodiments, the substituents (when there are multiple substituents, each independently) may be: halogen (such as fluorine, chlorine, bromine, iodine); haloalkyl (such as trifluoromethyl, etc.); alkoxy (such as methoxy, ethoxy, propoxy, butoxy, etc.); alkyl (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, etc.); alkynyl (such as ethynyl, etc.); aryl (such as phenyl, naphthyl, etc.); cyano; nitro; alkoxycarbonyl (such as tert-butyloxycarbonyl); or siloxy (for example tert-butyldimethylsiloxy).

[0043] In some embodiments, R 1 or R 2 It can be an optionally substituted aryl, in particular an optionally substituted phenyl, an optionally substituted naphthyl, or an optionally substituted anthracenyl. In some embodiments, the optionally substituted aryl (such as an optionally substituted phenyl, naphthyl, or anthracenyl) can be a halogen-substituted aryl (such as a halogen and / or alkynyl-substituted phenyl, naphthyl, or anthracenyl). In some embodiments, the optionally substituted phenyl (including the optionally substituted phenyl in the optionally substituted styryl) can be a halogen-substituted phenyl. In some embodiments, R 1 , R 2 Or the optionally substituted phenyl group may be wherein: n is 0, 1, 2, 3, 4, or 5, in particular 1, 2, 3, 4, or 5, more in particular 1; each X is independently selected from the substituents described above, in particular halogen or trihalomethyl, more in particular fluorine, chlorine, bromine, iodine, or trifluoromethyl. Halogen may be fluorine, chlorine, bromine, or iodine. Trihalomethyl may be trifluoromethyl.

[0044] In some embodiments, R 1 or R 2 It may be phenyl.

[0045] In some embodiments, R 1 , R 2The optionally substituted phenyl may be a halophenyl (e.g., 2-halophenyl, 3-halophenyl, or 4-halophenyl), a dihalophenyl (e.g., 2,3-dihalophenyl, 2,4-dihalophenyl, 2,5-dihalophenyl, 2,6-dihalophenyl, 3,4-dihalophenyl, or 3,5-dihalophenyl), a trihalophenyl (e.g., 2,3,4-trihalophenyl, 2,3,5-trihalophenyl, 2,3,6-trihalophenyl, 2,4,5-trihalophenyl, 2,4,6-trihalophenyl, or 3,4, 5-trihalophenyl), tetrahalophenyl (such as 2,3,4,5-tetrahalophenyl, 2,3,4,6-tetrahalophenyl, or 2,3,5,6-tetrahalophenyl), or pentahalophenyl (2,3,4,5,6-pentahalophenyl); wherein the halogen in the halophenyl, the two halogens in the dihalophenyl, the three halogens in the trihalophenyl, the four halogens in the tetrahalophenyl, or the five halogens in the pentahalophenyl can be (when there are multiple halogens, each independently) selected from the group consisting of fluorine, chlorine, bromine, and iodine.

[0046] In some embodiments, R 1 , R 2 Or the optionally substituted phenyl may be an alkyl substituted phenyl. 1 , R 2 Or the optionally substituted phenyl group may be an alkyl substituted phenyl group, in particular a phenyl group substituted by methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.

[0047] In some embodiments, R 1 , R 2 Or the optionally substituted phenyl may be an alkyl substituted phenyl. 1 , R 2 Or the optionally substituted phenyl group may be an alkyl substituted phenyl group, in particular a phenyl group substituted by methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.

[0048] In some embodiments, R 1 and R 2 At least one of them may be a phenyl group optionally substituted with a single or double halogen or trifluoromethyl group; that is, R 1 and R 2 At least one of the may be a phenyl group which is unsubstituted or substituted by one or two substituents, wherein the substituent (or the two substituents are each independently) is a halogen (i.e., fluorine, chlorine, bromine, or iodine) or a trifluoromethyl group.

[0049] In some embodiments, R 1 or R 2 Can be an optionally substituted heteroaryl. In some embodiments, R 1 , R 2Or the optionally substituted heteroaryl can be optionally substituted aza, oxa, thia, azaoxa, or azathia, a five-membered heterocyclic aromatic group, a six-membered heterocyclic aromatic group, a six-membered ring and a five-membered heterocyclic aromatic group (such as a benzo five-membered heterocyclic aromatic group), or a six-membered ring and a six-membered heterocyclic aromatic group (such as a benzo six-membered heterocyclic aromatic group).

[0050] In some embodiments, R 1 or R 2 It may be an optionally substituted pyrrolyl, furyl, thienyl, pyrazolyl, imidazolyl, Azolyl, iso oxazolyl, thiazolyl, isothiazolyl, pyridyl, pyranyl, thiopyranyl, pyridazinyl, pyrimidinyl, pyrazinyl, oxazinyl, thiazinyl, benzopyrrolyl (such as indolyl or isoindolyl), benzofuranyl, benzothiophenyl, benzopyrazolyl, benzimidazolyl (such as indazolyl), benzo Azolyl, benzyl oxazolyl, benzothiazolyl, benzisothiazolyl, benzopyridinyl (e.g., quinolyl or isoquinolyl), benzopyranyl (e.g., chromenyl or isochromenyl), benzothiopyranyl, benzopyridazinyl (e.g., oxazolyl or phthalazinyl), benzopyrimidinyl (quinazolinyl), benzopyrazinyl (quinoxalinyl), benzo The invention also includes but is not limited to oxazinyl, benzothiazinyl, purinyl, naphthyridinyl, or pteridinyl.

[0051] In some embodiments, R 1 or R 2 It may be optionally substituted pyridinyl (such as optionally substituted pyridin-2-yl, pyridin-3-yl, or pyridin-4-yl), pyrrolyl (such as optionally substituted 1H-pyrrol-2-yl or 1H-pyrrol-3-yl), quinolyl (such as optionally substituted quinol-2-yl, quinol-3-yl, or quinol-4-yl), indolyl (such as optionally substituted 1H-indol-2-yl or 1H-indol-3-yl), or thienyl (such as optionally substituted thien-2-yl or thien-3-yl), in particular pyridinyl, pyrrolyl, quinolyl, indolyl, or thienyl optionally substituted with methyl mono-substituted.

[0052] In some embodiments, R 1 or R 2 It may be an optionally substituted aromatic vinyl group, in particular an optionally substituted styryl group, an optionally substituted naphthyl vinyl group, or an optionally substituted anthracenyl group. 1 , R 2 Or the optionally substituted arylvinyl group may be: -C=C-Q, wherein Q is an optionally substituted aryl group, in particular an optionally substituted phenyl group, and may be an optionally substituted aryl group or an optionally substituted phenyl group as defined above. 1 , R 2or the optionally substituted arylvinyl group may be an optionally substituted (E)-arylvinyl group or optionally substituted (Z)-arylene vinyl In particular, optionally substituted (E)-arylvinyl. 1 , R 2 The optionally substituted aromatic vinyl group may be an optionally substituted (E)-styryl group or an optionally substituted (Z)-styryl group, particularly an optionally substituted (E)-styryl group.

[0053] In some embodiments, R 1 or R 2 Can be an optionally substituted cycloalkyl. 1 or R 2 Can be optionally substituted C 3-8 Cycloalkyl is in particular optionally substituted cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane or cyclooctane.

[0054] In some embodiments, R 1 or R 2 It may be an optionally substituted heterocycloalkyl. In particular, optionally substituted aza, oxa, thia, azaoxa, or azathia, a three-membered heterocycloalkyl, a four-membered heterocycloalkyl, a five-membered heterocycloalkyl, a six-membered heterocycloalkyl, a seven-membered heterocycloalkyl, or an eight-membered heterocycloalkyl. In some embodiments, R 1 , R 2 or the optionally substituted heterocycloalkyl group may be pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolidinyl, imidazolidinyl, Oxazolidinyl, isocyanate oxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, hexahydropyridazinyl, hexahydropyrimidinyl, piperazinyl, azinyl group, or a thiazinyl group.

[0055] In some embodiments, R 1 or R 2The phenyl group may be selected from halophenyl (e.g. fluorophenyl, chlorophenyl, bromophenyl, or iodophenyl, in particular 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, or 4-iodophenyl); dihalophenyl (e.g. difluorophenyl, dichlorophenyl, or chlorofluorophenyl, in particular 3,4-dichlorophenyl, 3,5-dibromophenyl, 2-chloro-4-fluorophenyl); haloalkylphenyl (e.g. trifluoromethylphenyl, in particular 2-trifluoromethylphenyl or 4-trifluoromethylphenyl); bis(haloalkyl)phenyl (e.g. bis(trifluoromethyl)phenyl, in particular 3,5-bis(trifluoromethyl)phenyl); alkoxyphenyl (e.g. methoxyphenyl, in particular 3-methoxyphenyl or 4-methoxyphenyl); phenyl, especially 3-methoxyphenyl); alkylphenyl (such as isopropylphenyl or tert-butylphenyl, especially 4-isopropylphenyl or 4-tert-butylphenyl); cyanophenyl (such as 4-cyanophenyl); nitrophenyl (such as 4-nitrophenyl); siloxyphenyl (such as (tert-butyldimethylsilyloxy)phenyl, especially 4-(tert-butyldimethylsilyloxy)phenyl); naphthyl (such as 1-naphthyl); heteroaryl (such as furanyl, thienyl, or piperidinyl optionally substituted with tert-butyloxycarbonyl, especially 2-furanyl, 2-thienyl, or tert-butyloxycarbonyl-4-piperidinyl); styryl; and cycloalkyl (such as cyclohexyl). In some embodiments, R 1 or R 2 The group consisting of a halophenyl group (e.g., a fluorophenyl group or a chlorophenyl group, in particular, a 4-fluorophenyl group or a 4-chlorophenyl group), a dihalophenyl group (e.g., a dichlorophenyl group, in particular, a 3,4-dichlorophenyl group), a haloalkylphenyl group (e.g., a trifluoromethylphenyl group, in particular, a 4-trifluoromethylphenyl group), a bis(haloalkyl)phenyl group (e.g., a bis(trifluoromethyl)phenyl group, in particular, a 3,5-bis(trifluoromethyl)phenyl group), an alkoxyphenyl group (e.g., a methoxyphenyl group, in particular, a 3-methoxyphenyl group), an alkylphenyl group (e.g., an isopropylphenyl group or a tert-butylphenyl group, in particular, a 4-isopropylphenyl group or a 4-tert-butylphenyl group), and a cycloalkyl group (e.g., a cyclohexyl group).

[0056] In some embodiments, R 1 and R 2 Each of the following may be independently selected from the group consisting of phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 3,4-dichlorophenyl, 3,5-dibromophenyl, 2-chloro-4-fluorophenyl, 2-trifluoromethylphenyl, 4-trifluoromethylphenyl, 3,5-bis(trifluoromethyl)phenyl, 3-methoxyphenyl, 4-methoxyphenyl, 4-methylphenyl, 4-ethynylphenyl, 4-isopropylphenyl, 4-tert-butylphenyl, 4-cyanophenyl, 4-nitrophenyl, 4-(tert-butyldimethylsilyloxy)phenyl, 1-naphthyl, 2-furyl, 2-thienyl, tert-butyloxycarbonyl-4-piperidinyl, styryl, and cyclohexyl. In some embodiments, R 1 and R 2The groups may be each independently selected from the group consisting of a phenyl group, a 4-fluorophenyl group, a 4-chlorophenyl group, a 3,4-dichlorophenyl group, a 4-trifluoromethylphenyl group, a 3-methoxyphenyl group, a 4-isopropylphenyl group, a 4-tert-butylphenyl group, and a cyclohexyl group.

[0057] In some embodiments, R 1 and R 2 In some embodiments, R 1 and R 2 different.

[0058] use

[0059] On the one hand, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (or drug) comprising it for the preparation of a drug for treating or preventing stroke and / or inflammatory diseases. On the one hand, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (or drug) comprising it for the treatment or prevention of stroke and / or inflammatory diseases. On the one hand, there is provided a pharmaceutical composition (or drug) for the treatment or prevention of stroke and / or inflammatory diseases, including a compound of formula (I) or a pharmaceutically acceptable salt thereof. On the one hand, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (or drug) comprising it for the treatment or prevention of stroke and / or inflammatory diseases. On the one hand, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (or drug) comprising it for the treatment or prevention of stroke and / or inflammatory diseases. On the one hand, there is provided a method for treating or preventing stroke and / or inflammatory diseases, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (or drug) comprising it.

[0060] On the one hand, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (or drug) comprising it for the preparation of a drug for neuroprotection, removal of reactive oxygen species, and / or alleviation of oxidative stress. On the one hand, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (or drug) comprising it for neuroprotection, removal of reactive oxygen species, and / or alleviation of oxidative stress. On the one hand, there is provided a pharmaceutical composition (or drug) for neuroprotection, removal of reactive oxygen species, and / or alleviation of oxidative stress, including a compound of formula (I) or a pharmaceutically acceptable salt thereof. On the one hand, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof for neuroprotection, removal of reactive oxygen species, and / or alleviation of oxidative stress, or a pharmaceutical composition (or drug) comprising it. On the one hand, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (or drug) comprising it for neuroprotection, removal of reactive oxygen species, and / or alleviation of oxidative stress. In one aspect, a method for neuroprotection, scavenging reactive oxygen species, and / or alleviating oxidative stress is provided, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition (or drug) comprising the same.

[0061] In some embodiments, the stroke is ischemic stroke, particularly cerebral ischemia reperfusion caused stroke, also particularly cerebral ischemia reperfusion injury. In some embodiments, the stroke is ischemia, particularly focal transient ischemia, the brain damage caused. In some embodiments, the stroke includes organic brain damage, neurobehavioral deficits or neuronal cell damage, particularly organic brain damage, neurobehavioral deficits or neuronal cell damage caused by ischemia reperfusion. In some embodiments, the ischemia (such as ischemic stroke ischemic stroke) is caused by local blood circulation disorder, particularly the local blood circulation disorder caused by thrombosis, inflammation, hypertension, atherosclerosis or vascular malformation.

[0062] In some embodiments, the inflammatory disease is inflammation, in particular cellular inflammatory response. In some embodiments, the inflammatory disease or inflammation is inflammation after cerebral ischemia. In some embodiments, the inflammatory disease or inflammation is inflammation caused by ischemia, in particular inflammation caused by cerebral ischemia; or inflammation caused by lipopolysaccharide reduction; or inflammation caused by acute stress.

[0063] In some embodiments, the inflammatory disease (or inflammation) is inflammation of one or more of the skin, muscles, bones, joints, cartilage, cerebrum, cerebellum, brain stem, spinal cord, eyes, ears, nose, tongue, heart, blood vessels, throat, trachea, lungs, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, kidneys, bladder, urethra, ovaries, uterus, testicles, spleen, thymus, lymphatic vessels, or lymph nodes.

[0064] In some embodiments, the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, in the medicament or pharmaceutical composition, the compound of formula (I) or a pharmaceutically acceptable salt thereof is an active pharmaceutical ingredient, particularly the only active pharmaceutical ingredient.

[0065] In some embodiments, the drug or pharmaceutical composition is an oral administration preparation, a parenteral administration preparation, an inhalation spray administration preparation, a drop administration preparation, or an implant administration preparation. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered at a dose of 0.1-50 mg / kg patient weight, particularly 0.1-10 mg / kg, 0.1-5 mg / kg, 0.5-10 mg / kg, or 0.5-5 mg / kg patient weight, or particularly 10-50 mg / kg, 15-50 mg / kg, 20-50 mg / kg patient weight. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered at a dose of 0.1-50 μM / kg patient body weight, particularly 0.1-10 μM / kg, 0.1-5 μM / kg, 0.5-10 μM / kg, or 0.5-5 μM / kg patient body weight, or particularly 10-50 μM / kg, 15-50 μM / kg, 20-50 μM / kg patient body weight.

[0066] Example

[0067] The reagents used in the examples of this application are as follows:

[0068] -2,3,5-Triphenyltetrazolium chloride (TTC) was purchased from Biosharp (catalog number BS095-25g)

[0069] -4% paraformaldehyde was purchased from Abisson (Cat. No. abs9179)

[0070] -Poly-L-lysine was purchased from Sigma (Cat. No. P4832)

[0071] -DMEM high glucose medium was purchased from Gibco (Cat. No. C11995500bt)

[0072] -Pancreatin-EDTA was purchased from Gibco (Cat. No. 25200056)

[0073] - Fetal bovine serum was purchased from Gibco (Cat. No. 10099-141C)

[0074] - Penicillin-streptomycin was purchased from Gibco (Cat. No. 15140122)

[0075] -Neurobasal TMThe culture medium was purchased from Gibco (Cat. No. 21103049)

[0076] -B-27 TM Supplement (50×) was purchased from Gibco (Cat. No. 17504044)

[0077] -GlutaMAX was purchased from Gibco (Cat. No. 35050-061)

[0078] -cck-8 was purchased from Biyuntian Company (item number C0038)

[0079] The instruments used in the specific steps of the embodiments of the present application are as follows:

[0080] -NMR: Agilent 600 MHz NMR instrument was used.

[0081] -Absorbance determination: measured by Molecular Devices multifunctional microplate reader.

[0082] - Cell culture: Use a Thermos CO2 incubator.

[0083] Preparation Example 1 Preparation of the compound of formula (I)

[0084] The preparation method of the compound of formula (I) can be as shown in formula (i-1) or formula (i-2).

[0085]

[0086] The method for preparing the compound of formula (I) may include preparing the compound of formula (I) from a compound of formula (Ia) (or a compound of formula (Ia')) and a compound of formula (Ib), particularly in the presence of a catalyst. For example, the compound of formula (Ia) (or a compound of formula (Ia')), a compound of formula (Ib), and one or more catalysts may be mixed in a solvent simultaneously or sequentially for reaction.

[0087]

[0088] The catalyst may include an amine and an acid. The amine may be 1,2-diphenylethane-1,2-diamine, such as racemic 1,2-diphenylethane-1,2-diamine; or a compound of formula (C1) or formula (C2), wherein X 1 may be hydrogen or methoxy, and X 2 The acid may be trifluoroacetic acid, o-hydroxybenzoic acid, or a compound of formula (A1). The catalyst may optionally further include a thiol. The thiol may be a compound of formula (T1), wherein X 3 , X 4 , X 5 They may each independently be hydrogen, hydroxy, carboxyl, or methoxycarbonyl.

[0089]

[0090] In some embodiments, the catalyst comprises an amine and an acid, wherein the amine is a racemic mixture of (1R,2R)-1,2-diphenylethane-1,2-diamine and (1S,2S)-1,2-diphenylethane-1,2-diamine, and the acid is trifluoroacetic acid.

[0091] In some embodiments, the solvent is toluene, o-xylene, mesitylene, or tetrahydrofuran, particularly toluene.

[0092] The following is an exemplary illustration of the preparation methods of some representative compounds of formula (I) of the present application. Those skilled in the art should be able to infer the preparation methods of other compounds of formula (I) based on the specific preparation methods of the following compounds, combined with conventional preparation and purification methods in the art.

[0093] (E)-2-Benzylidene-9-phenylhexahydro-5H-cyclopenta[c]pyrazolo[1,2-a]pyrazole-1,5(2H)-dione (1) Preparation

[0094] Compound 1 is shown below. It is a compound of formula (I), wherein: R 1 is phenyl; and R 2 It is phenyl.

[0095]

[0096] (E)-5-benzylidenecyclopentanone (0.15mmol, 25.5mg, 1.5 equivalents), (Z)-2-benzylidene-5-oxypyrazolidine-2-cation-1-anion (0.1mmol, 17.4mg, 1 equivalent), racemic 1,2-diphenylethylenediamine (0.02mmol, 4.2mg, 20mol%) were dissolved in 1mL of toluene, and then trifluoroacetic acid (0.02mmol, 1.5μL, 20mol%) was added and placed in a 50°C oil bath and stirred for 24 hours.

[0097] The reaction was monitored by thin layer chromatography. After the reaction was completed, the solvent was removed under reduced pressure, and then the product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 7 mg of yellow solid, which was compound 1. Yield: 20%.

[0098] use 1 H NMR and 13 The structure of compound 1 was determined by C NMR and the molecular weight was determined by HRMS-EI. The data are as follows. 1 H NMR spectra and 13 C NMR spectra are shown in Figure 1 as shown in (a) and (b).

[0099] 1 H NMR (600MHz, CDCl3) δ7.58(d,J=7.2Hz,2H),7.53(t,J=2.6Hz,1H),7.46–7.34(m,6H),7.27(d,J=6.5Hz,2H),4.93(s, 1H), 4.17 (s, 1H), 3.59 (d, J = 18.3Hz, 1H), 3.47 (ddd, J = 21.9, 8.0, 3.1Hz, 2H), 3.06 (s, 2H), 2.42 (s, 1H), 2.11 (s, 1H).

[0100] 13 C NMR (150MHz, CDCl3) δ204.56,167.16,136.07,135.95,134.72,132.78,131.09,13 0.19,129.08,128.92,128.72,128.34,71.36,61.33,52.16,45.87,34.32,33.82.

[0101] (E)-2-((4-tert-butyl)benzylidene)-9-(4-chlorophenyl)hexahydro-5H-cyclopenta[c]pyrazolo[1,2-a] Preparation of pyrazole-1,5(2H)-dione (38) Compound 38 is shown below. It is a compound of formula (I), wherein: R 1 is 4-chlorophenyl; and R 2 It is 4-tert-butylphenyl.

[0102]

[0103] (E)-5-(4-tert-butylbenzylidene)cyclopentanone (0.15mmol, 33.9mg, 1.5 equivalents), (Z)-2-(4-chlorobenzylidene)-5-oxypyrazolidine-2-cation-1-anion (0.1mmol, 20.85mg, 1 equivalents), racemic 1,2-diphenylethylenediamine (0.02mmol, 20mol%) were dissolved in 1mL of toluene, and then trifluoroacetic acid (0.02mmol, 1.5μL, 20mol%) was added and stirred in a 50°C oil bath for 24 hours.

[0104] The reaction was monitored by thin layer chromatography. After the reaction was completed, the solvent was removed under reduced pressure, and then the product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 11.5 mg of a white solid, which was compound 38. Yield: 26%.

[0105] use 1 H NMR and 13 C NMR confirmed the structure of compound 38. 1 H NMR spectra and 13C NMR spectra are shown in Figure 2 as shown in (a) and (b).

[0106] 1 H NMR (600MHz, CDCl3) δ7.52 (dd, J=6.2, 2.1Hz, 3H), 7.46 (d, J=8.5Hz, 2H), 7.3 7(d,J=8.5Hz,2H),7.25(t,J=3.7Hz,2H),4.83(s,1H),4.02(s,1H),3.62(d,J =18.3Hz,1H),3.44(ddd,J=18.9,8.1,3.0Hz,1H),3.39(dd,J=8.2,4.2Hz,1H) ,3.09(s,1H),2.98(s,1H),2.50(s,1H),1.68(d,J=14.3Hz,1H),1.33(s,9H).

[0107] 13 C NMR (150MHz, CDCl3) δ204.09,154.05,153.90,136.43,134.87,134.64,131.85,131.71,1 31.10,129.50,129.25,125.98,70.72,61.85,51.97,46.60,34.98,34.97,33.27,31.10.

[0108] Experimental Example 1 Test of Anti-Oxygen Glucose Deprivation (OGD)-Induced Neurological Damage Activity

[0109] Experimental modeling

[0110] Plate coating

[0111] Poly-L-lysine (PLL) was used to coat 96-well plates to facilitate the attachment of mouse primary neurons in subsequent experiments.

[0112] Add poly-lysine solution to a 96-well plate, 100 μL per well. Then place the 96-well plate in a CO2 incubator overnight to allow poly-lysine to bind to the surface of the plate. Afterwards, recover the PLL and rinse the coated 96-well plate 3 times with sterile ultrapure water to remove excess PLL. Place the coated 96-well plate in a CO2 incubator to dry for later use.

[0113] Primary mouse neuronal cell collection

[0114] Pregnant C57 mice at E15–17 were killed by cervical dislocation and disinfected with 75% alcohol. The lower abdomen of the pregnant mice was cut open in two layers, skin and muscle, and the uterus was removed and temporarily immersed in ice-cold sterile DMEM high-glucose medium. The fetuses were removed from the uterus one by one, and the fetal brains were extracted in batches. The fetal meninges were peeled off under a stereomicroscope, and the fetal cerebral cortex tissue was separated and collected.

[0115] The obtained mouse cerebral cortex tissue was digested with 0.25% trypsin at 37°C for 5 min to dissociate the cells. The digestion was terminated with seed plate solution, the cells were dispersed, filtered with a 70 μM cell strainer, and the cells were counted. 4 pcs / well, 24-well plate 10 5 The cells were seeded in the well plates at a cell density of 100 cells / well.

[0116] Transfer the plate to a CO2 incubator and incubate at 37°C, 5% CO2 for 4–6 hours before replacing the medium with maintenance medium. After 4–5 days of incubation, perform a half-medium change and continue incubation for 7–9 days.

[0117] Oxygen glucose deprivation reperfusion (OGD-R) model

[0118] For the primary mouse neuronal cells cultured for 7-9 days, the maintenance medium was replaced with DMEM sugar-free medium, and the cells were cultured in an anaerobic environment (5% CO2, 95% N2) for 2 hours to induce neuronal cell damage caused by OGD.

[0119] Experimental groups and drug administration

[0120] After culturing in the above anaerobic environment for 2 hours, the medium was replaced with DMEM high-glucose medium supplemented with the following components as a maintenance medium, and the cells were transferred to 37° C. and 5% CO 2 to continue culturing for up to 24 hours.

[0121] - Normal group: No OGD modeling was performed, and no positive control or compound of formula (I) was added to the culture medium.

[0122] - Vehicle control group: no positive control or compound of formula (I) was added.

[0123] -NA-1 control group: NA-1 (positive control) was added at a final concentration of 2.5 μM, 5.0 μM, 10 μM, 20 μM, or 40 μM.

[0124] - Edaravone control group: Edaravone (positive control) was added at a final concentration of 2.5 μM, 5.0 μM, 10 μM, 20 μM, or 40 μM.

[0125] - Compound group: Add the compound of formula (I) to a final concentration of 2.5 μM, 5.0 μM, 10 μM, 20 μM, or 40 μM.

[0126] 1.1 Cell viability detection (CCK-8 assay)

[0127] After the above administration and culture for 24 hours, CCK-8 was added to the 96-well plate, 10 μL per well, and cultured for 2.5 hours at 37°C and 5% CO2. The absorbance of each well at 450nm was measured using a multifunctional microplate reader to obtain cell viability data. The higher the cell viability value, the stronger the protective effect of the sample on the cells.

[0128] result

[0129] like Figure 3 As shown in (a), compound 1 showed significant protective effects on cells at all experimental concentrations (2.5μM, 5μM, 10μM, 20μM, and 40μM) compared to the vehicle control group. As the concentration increased, the protective effect of compound 1 on cells became stronger. Figure 3 As shown in (b), whether it is a low concentration (such as 1 μM) or a high concentration (such as 40 μM), the protective effect of compound 1 on cells is significantly higher than that of NA-1 or edaravone at the same concentration. Among them, the protective effect of 5 μM compound 1 on cells is equivalent to that of 40 μM NA-1 on cells, and is better than that of 40 μM edaravone on cells. The compound of formula (I) can protect cells, especially nerve cells, and can inhibit or slow down the decline in cell viability caused by damage.

[0130] The cell viability values ​​of each compound of formula (I) at 10 μM, 20 μM, and 40 μM measured by CCK-8 are shown in Table 1.

[0131] Table 1

[0132]

[0133]

[0134] n = 6; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0135] Median effective concentration (EC5 0 ) value determination

[0136] The EC of the protective effect of the compound on cells was calculated based on the above cell viability test results.50 Table 2 shows the EC values ​​of each compound. 50 Value. Figure 3 As shown in (c), compound 38 also showed significant cell protection, and its EC 50 The value is as low as 1.21±0.05μM, indicating that it can have a significant effect at low concentrations.

[0137] Table 2

[0138]

[0139] 1.2 Detection of caspase-3 expression, a marker of apoptosis

[0140] Caspase-3 is a cysteine-aspartic acid protease that plays a key role in cell apoptosis and is widely used as a biomarker of apoptosis.

[0141] Western blot analysis

[0142] The cells were depleted of culture medium and washed with ice-cold PBS. After PBS was depleted, 80 μL of ice-cold RIPA lysis buffer containing protease inhibitors was added to lyse the cells with gentle shaking at 4°C for 30 min. The precipitate was removed by centrifugation at 13,100 rpm for 30 min and the supernatant was taken. According to the instructions, the total protein content in the supernatant was measured using the BCA protein concentration assay kit, and the protein concentration was adjusted to 3 μg / μL using the previously prepared RIPA lysis buffer. Then 5× SDS–PAGE loading buffer was added to the supernatant, denatured at 95°C for 10 min, and centrifuged at 13,100 rpm for 1 min. The samples were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with EveryBlot Blocking Buffer for 5 min at room temperature and incubated with primary antibodies (caspase-3, focal adhesion protein) at 4°C overnight. The HRP-conjugated secondary antibody was incubated with the PVDF membrane at room temperature for 45 min. After the addition of ECL substrate, the band intensity was detected and analyzed using a chemiluminescence imaging system (Viber, France). Vinculin was used as an internal control.

[0143] The results are as follows Figure 4 As shown. Figure 4 As shown in (b), compound 1 can inhibit neuronal cell apoptosis induced by oxygen-glucose deprivation reperfusion, and the inhibitory effect increases with the increase of the concentration of compound 1. Edaravone cannot inhibit neuronal cell apoptosis induced by oxygen-glucose deprivation reperfusion. The compound of formula (I) has the activity of inhibiting cell apoptosis.

[0144] Experimental Example 2 LPS-induced microglial and neutrophil inflammatory activity test

[0145] 2.1 LPS-induced microglial inflammatory response

[0146] Acquisition of astrocytes

[0147] Mice within 24 hours of birth were placed in ice ethanol and anesthetized at low temperature for 5 minutes. The skin and skull were broken with forceps along the line between the eyes and ears, the cranial cavity was opened, the brain stem at the base of the skull was separated, and the brain was taken out and placed in pre-cooled DMEM culture medium. The olfactory bulb (located on the ventral side of the brain) was removed under a microscope, the meninges were torn open to expose the ventral side of the brain, the cerebral cortex was removed with forceps and the brain stem was retained, and the brain stem was immersed in a new 6 cm dish containing pre-cooled DMEM culture medium. The culture medium was discarded and trypsin was added for digestion at 37°C for 8 minutes. 3 mL of DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin was used for centrifugation and washing to wash away the trypsin to obtain a cell suspension.

[0148] 2. Culture of Microglia

[0149] The cells were counted and diluted to 500 × 10 3 / T75 flask, cultured in a T75 culture flask coated with poly-lysine. Change the medium every 3 days, rinse twice with PBS from the second time onwards, and shake the culture flask horizontally about 50 times when changing the culture medium. After 15-20 days, high-brightness microglia will grow on the upper layer of astrocytes. Place the culture medium on a 37°C constant temperature shaker and shake at 280 rpm for 1 hour. Aspirate the supernatant containing microglia, count and culture in a 24-well plate treated with poly-lysine at a density of 5×10 3 cells / well. Incubate overnight.

[0150] 2.2 LPS-induced neutrophil inflammatory response

[0151] Bone marrow cell harvesting

[0152] After the mice were euthanized, they were disinfected with 70% alcohol. Then, the bone marrow cavity of the mice was surgically exposed, the muscles were carefully removed, the hip joints were dislocated, and the femurs and tibias were removed. The obtained bones were rinsed with RPMI 1640 medium containing pre-cooled 10% FBS and 1% penicillin-streptomycin, placed on ice and further processed with a scalpel and needle, and the bone marrow cells were transferred to a centrifuge tube with a 100μm cell sieve and rinsed with EDTA solution to prevent blood coagulation.

[0153] Neutrophil isolation and collection

[0154] Prepare a density gradient centrifuge (3 mL of separation solution with a density of 1.119 g / mL, and 3 mL of separation solution with a density of 1.077 g / mL) in a 15 mL centrifuge tube and equilibrate at room temperature. Carefully place the bone marrow cell suspension on top of the 1.077 g / mL separation solution, centrifuge at 800 g for 30 minutes at room temperature, and collect neutrophils at the interface of the 1.119 g / mL and 1.077 g / mL separation solutions.

[0155] The collected neutrophils were washed twice with RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin, centrifuged at 1000 g for 5 minutes at 4°C, and then diluted.

[0156] Experimental grouping, drug administration and testing

[0157] The microglia and neutrophils obtained above were treated with a culture medium containing the following components, and cultured at 37° C. and 5% CO 2 for 24 hours. The supernatant was collected and inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA).

[0158] - Blank control group: neither LPS nor the compound of formula (I) was added.

[0159] - Vehicle control group: LPS was added at a final concentration of 0.5 μg / mL, but no compound of formula (I) was added.

[0160] - Compound group: LPS at a final concentration of 0.5 μg / mL and the compound of formula (I) of the present application at a final concentration of 10 μM, 20 μM, or 40 μM were added.

[0161] result

[0162] Compared with the model control group, the lower the value of inflammatory factors, the stronger the inhibitory effect of the compound on cell inflammation.

[0163] like Figure 5 and Figure 6 As shown, compound 1 can inhibit the release of IL-6 and IL-1β from microglia and IL-6 from neutrophils at concentrations of 10 μM, 20 μM, and 40 μM, and has an outstanding anti-inflammatory effect; compound 38 can inhibit the release of IL-6 from neutrophils at concentrations of 0.25 μM, 2.5 μM, and 5 μM, and can achieve an anti-inflammatory effect at a lower concentration than compound 1. The compound of formula (I) has anti-inflammatory activity.

[0164] Experimental Example 3 Activity test of intraperitoneal injection intervention for cerebral ischemia injury

[0165] Intraluminal middle cerebral artery occlusion / reperfusion (MCAO / R) model in mice

[0166] Prepare C57BL / 6J mice. The mice were fasted and deprived of water the night before the experiment. The next day, they were anesthetized with 1% sodium phenobarbital. After the skin of the mice was prepared, the neck position was adjusted to fully expose the neck. The skin was cut along the midline of the neck, and the muscles and fascia were bluntly separated to fully expose the left common carotid artery, internal carotid artery and external carotid artery. The common carotid artery was ligated with a slipknot at the proximal end, and two knots were ligated at the distal end of the external carotid artery. A slipknot was tied at the proximal end of the external carotid artery 1 mm away from the bifurcation of the common carotid artery, and it was not tied tightly.

[0167] infarction

[0168] Use ophthalmic scissors to make a triangular incision on the blood vessel distal to the slipknot of the external carotid artery of the mouse, and slowly insert the silicone thread plug from the incision toward the common carotid artery. Cut the blood vessel between the two knots at the distal end of the external carotid artery, and insert the thread plug into the internal carotid artery. Stop moving forward and fix the thread plug when resistance is felt, and block the artery for 90 minutes.

[0169] Reperfusion

[0170] After the infarction is over, the suture is slowly and evenly pulled out, and the stump of the external carotid artery is ligated to restore blood flow (reperfusion).

[0171] Experimental animal grouping and drug administration

[0172] After reperfusion, the mice were injected with a solution containing the compound of the present application or an equal amount of vehicle in the following groups.

[0173] - Sham operation group: Sham operation was performed without infarction and reperfusion. The vehicle was administered at the time of drug administration.

[0174] -Vehicle control group: vehicle was administered at the time of drug administration.

[0175] - Low-dose group: 20 mg of Compound 1 per kg of mouse body weight was administered.

[0176] - High-dose group: 40 mg of Compound 1 per kg of mouse body weight was administered.

[0177] After administration, the mice were returned to the cage for monitoring and temperature maintenance. After the mice woke up, 0.5 mL of normal saline was injected intraperitoneally.

[0178] 3.1 2,3,5-Triphenyltetrazolium chloride (TTC) staining

[0179] TTC staining is a method used to evaluate cerebral ischemic injury. It uses TTC to react with succinate dehydrogenase in the mitochondria of living cells to generate red formazan, thereby reflecting cell viability.

[0180] The mice that had completed the evaluation of the degree of neurological damage were killed by cervical dislocation. The complete brain was removed and frozen at -20°C for 20 minutes. The frozen brain was sliced ​​coronally on ice with a thickness of about 2 mm, for a total of 7 slices. The obtained brain slices were placed in a well plate, added to the preheated 2% TTC solution, and incubated at 37°C in the dark for 20 minutes. The TTC solution was aspirated, 4% paraformaldehyde was added for fixation, and a small amount of glycerol was added for storage at 37°C.

[0181] The next day, the TTC-stained mouse brain slices were photographed and processed using ImageJ v1.48 software. The red-stained tissues were normal tissues, and the unstained areas were infarcted areas.

[0182] The infarct rate was calculated using the following formula and corrected for the non-infarcted side brain tissue volume.

[0183] Infarction rate % = (sum of non-infarcted brain tissue area on the infarcted side - sum of non-infarcted brain tissue area on the infarcted side) / sum of non-infarcted brain tissue area × 100

[0184] 3.2 Assessment of neurological impairment

[0185] 24 hours after the mice were intraperitoneally injected with normal saline, the Longa score for nerve injury and the modified neurological score (mNSS) were used to evaluate the degree of neurological damage in the mice.

[0186] result

[0187] like Figure 7 As shown in (a) to (d), compound 1 can significantly reduce the cerebral infarction volume and neurological injury score after reperfusion injury in mice with stroke caused by MCAO / R model, and there is a significant difference (p<0.05). Figure 7 As shown in (e), compound 1 increased the survival rate of mice within 24 hours.

[0188] These protective effects all showed obvious dose dependence, that is, the protective effect of the high-dose group (administered with 40 mg / kg of compound 1) was better than that of the low-dose group (administered with 20 mg / kg of compound 1).

[0189] The significant reduction of cerebral infarct volume and neurological injury score by administration of the compound of formula (I) indicates that the compound of formula (I) has a significant effect in improving neurological injury after cerebral ischemia-reperfusion.

[0190] The above-mentioned implementation modes are only preferred implementation modes of the present application and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present application shall fall within the scope of protection required by the present application.

Claims

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, in the preparation of a medicament for treating or preventing stroke and / or inflammatory diseases, neuroprotection, scavenging free radicals, and / or alleviating oxidative stress, in, R 1 is an optionally substituted aryl, heteroaryl, arylvinyl, cycloalkyl, or heterocycloalkyl; and R 2 is an optionally substituted aryl, heteroaryl, arylvinyl, cycloalkyl, or heterocycloalkyl.

2. The use according to claim 1, characterized in that R 1 is aryl, heteroaryl, arylvinyl, cycloalkyl, or heterocycloalkyl optionally substituted with one or more substituents; and R 2 is an aryl group, a heteroaryl group, an arylvinyl group, a cycloalkyl group, or a heterocycloalkyl group which is optionally substituted by one or more substituents, wherein the substituents are each independently selected from the group consisting of fluorine, chlorine, bromine, iodine, trifluoromethyl, methoxy, ethoxy, propoxy, butoxy, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, ethynyl, phenyl, naphthyl, cyano, nitro, tert-butyloxycarbonyl, and tert-butyldimethylsilyloxy.

3. The use according to claim 2, characterized in that R 1 is phenyl optionally substituted by one or more of said substituents; and R 2 is phenyl which is optionally substituted by one or more of the substituents mentioned above.

4. The use according to any one of claims 1 to 3, characterized in that: R 1 and R 2 At least one of the groups is a phenyl group optionally substituted with mono- or di-halogen or trifluoromethyl.

5. The use according to claim 1, characterized in that R 1 is selected from the group consisting of phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 3,4-dichlorophenyl, 3,5-dibromophenyl, 2-chloro-4-fluorophenyl, 2-trifluoromethylphenyl, 4-trifluoromethylphenyl, 3,5-bis(trifluoromethyl)phenyl, 3-methoxyphenyl, 4-methoxyphenyl, 4-methylphenyl, 4-ethynylphenyl, 4-isopropylphenyl, 4-tert-butylphenyl, 4-cyanophenyl, 4-nitrophenyl, 4-(tert-butyldimethylsilyloxy)phenyl, 1-naphthyl, 2-furyl, 2-thienyl, tert-butyloxycarbonyl-4-piperidinyl, styryl, and cyclohexyl.

6. The use according to claim 1 or 5, characterized in that R 2 is selected from the group consisting of phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 3,4-dichlorophenyl, 3,5-dibromophenyl, 2-chloro-4-fluorophenyl, 2-trifluoromethylphenyl, 4-trifluoromethylphenyl, 3,5-bis(trifluoromethyl)phenyl, 3-methoxyphenyl, 4-methoxyphenyl, 4-methylphenyl, 4-ethynylphenyl, 4-isopropylphenyl, 4-tert-butylphenyl, 4-cyanophenyl, 4-nitrophenyl, 4-(tert-butyldimethylsilyloxy)phenyl, 1-naphthyl, 2-furyl, 2-thienyl, tert-butyloxycarbonyl-4-piperidinyl, styryl, and cyclohexyl.

7. The use according to any one of claims 1 to 6, characterized in that The drug is a drug for treating or preventing stroke; Optionally, the stroke includes organic brain damage, neurobehavioral deficits, or neuronal cell damage.

8. The use according to claim 7, characterized in that The stroke is an ischemic stroke, in particular a stroke caused by cerebral ischemia-reperfusion, and in particular a cerebral ischemia-reperfusion injury; Optionally, the ischemia is caused by a local blood circulation disorder, in particular, the local blood circulation disorder caused by thrombosis, inflammation, hypertension, atherosclerosis, or vascular malformation.

9. The use according to any one of claims 1 to 6, characterized in that: The drug is a drug for treating or preventing inflammatory diseases; Optionally, the inflammatory disease is inflammation caused by ischemia, in particular inflammation caused by cerebral ischemia; or inflammation caused by lipopolysaccharide reduction; or inflammation caused by fighting against acute stress. Optionally, the inflammatory disease is inflammation of one or more of the skin, muscles, bones, joints, cartilage, cerebrum, cerebellum, brain stem, spinal cord, eyes, ears, nose, tongue, heart, blood vessels, throat, trachea, lungs, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, kidneys, bladder, urethra, ovaries, uterus, testicles, spleen, thymus, lymphatic vessels, or lymph nodes.

10. The use according to any one of claims 1 to 6, characterized in that: The drug is a drug for neuroprotection, free radical scavenging, and / or oxidative stress alleviation.

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