Patent Application
20230219911
The present invention relates to the field of medicinal chemistry, and in particular to a series of novel benzothiazole derivatives and pharmaceutically acceptable salts, solvates or prodrugs thereof, a preparation method for the same, as well as a pharmaceutical composition comprising the compound. The present invention also relates to the use of such compounds in the preparation of a medicament for treating and/or preventing a sEH-mediated disease, and in particular in the preparation of a medicament for treating inflammatory diseases, cardiovascular and cerebrovascular diseases, diabetes, diabetic complications, diabetes-related diseases, fibrotic diseases, neurological and mental diseases, pains, ulcerative diseases, and the like.
Epoxide hydrolase (EH) is ubiquitous in mammals, insects, plants and microorganisms, and is an enzyme widely distributed in nature. Currently, epoxide hydrolases are classified into eight types according to their activities, biochemical characteristics, and current research results. Soluble epoxide hydrolase (sEH) is one of the in-depth studied epoxide hydrolase at this stage. It was first discovered in 1973 by Gill et al. when they were studying the metabolism of terpenoid epoxides similar to insect juvenile hormones, named for its location in soluble components of the cell such as the cytoplasm and peroxisome. A large number of researches in recent years have shown that the soluble epoxide hydrolase is a potential therapeutic target for many diseases such as hypertension, lung disease, diabetes, pains, inflammation, and other immune system diseases.
Human sEH is encoded by the EPHX2 gene, which is located in the P21-P12 region of human chromosome 8, and consists of 19 exons of 27-265 bp and 18 introns, with a full length of about 45 kb and a total of 555 amino acids encoded. sEH is expressed in almost all organs, and has been found in liver, kidney, lung, heart, brain, spleen, adrenal gland, intestine, bladder, vascular endothelium, smooth muscle, leucocytes, and ovarian cells. It has the highest activity in the liver, followed by the kidney. sEH plays an important role in the metabolism of lipid epoxides. Its endogenous substrates include epoxyeicosatrienoic acids (EETs). EETs are a class of endogenous chemical mediators that have been shown to have a variety of physiological functions, such as dilating blood vessels, lowering blood pressure, regulating lipid metabolism and insulin resistance, etc. (Q. Wang, W. Pang, Z. CuI, American Journal of physiology-Renal physiology, 2013, 304: 168-176). Studies have found that EETs in vitro can be rapidly hydrolyzed into dihydroxyeicosatrienoc acids (DHETs) by the action of sEH, thereby losing their activities. Thus, how to inhibit the activity of sEH becomes a key issue. In recent years, studies have shown that the activity of sEH can be reduced by small molecule inhibitors or gene knockout technology to stabilize or increase the content of EETs.
The pharmacological effect obtained by inhibiting sEH can be measured by the level of intracellular EETs. The research on sEH inhibitors mainly focuses on the effects on blood vessels and inflammation. EETs are activators that inhibit nuclear transcription factor (NF-wB). In the animal model of inflammation, the inhibition of sEH can improve and control the inflammation. Furthermore, in the animal model of inflammation, tobacco smoke-induced airway inflammation and monocrotaline-induced pulmonary hypertension were also reduced in severity by inhibiting sEH. sEH inhibitors also have certain analgesic effects. In the animal model experiment of cerebral ischemia, the inhibition of sEH activity can inhibit the ischemic inflammatory response, stimulate the apoptosis of anti-neuronal gene expression, and reduce the effect of cerebral infarction (A. Cautaux, F. Adam, C. J. Willer, Joint Bone Spine, 2005, 72: 359-371).
Studies have found that there is a key aspartic acid residue in the catalytic site of sEH, so the early research on sEH inhibitors mainly focused on modification reagents for carboxyl. Dicyclohexylcarbodiimide (DCC), as a common reagent in the syntheses of amide bonds and peptides, can react with a carboxylic acid to form the corresponding ester. Thus, it is unexpectedly found that DCC has a good inhibitory activity on sEH. However, according to the subsequent pharmacokinetic analysis, DCC will be hydrolyzed in solution into the corresponding urea, which is the main active substance for inhibiting sEH, so most of the early sEH inhibitors contain urea structures (C. Morisseau, B. D. Hammock, Annual Review of Pharmacology and Toxicology, 2005, 45: 311 to 333). t-AUCB is a sEH inhibitor jointly discovered by the University of California and UCD Cancer Center. Studies have found that this compound has a relatively high inhibitory activity on sEH in various mammals and humans.
At present, some compounds with outstanding activities and good therapeutic effects are being in the preclinical and clinical stages, such as AR9281, GSK2256294, and EC5026. EC5026 is a urea-based sEH inhibitor developed by the team of Bruce Hammock from the University of California. This drug is currently undergoing Phase Ia clinical trials, and its indication involves the neuropathic pains.
On the basis of the references, the inventors have designed and synthesized a series of benzothiazole derivatives, and the results from the in vitro sEH kinase inhibitory activity test have shown that they all have inhibitory activities.
The present invention relates to a compound of general formula I or its stereoisomers as well as pharmaceutically acceptable salts, solvates or prodrugs thereof,
wherein,
X is CH or N;
Y is CH or N;
Z is CH, N or (one or direct) bond;
Q is O, S, NH or NCH3;
R1 is hydrogen, —(CH2)mNR3R4, —C(O)NH(CH2)nR5, —C(O)(CH2)nR5 or —S(O)2NH(CH2)nR5;
m is an integer from 2 to 4 (e.g., 2, 3 or 4);
R2 is hydrogen, —C(O)NH(CH2)nR5, —C(O)(CH2)nR5, —S(O)2NH(CH2)nR5 or —(CH2)mNR3R4; and
R1 and R2 are not hydrogen at the same time;
n is an integer from 0 to 4 (e.g., 0, 1, 2, 3 or 4);
R3 and R4 are same or different, and independently selected from hydrogen, (C1-C6)alkyl, (C3-C7)cycloalkyl, (C1-C6)alkylacyl, (C1-C6)alkoxyl, (C2-C6)alkenyl or (C2-C6)alkynyl; or
R3 and R4 together with the nitrogen atom to which they are attached form a 4 to 10 (e.g., 4, 5, 6, 7, 8, 9 or 10) membered heterocyclic group or 5 to 10 (e.g., 5, 6, 7, 8, 9 or 10) membered heteroaryl group, wherein the heterocyclic group or heteroaryl group optionally contains 0 to 4 (e.g., 0, 1, 2, 3 or 4) heteroatom(s) selected from N, O, and/or S in addition to the nitrogen atom to which R3 and R4 are attached, the heterocyclic group optionally comprises 0 to 2 carbon-carbon double bond(s) or carbon-carbon triple bond(s) in addition to the nitrogen atom to which R3 and R4 are attached, and the heterocyclic group or heteroaryl group is optionally substituted by 1 to 3 (e.g., 1, 2 or 3) same or different R6;
R5 is hydrogen, (C1-C6)alkyl, (C3-C10)cycloalkyl, (C6-C10)aryl or 5 to 10 (e.g., 5, 6, 7, 8, 9 or 10) membered heteroaryl group, wherein the heteroaryl group contains 1 to 3 (e.g., 1, 2 or 3) heteroatom(s) selected from N, O or S, and the aryl or heteroaryl group is optionally substituted by 1 to 3 (e.g., 1, 2 or 3) same or different R7;
R8 is hydrogen, —C(O)(CH2)qR9, —(CH2)qQC(O)OR9 or —C(O)NH(CH2)qR9;
R9 is (C1-C6)alkyl, (C3-C7)cycloalkyl, (C6-C10)aryl or 5 to 10 membered (e.g., 5, 6, 7, 8, 9 or 10) heteroaryl group, wherein the aryl or heteroaryl group is optionally substituted by 1 to 3 (e.g., 1, 2 or 3) same or different Rio;
q is an integer from 0 to 4 (e.g., 0, 1, 2, 3, 4);
R6, R7, and Rio are independently 1 to 3 (e.g., 1, 2 or 3) same or different substituent(s) selected from hydrogen, hydroxyl, halogen, nitro, amino, cyano, azido, mercapto, (C1-C6)alkyl, (C1-C6)alkoxyl, (C1-C6)alkyl or (C1-C6)alkoxyl optionally substituted by hydroxyl, amino or halogen, (C1-C6)alkylthiol, allyl, amino substituted by mono or di(C1-C6 alkyl), (C1-C6)alkylamido, free, salified, esterified, or amidated carboxyl, (C1-C6)alkylsulfinyl, (C1-C6)alkylsulfonyl, (C1-C6)alkylacyl, carbamoyl or carbamoyl substituted by mono or di(C1-C6 alkyl).
Preferably, the present invention relates to a compound of general formula I and its stereoisomers as well as pharmaceutically acceptable salts, solvates or prodrugs thereof,
wherein,
Z is CH;
R1 is hydrogen or —(CH2)mNR3R4;
R2 is hydrogen, —C(O)NH(CH2)nR5 or —C(O)(CH2)nR5;
R1 and R2 are not hydrogen at the same time;
R3 and R4 are same or different, and independently selected from hydrogen, (C1-C6)alkyl, (C3-C7)cycloalkyl, (C1-C6)alkylacyl, (C1-C6)alkoxyl, (C2-C6)alkenyl or (C2-C6)alkynyl; or
R3 and R4 together with the nitrogen atom to which they are attached form a 4 to 10 membered heterocyclic group, wherein the heterocyclic group optionally contains 1 to 4 heteroatom(s) selected from N, O, and/or S in addition to the nitrogen atom to which R3 and R4 are attached, and optionally substituted by 1 to 3 same or different R6.
More preferably, the present invention relates to a compound of general formula I and its stereoisomers as well as pharmaceutically acceptable salts, solvates or prodrugs thereof, wherein
R5 is (C3-C10)cycloalkyl, phenyl or a 5 to 10 membered heteroaryl group containing 1 to 3 heteroatom(s) selected from N, O or S; more preferably (C3-C7)cycloalkyl, phenyl, indolyl, quinolyl, pyridyl, pyrimidinyl, furyl, thienyl, pyrrolyl, wherein aryl or heteroaryl group is optionally substituted by 1 to 3 same or different R7;
More preferably, the present invention relates to a compound of general formula I and its stereoisomers as well as pharmaceutically acceptable salts, solvates or prodrugs thereof,
wherein,
R1 is hydrogen or —(CH2)mNR3R4;
R3 and R4 are same or different, and independently selected from hydrogen, (C1-C6)alkyl or (C3-C7)cycloalkyl or together with the nitrogen atom to which they are attached form
R2 is hydrogen, —C(O)NH(CH2)nR5 or —C(O)(CH2)nR5.
Particularly preferably, the present invention relates to a compound of general formula I and its stereoisomers as well as pharmaceutically acceptable salts, solvates or prodrugs thereof,
wherein,
R9 is phenyl optionally substituted by 1 to 3 same or different Rio.
Particularly preferably, the present invention relates to the following compound of general formula I and its stereoisomers as well as pharmaceutically acceptable salts, solvates or prodrugs thereof:
According to some general methods in the field of the present invention, the compound of general formula I of the present invention can form its pharmaceutically acceptable salt with an acid. The preferred acid is hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, trifluoroacetic acid, and aspartic acid.
The present invention also includes the prodrugs of the derivatives of the present invention. The prodrugs of the derivatives of the present invention are derivatives of general formula (I), which themselves may have a weaker activity or even no activity, but can be converted into the corresponding biologically active forms under physiological conditions (for example, by metabolism, solvolysis or other means) upon administration.
Unless otherwise indicated, as used in the present invention, the term “halogen” refers to fluoro, chloro or bromo; the term “alkyl” refers to linear or branched alkyl; the term “cycloalkyl” refers to substituted or unsubstituted cycloalkyl; the term “alkoxyl” refers to linear or branched alkoxyl; the term “alkenyl” refers to linear or branched alkenyl; the term “alkynyl” refers to linear or branched alkynyl; the term “aryl” refers to a phenyl group with or without substituent(s); the term “heteroaryl group” refers to a monocyclic or polycyclic ring system which contains one or more heteroatom(s) selected from N, O, and S, and which is aromatic, such as imidazolyl, pyridyl, pyrazolyl, furyl, thienyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, naphthyl, quinolyl, isoquinolyl, benzimidazolyl, and benzoxazolyl.
The present invention also includes a pharmaceutical composition comprising the compound of general formula I and pharmaceutically acceptable salts and/or solvates thereof as active ingredient as well as a pharmaceutically acceptable carrier. The compound of the present invention can be used in combination with other active ingredients as long as they do not bring about other adverse effects such as allergic reactions.
The carrier used in the pharmaceutical composition of the present invention is a common type available in the pharmaceutical field, including: adhesives, lubricants, disintegrants, cosolvents, diluents, stabilizers, suspending agents, pigments, flavoring agent, and the like for oral preparations; pH regulators, osmotic pressure regulators, solubilizers, stabilizers, and the like for injection preparations; and substrates, diluents, lubricants, preservatives, and the like for topical preparations. The pharmaceutical preparations may be administrated orally, parenterally (e.g., intravenously, subcutaneously, intraperitoneally, etc.) or topically (e.g., ocularly, nasally, sublingually, transdermally, etc.). If a certain drug is unstable under stomach conditions, it may be formulated into an enteric-coated tablet.
Through in vivo and in vitro inhibitory activity tests, it has been found that the compound of the present invention has a significant inhibitory activity on sEH, and can stabilize the level of EETs in vivo. The results of pharmacodynamic studies have shown that the compound of the present invention can exert the functions such as inhibiting the paw swelling and pain induced by carrageenan as well as the release of inflammatory factors induced by lipopolysaccharide, protecting the traumatic brain injury, maintaining the blood glucose homeostasis, improving the diabetic retinopathy, resisting against the tissue fibrosis, and relieving the depression, and thus can be used to treat sEH-mediated inflammatory diseases, cardiovascular and cerebrovascular diseases, diabetes and complications thereof, fibrotic diseases, neurological and mental diseases, pains, ulcerative diseases, etc.
The precise dosage of a compound of the present invention required for treating the sEH-mediated condition will vary from subject to subject, depending on the species, age, and general conditions of the subject, the severity of the disease to be treated, the particular compound used, and the administration manner (such as the administration route and frequency), etc. An appropriate effective amount can be determined by one of ordinary skill in the art through conventional experimentation.
The dosage of the compound may be about 0.1-100 mg/kg body weight/day, preferably 1-50 mg/kg body weight/day. It will be appreciated that the dosage may vary depending on the requirements of the patient, the severity of the hypertensive, cardiovascular, inflammatory, and diabetes-related diseases to be treated, and the particular compound employed. Furthermore, it will be appreciated that the initial dosage administrated may be increased beyond the upper limit in order to rapidly achieve the desired blood level or the initial dosage may be less than the optimal value. The daily dosage may be gradually increased during treatment, depending on the specific conditions. If necessary, the daily dosage may also be divided into multiple doses, for example 2-4 times a day.
The mammals mean humans or animals.
The amount of the active ingredient (i.e., the compound according to the present invention) in the pharmaceutical composition and its unit dosage form may vary depending on the particular application, the potency and desired concentration of the particular compound. Generally, the content of the active ingredient will be between 0.5% and 90%, based on the total weight of the composition.
In the combination therapy, the compound of the present invention and other compound(s) may be administrated simultaneously or at intervals. When administrated simultaneously, the compound of the present invention and other compound(s) may be combined in a single pharmaceutical composition or in separate compositions.
The compounds of the present invention and preparation methods thereof are further explained and illustrated in the Examples and preparations provided hereinafter. It should be understood that the scope of the following Examples and preparations shall not limit the scope of the present invention in any way.
The following synthetic routes describe the preparations for the derivatives of general formula I in the present invention. All starting materials are prepared by the methods described in these synthetic routes or by the methods well known to one of ordinary skill in the field of organic chemistry or are commercially available. The final compounds in the present invention are all prepared by the methods described in these synthetic routes or by the methods analogous thereto, which are well known to one of ordinary skill in the field of organic chemistry. All the variables applicable in these synthetic routes are as defined below or as defined in the claims.
According to the compound of general formula I in the present invention, in Routes 1 to 6, the following compounds are exemplified, wherein the definitions for substituents R1, R5, R9, X, Y, Z, etc. are the same as those in the claims.
In Route 1, using 2-amino-6-nitrobenzothiazole as the starting material, DMAP as the base, and Boc anhydride for Boc protection, Intermediate B is obtained, followed by reduction reaction under the condition of hydrazine hydrate and activated carbon to obtain Intermediate C, followed by substitution reaction with phenyl chloroformate to obtain Intermediate D, followed by nucleophilic substitution reaction with R5-substituted amine to obtain Intermediate E, followed by Boc deprotection reaction with trifluoroacetic acid, to generate Compound I-i of general formula I.
In Route 2, Intermediate C is subjected to a nucleophilic substitution reaction with R1-substituted chlorinated compound under the condition of potassium carbonate as the base to obtain Intermediate F, followed by nucleophilic substitution reaction with R5-substituted phenyl chloroformate G to obtain Intermediate H, followed by Boc deprotection reaction under the condition of trifluoroacetic acid, to generate Compound I-ii of general formula I.
In Route 3, Compound I-ii and R9-substituted chlorinated compound are used to prepare Compound I-iii of general formula I under the condition of triethylamine as acid-binding agent; or Compound I-ii and R9-substituted phenyl chloroformate are used to generate urea under the condition of DIPEA as the base, to prepare Compound I-iv of general formula I.
In Route 4, using 2-amino-5-nitrophenol (J) as the starting material, and a solution of boron trifluoride in diethyl ether is used for a tube-sealed reaction, Intermediate K is obtained, followed by reduction reaction under the condition of hydrogen and palladium on carbon to obtain Intermediate L, followed by nucleophilic substitution reaction with R1-substituted chlorinated compound under the condition of potassium carbonate as the base to obtain Intermediate M, followed by nucleophilic substitution reaction with R5-substituted phenyl chloroformate G to prepare Compound I-v. Compound I-v and R9-substituted chlorinated compound are used to prepare Compound I-vi of general formula I under the condition of triethylamine as acid-binding agent; or Compound I-v and R9-substituted phenyl chloroformate are used to generate urea under the condition of DIPEA as the base, to prepare Compound I-vii of general formula I.
In Route 5, 2-chloro-5-aminopyridine (N) is used as the starting material and reacted with potassium thiocyanate and bromine, to obtain Intermediate O, which is subjected to nucleophilic substitution reaction with sodium hydroxide as the base to obtain Intermediate P, followed by nucleophilic substitution reaction with R5-substituted phenyl chloroformate G, to prepare Compound I-viii. Compound I-viii and R9-substituted chlorinated compound are used to prepare Compound I-ix of general formula I under the condition of triethylamine as acid-binding agent; or Compound I-viii and R9-substituted phenyl chloroformate are used to generate urea under the condition of DIPEA as the base, to prepare Compound I-x of general formula I.
In Route 6, 4,6-dichloropyridin-3-amine, 2,4-dichloropyrimidin-5-amine or 3,5-dichloropyridin-2-amine (Q) is used as the starting material and subjected to substitution reaction with phenyl isothiocyanate, to obtain Intermediate R; Intermediate R is reacted under the condition of sodium methoxide as the base, to obtain Intermediate S; Intermediate S is reacted in a 70% sulfuric acid solution, to obtain Intermediate T; Intermediate T is subjected to Buchward-Hartwig reaction with R1-substituted primary amine, to obtain Intermediate U; and then Intermediate U is subjected to nucleophilic substitution reaction with R5-substituted phenyl chloroformate G, to prepare Compound I-xi. Compound I-xi and R9-substituted chlorinated compound are used to prepare Compound I-xii of general formula I under the condition of triethylamine as acid-binding agent.
#: Compared with the sham group, p<0.05; *: Compared with the UUO group, p<0.05.
#: Compared with the sham group, p<0.05; *: Compared with the UUO group, p<0.05.
*: Compared with the model group, p<0.05.
*: Compared with the model group, p<0.05.
*: Compared with the model group, p<0.05.
*: Compared with the model group, p<0.05.
*: Compared with the blank solvent group, p<0.05.
In the following Examples, the methods for preparing some of said compounds are described. It should be understood that the following methods and other methods known to one of ordinary skill in the art can be applied to the preparations of all compounds described herein. The Examples are intended to illustrate the scope of the invention, rather than limiting the scope. The proton nuclear magnetic resonance spectrum for the compound was determined by Bruker ARX-400 or Bruker ARX-600, and the mass spectrum was determined by Agilent 1100 LC/MSD; and the reagents used were analytical or chemically pure.
To a solution of 2-amino-6-nitrobenzothiazole (A) (purchased from Shanghai Bide Pharmatech Ltd.) (10 g, 51.2 mmol) and 4-dimethylaminopyridine (purchased from Beijing Ouhe Technology Co., Ltd.) (3.2 g, 26.2 mmol) in DMF (100 mL), di-tert-butyl dicarbonate (purchased from Shanghai Bide Pharmatech Ltd.) (16.8 g, 77.0 mmol) was added, and the resulting solution was stirred at 90° C. for 18 h. The resulting mixture was poured into stirred ice water (500 mL), then the yellow solid was filtered and dried under reduced pressure. The crude product was purified by flash column chromatography, to obtain 15.3 g of Intermediate B. Yield: 85%. MS (ESI) m/z: 296.3 [M+H]+.
Intermediate B (12.0 g, 40.5 mmol) was dissolved in 1,4-dioxane (120 mL), and FeCl3·H2O (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (1.0 g, 6.2 mmol), activated carbon (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (0.15 g, 12.5 mmol), and 80% hydrazine hydrate (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (35 mL) were added to the solution, respectively. The mixture solution was heated to 90° C. for 12 h. The mixture solution was filtered through celite, the filter cake was washed with 1,4-dioxane (100 mL), and the filtrate was concentrated under vacuum, to obtain 9.3 g of Intermediate C. Yield: 73.3%. MS (ESI) m/z: 266.2 [M+H]+.
Intermediate C (10 g, 37.7 mmol) was dissolved in acetone (100 mL), and phenyl chloroformate (purchased from Shanghai Bide Pharmatech Ltd.) (9.0 g, 57.7 mmol) was dropwise added to the solution under stirring. After dropwise addition, the reaction solution was stirred at room temperature for 2 h. The reaction mixture solution was filtered by suction, to obtain 9.4 g of Intermediate D. Yield: 83.6%. MS (ESI) m/z: 386.1 [M+H]+.
Intermediate D (0.5 g, 1.3 mmol) was dissolved in 1,4-dioxane (5 mL), and p-chloroaniline (purchased from Beijing Ouhe Technology Co., Ltd.) (0.15 g, 1.5 mmol) and triethylamine (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (0.13 g, 1.3 mmol) were added to the solution. The mixture was heated to 60° C. and stirred for 3 h. The reaction solution was filtered by suction, and the filter cake was washed with diethyl ether, and dried, to obtain 0.3 g of Intermediate E. Yield: 86.1%.
Intermediate E (0.3 g) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (purchased from Reagent Station of Shenyang Pharmaceutical University) (3 mL) was added to the solution. After stirring overnight at room temperature, the mixture solution was concentrated under reduced pressure and water was added (5 mL), and filtered by suction after adjusting the pH to 8 with saturated sodium bicarbonate solution. The filter cake was further purified by silica-gel column chromatography, to obtain 0.15 g of Intermediate I. Yield: 77%.
Using sodium cyanate (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate E was synthesized in accordance with the synthesis process of step 1.4 in Example 1, then 1-(2-aminobenzo[d]thiazol-6-yl)urea was synthesized in accordance with the synthesis process of step 1.5 in Example 1. Yield: 65%.
Using aniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate E was synthesized in accordance with the synthesis process of step 1.4 in Example 1, then 1-(2-aminobenzo[d]thiazol-6-yl)-3-phenylurea was synthesized in accordance with the synthesis process of step 1.5 in Example 1. Yield: 70%.
Using adamantanamine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate E was synthesized in accordance with the synthesis process of step 1.4 in Example 1, then 1-(2-aminobenzo[d]thiazol-6-yl)-3-[(3s,5s,7s)-adamantan-1-yl]urea was synthesized in accordance with the synthesis process of step 1.5 in Example 1. Yield: 68%.
Using p-chlorobenzylamine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate E was synthesized in accordance with the synthesis process of step 1.4 in Example 1, then 1-(2-aminobenzo[d]thiazol-6-yl)-3-(4-chlorobenzyl)urea was synthesized in accordance with the synthesis process of step 1.5 in Example 1. Yield: 54%.
Using cyclohexylamine (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate E was synthesized in accordance with the synthesis process of step 1.4 in Example 1, then 1-(2-aminobenzo[d]thiazol-6-yl)-3-cyclohexylurea was synthesized in accordance with the synthesis process of step 1.5 in Example 1. Yield: 89%.
1-(2-Aminobenzo[d]thiazol-6-yl)-3-(4-chlorophenyl)urea (0.2 g, 0.4 mmol) was dissolved in 1,4-dioxane (3 mL), and DIPEA (purchased from Shanghai Bide Pharmatech Ltd.) (0.1 g, 0.7 mmol) was added. At room temperature, phenyl (4-chlorophenyl)carbamate (0.1 g, 0.4 mmol) was added to the solution, and stirred overnight at room temperature. After the reaction was completed, water (10 mL) was added, and a solid was precipitated, which was filtered by suction. The filter cake was washed, dried, and purified by column chromatography, to obtain 0.08 g of white solid. Yield: 76%.
Intermediate C (2.0 g, 7.5 mmol) was dissolved in 1,4-dioxane (20 mL), and potassium carbonate (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (3.1 g, 22.4 mmol) and 4-(2-chloroethyl)morpholine (purchased from Shanghai Bide Pharmatech Ltd.) (1.2 g, 11.2 mmol) were added to the solution. The reaction solution was heated to reflux and reacted under stirring for 8 h. After the reaction was cooled to room temperature, water (80 mL) was added thereto, and extracted with ethyl acetate (2×80 mL). The organic layers were combined, washed with brine, dried, concentrated and dried under reduced pressure, to obtain 1.5 g of Intermediate F. Yield: 60%.
At room temperature, 4-chloroaniline (purchased from Beijing Ouhe Technology Co., Ltd.) (1.4 g, 11.2 mmol) and sodium carbonate (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (0.7 g, 6.7 mmol) were added to a mixture solution of ethyl acetate-tetrahydrofuran-water (3:1:1=10 mL), and a solution of phenyl chloroformate (purchased from Shanghai Bide Pharmatech Ltd.) (1.9 g, 12.3 mmol) in ethyl acetate (3 mL) was dropwise added in ice-bath. After dropwise addition, the system was reacted at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and water (10 mL) was added to the residue. After adjusting the pH to 5 with 1 M hydrochloric acid solution, the solution was filtered by suction. The filter cake was washed with water (10 mL), and dried, to obtain 2.5 g of white solid. Yield: 91%.
At room temperature, Intermediate F (0.5 g, 0.6 mmol) and Intermediate G (0.4 g, 0.7 mmol) were added to 1,4-dioxane (4 mL), and DIPEA (purchased from Shanghai Bide Pharmatech Ltd.) (0.3 g, 2.3 mmol) was dropwise added to the reaction solution. After dropwise addition, the system was heated to 60° C. and reacted for 3 h. After the reaction was completed, the solution was cooled to room temperature, and the solvent was concentrated under reduced pressure. To the residue water (10 mL) was added. After adjusting the pH to 10 with 2.5% NaOH solution, the solution was filtered by suction, and dried, to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=20:1), to obtain 0.35 g of white solid. Yield: 62%.
Intermediate H (0.3 g, 0.6 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (purchased from Reagent Station of Shenyang Pharmaceutical University) (3 mL) was added to the solution. After stirring overnight at room temperature, the mixture solution was concentrated under reduced pressure and water was added (3 mL), and filtered by suction after adjusting the pH to 8 with saturated sodium bicarbonate solution. The filter cake was further purified by silica-gel column chromatography, to obtain 0.15 g of yellow solid. Yield: 80%.
Using 4-acetylaniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-acetylphenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 54%.
Using 4-acetamidoaniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-acetamidophenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 69%.
Using 4-chlorophenylacetic acid (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then N-(4-{3-(2-aminobenzo[d]thiazol-6-yl)-3-[2-(4-morpholinyl)ethyl]ureido}phenyl)acetamide was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 72%.
Using 4-chloroaniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-chlorophenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 57%.
Using 4-trifluoromethylaniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-trifluoromethylphenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 43%.
Using 2-aminopyridine (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(pyridin-2-yl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 38%.
Using 2-naphthylamine (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(naphthalen-1-yl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 59%.
Using 3-bromo-4-fluoroaniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(3-bromo-4-fluorophenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 75%.
Using 4-trifluoromethoxyaniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-trifluoromethoxyphenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 52%.
Using p-ethylaniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-ethylphenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 85%.
Using 4-isopropoxyaniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-isopropoxyphenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 91%.
Using 4-isopropylaniline (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, the key Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, then Intermediate H was synthesized in accordance with the synthesis process of step 8.3 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-isopropylphenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 78%.
Using 2-chloro-N,N-dimethylethyl-1-amine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate F was synthesized in accordance with the synthesis process of step 8.1 in Example 8, then using p-chloroaniline as raw material, Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(N,N-diethylamino)ethyl]-3-(4-chlorophenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 54%.
Using 1-(2-chloroethyl)-4-methylpiperidine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate F was synthesized in accordance with the synthesis process of step 8.1 in Example 8, then using p-chloroaniline as raw material, Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-methylpiperidin-1-yl)ethyl]-3-(4-chlorophenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 65%.
Using 1-(2-chloroethyl)-4-methylpiperazine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate F was synthesized in accordance with the synthesis process of step 8.1 in Example 8, then using p-chloroaniline as raw material, Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(4-methylpiperazin-1-yl)ethyl]-3-(4-trifluoromethylphenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 43%.
Using 1-(2-chloroethyl)pyrrolidine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate F was synthesized in accordance with the synthesis process of step 8.1 in Example 8, then using p-chloroaniline as raw material, Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(pyrrolidin-1-yl)ethyl]-3-(4-chlorophenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 51%.
Using 2-chloro-N,N-dimethylethyl-1-amine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate F was synthesized in accordance with the synthesis process of step 8.1 in Example 8, then using p-chloroaniline as raw material, Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, and then 11-(2-aminobenzo[d]thiazol-6-yl)-1-[2-(N,N-dimethylamino)ethyl]-3-(4-chlorophenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 68%.
Using 4-(3-chloropropyl)morpholine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate F was synthesized in accordance with the synthesis process of step 8.1 in Example 8, then using p-chloroaniline as raw material, Intermediate G was synthesized in accordance with the synthesis process of step 8.2 in Example 8, and then 1-(2-aminobenzo[d]thiazol-6-yl)-1-[3-(4-morpholinyl)propyl]-3-(4-chlorophenyl)urea was synthesized in accordance with the synthesis process of step 8.4 in Example 8. Yield: 76%.
1-(2-Aminobenzo[d]thiazol-6-yl)-3-(4-chlorophenyl)-1-[2-(4-morpholinyl)ethyl]urea (0.2 g, 0.4 mmol) was dissolved in ethyl acetate (3 mL), and potassium carbonate (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (0.1 g, 0.7 mmol) was added. At room temperature, phenylacetic acid (0.1 g, 0.4 mmol) was dropwise added to the solution, and stirred overnight at room temperature. After the reaction was completed, water (5 mL) was added, and extracted with ethyl acetate (2×10 mL). The organic layer was concentrated under reduced pressure, and purified by column chromatography, to obtain 0.08 g of white solid. Yield: 76%.
Using ethyl formate (purchased from Beijing Ouhe Technology Co., Ltd.) as raw material, 1-[2-(2-ethoxyformamido)benzo[d]thiazol-6-yl]-1-[2-(4-morpholinyl)ethyl]-3-(4-chlorophenyl)urea was prepared in accordance with the synthesis process in Example 27. Yield: 45%.
Using ethyl bromoacetate (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, 1-[2-(2-ethoxyformyl)ethylamino-1-yl]benzo[d]thiazol-6-yl]-1-[2-(4-morpholinyl)ethyl]-3-(4-chlorophenyl)urea was prepared in accordance with the synthesis process in Example 27. Yield: 32%.
Using acetyl chloride (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, 1-(2-acetamidobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-chlorophenyl)urea was prepared in accordance with the synthesis process in Example 27. Yield: 82%.
Using tert-butoxyformyl chloride (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, tert-butyl (6-{2-(4-chlorophenyl)-N-[2-(4-morpholinyl)ethyl]acetamido}benzo[d]thiazol-2-yl)carbamate was prepared in accordance with the synthesis process in Example 27. Yield: 60%.
Using benzoyl chloride (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, 1-(2-phenylformamidobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-chlorophenyl)urea was prepared in accordance with the synthesis process in Example 23. Yield: 75%.
Using phenylpropionyl chloride (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, 1-(2-phneylpropionamidobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-chlorophenyl)urea was prepared in accordance with the synthesis process in Example 27. Yield: 68%.
Using phenylacetyl chloride (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, 1-(2-phenylacetamidobenzo[d]thiazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-trifluoromethylphenyl)urea was prepared in accordance with the synthesis process in Example 27. Yield: 73%.
2-Amino-5-nitrophenol (J) (purchased from Shanghai Bide Pharmatech Ltd.) (10 g, 64.9 mmol) was dissolved in 1,4-dioxane (100 mL), and cyanoguanidine (purchased from Beijing Ouhe Technology Co., Ltd.) (17 g, 202.3 mmol) and BF3·Et2O (purchased from Shanghai Bide Pharmatech Ltd.) (28 g, 202.3 mmol) were successively added thereto. The tube was sealed quickly, and reacted at 80° C. for 6 h. After the reaction solution was cooled to room temperature, 2 N sodium bicarbonate solution was added to the solution, and extracted with ethyl acetate (3×100 mL). The organic layers were combined, washed with brine (100 mL), dried over sodium sulfate, concentrated and dried under reduced pressure, to obtain 8.4 g of Intermediate K as brown solid. Yield: 70%.
Intermediate K (8.0 g, 53.7 mmol) was dissolved in methanol (80 mL), and Pd/C (purchased from Shanghai Bide Pharmatech Ltd.) (0.8 g, 5.4 mmol) was added to the solution. The reaction was maintained by feeding hydrogen at room temperature for 12 h. The mixture solution was filtered through celite, the filter cake was washed with methanol (40 mL), and the filtrate was concentrated under vacuum, to obtain 5.4 g of Intermediate L. Yield: 60%. MS (ESI) m/z: 150.2 [M+H]+.
Intermediate L (2.0 g, 13.4 mmol) was dissolved in 1,4-dioxane (20 mL), and potassium carbonate (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (3.1 g, 22.4 mmol) and 4-(2-chloroethyl)morpholine (purchased from Shanghai Bide Pharmatech Ltd.) (1.2 g, 11.2 mmol) were added to the solution. The reaction solution was heated to reflux and reacted under stirring for 8 h. After the reaction was cooled to room temperature, water (80 mL) was added thereto, and extracted with ethyl acetate (2×80 mL). The organic layers were combined, washed with brine (60 mL), dried over sodium sulfate, concentrated and dried under reduced pressure, to obtain 1.5 g of Intermediate M as brown solid. Yield: 72%.
At room temperature, Intermediate M (0.50 g, 0.6 mmol) and phenyl p-toluene methylcarbamate (G) (0.4 g, 0.7 mmol) were added to 1,4-dioxane (4 mL), and DIPEA (purchased from Shanghai Bide Pharmatech Ltd.) (0.3 g, 2.3 mmol) was dropwise added to the reaction solution. After dropwise addition, the system was heated to 60° C. and reacted for 3 h. After the reaction was completed, the solution was cooled to room temperature, and the solvent was concentrated under reduced pressure. To the residue water (10 mL) was added. After adjusting the pH to 10 with 2.5% NaOH solution, the solution was filtered by suction, and dried, to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=20:1), to obtain 0.35 g of white solid. Yield: 62%.
1-(2-Aminobenzo[d]oxazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-methylphenyl)urea (0.2 g, 0.4 mmol) was dissolved in ethyl acetate (3 mL), and potassium carbonate (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (0.1 g, 0.7 mmol) was added. At room temperature, acetyl chloride (0.1 g, 0.4 mmol) was dropwise added to the solution, and stirred overnight at room temperature. After the reaction was completed, water (5 mL) was added, and extracted with ethyl acetate (2×10 mL). The organic layer was concentrated under reduced pressure, and purified by column chromatography, to obtain 0.08 g of white solid. Yield: 76%.
Using p-chlorophenylacetyl chloride (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, 1-(2-phenylacetamidobenzo[d]oxazol-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-methylphenyl)urea was prepared in accordance with the synthesis process in Example 36. Yield: 60%.
2-Chloro-5-aminopyridine (N) (10 g, 78.1 mmol) and potassium thiocyanate (purchased from Shanghai Bide Pharmatech Ltd.) (15 g, 312.4 mmol) were dissolved in glacial acetic acid (100 mL), and bromine (purchased from Reagent Station of Shenyang Pharmaceutical University) (12 g, 156.2 mmol) was dropwise added thereto in ice bath. After dropwise addition, the reaction solution was heated to room temperature and reacted for 12 h. After adjusting the pH to 10 by adding ammonia water into the solution, a yellow solid was precipitated and filtered by suction, to obtain 12.2 g of Intermediate 0 as brown solid. Yield: 83%.
Intermediate O (2.0 g, 13.4 mmol) was dissolved in 1,4-dioxane (20 mL), and sodium hydroxide (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (2.5 g, 22.4 mmol) and 2-morpholino-1-amine (purchased from Shanghai Bide Pharmatech Ltd.) (1.2 g, 11.2 mmol) were added to the solution. The reaction solution was heated to reflux and reacted under stirring for 8 h. After the reaction was cooled to room temperature, water (30 mL) was added thereto, and extracted with ethyl acetate (2×30 mL). The organic layers were combined, washed with brine (60 mL), dried over sodium sulfate, concentrated and dried under reduced pressure, to obtain 1.3 g of Intermediate P as brown solid. Yield: 54%.
At room temperature, Intermediate P (0.5 g, 0.6 mmol) and phenyl p-fluorobenzylcarbamate (G) (0.4 g, 0.7 mmol) were added to 1,4-dioxane (4 mL), and DIPEA (purchased from Shanghai Bide Pharmatech Ltd.) (0.30 g, 2.3 mmol) was dropwise added to the reaction solution. After dropwise addition, the system was heated to 60° C. and reacted for 3 h. After the reaction was completed, the solution was cooled to room temperature, and the solvent was concentrated under reduced pressure. To the residue water (10 mL) was added. After adjusting the pH to 10 with 2.5% NaOH solution, the solution was filtered by suction, and dried, to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=20:1), to obtain 0.3 g of white solid. Yield: 51%.
1-(2-Aminothiazo[5,4-b]pyridin-5-yl)-3-(4-fluorophenyl)-1-[2-(4-morpholinyl)ethyl]urea (0.2 g, 0.4 mmol) was dissolved in ethyl acetate (3 mL), and potassium carbonate (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (0.1 g, 0.7 mmol) was added to the solution, and stirred overnight at room temperature. After the reaction was completed, water (10 mL) was added, and extracted with ethyl acetate (2×30 mL). The organic layer was concentrated under reduced pressure, and purified by column chromatography, to obtain 0.08 g of white solid. Yield: 65%.
At room temperature, 1-(2-aminobenzo[d]thiazol-6-yl)-3-(4-chlorophenyl)-1-[2-(4-morpholinyl)ethyl]urea (0.5 g, 0.6 mmol) and phenyl benzylaminocarbamate (purchased from Shanghai Bide Pharmatech Ltd.) (0.4 g, 0.7 mmol) were added to 1,4-dioxane (4 mL), and triethylamine 0.3 g (2.3 mmol) was dropwise added to the reaction solution. After dropwise addition, the system was heated to 60° C. and reacted for 3 h. After the reaction was completed, the solution was cooled to room temperature, and the solvent was concentrated under reduced pressure. To the residue water (10 mL) was added. After adjusting the pH to 10 with 2.5% NaOH solution, the solution was filtered by suction, and dried, to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=20:1), to obtain 0.25 g of white solid. Yield: 40%.
4,6-Dichloropyridin-3-amine (Q) (5 g, 30.7 mmol) and phenyl isothiocyanate (purchased from Shanghai Bide Pharmatech Ltd.) (4.5 mL, 33.7 mmol) were dissolved in acetone (75 mL), heated to 60° C., and reacted for 4 h. The reaction solution was concentrated under reduced pressure, to obtain 8 g of brown solid. Yield: 80%.
Intermediate R (9 g, 27.7 mmol) was added to NMP (45 mL), and at 0° C., sodium methoxide (purchased from Shanghai Bide Pharmatech Ltd.) (3.0 g, 55.8 mmol) was added to the solution. After addition, the reaction solution was heated to 120° C. and reacted for 4 h. Water (100 mL) was added to the reaction solution, and a brown solid was precipitated, which was filtered by suction to obtain 7 g of brown solid. Yield: 87%.
Intermediate S (5 g, 17.3 mmol) was dissolved in 70% sulfuric acid solution (purchased from Reagent Station of Shenyang Pharmaceutical University) (15 mL), and heated to 110° C. and reacted for 4 h. The reaction solution was concentrated under reduced pressure, adjusted to pH=8 with sodium bicarbonate solution (20 mL), and filtered by suction to obtain 1.8 g of off-white solid. Yield: 56%.
Intermediate T (0.8 g, 25.1 mmol) and cesium carbonate (purchased from Shanghai Bide Pharmatech Ltd.) (2.2 g, 75.3 mmol) were dissolved in 1,4-dioxane (10 mL), and palladium acetate (purchased from Shanghai Bide Pharmatech Ltd.) (0.1 g, 2.51 mmol) and xantphos (purchased from Shanghai Bide Pharmatech Ltd.) (0.35 g, 50.2 mmol) were added to the solution. With feeding nitrogen gas, the reaction solution was heated to 100° C. and reacted for 12 h. After removing the insoluble matters by suction filtration, the filtrate was purified by column chromatography, to obtain 0.5 g of white solid. Yield: 66%.
At room temperature, Intermediate U (0.50 g, 0.6 mmol) and phenyl p-trifluoromethoxybenzylcarbamate (G) (0.4 g, 0.7 mmol) were added to 1,4-dioxane (4 mL), and DIPEA (purchased from Shanghai Bide Pharmatech Ltd.) (0.3 g, 2.3 mmol) was dropwise added to the reaction solution. The system was heated to 60° C. and reacted for 3 h. After the reaction was completed, the solution was cooled to room temperature, and the solvent was concentrated under reduced pressure. To the residue water (10 mL) was added. After adjusting the pH to 10 with 2.5% NaOH solution, the solution was filtered by suction, and dried, to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=20:1), to obtain 0.35 g of white solid. Yield: 60%.
1-(2-Aminothiazo[4,5-c]pyridin-6-yl)-1-[2-(4-morpholinyl)ethyl]-3-(4-trifluoromethoxyphenyl)urea (0.2 g, 0.4 mmol) was dissolved in ethyl acetate (3 mL), and potassium carbonate (purchased from Shenyang Aiketongsheng Chemical Reagent Co., Ltd.) (0.1 g, 0.7 mmol) was added. At room temperature, cyclopropionyl chloride (0.15 g, 0.4 mmol) was dropwise added to the solution, and stirred overnight at room temperature. After the reaction was completed, water (10 mL) was added, and extracted with ethyl acetate (2×30 mL). The organic layer was concentrated under reduced pressure, and purified by column chromatography, to obtain 0.06 g of white solid. Yield: 62%.
Using ethyl chloroacetate (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, 1-{[2-(2-ethoxyformyl)ethylamino-1-yl]thiazo[4,5-c]pyridin-6-yl}-1-[2-(4-morpholinyl)ethyl]-3-(4-trifluoromethoxyphenyl)urea was prepared in accordance with the synthesis process in Example 42. Yield: 42%.
Using 2,4-dichloropyrimidin-5-amine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate U was synthesized in accordance with the synthesis processes of steps 41.1 to 41.4 in Example 41, and then 1-(2-aminothiazo[5,4-d]pyrimidin-5-yl)-1-[2-(4-morpholinyl)ethyl]-3-(pyridin-3-yl)urea was synthesized in accordance with the synthesis process of step 41.5 in Example 41. Yield: 59%.
Using acetyl chloride (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, 1-(2-acetamidothiazo[5,4-d]pyrimidin-5-yl)-1-[2-(4-morpholinyl)ethyl]-3-(pyridin-3-yl)urea was prepared in accordance with the synthesis process in Example 42. Yield: 42%.
Using phenylpropionyl chloride (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, 1-(2-phenylpropionamidothiazo[5,4-d]pyrimidin-5-yl)-1-[2-(4-morpholinyl)ethyl]-3-(pyridin-3-yl)urea was prepared in accordance with the synthesis process in Example 42. Yield: 56%.
Using 3,5-dichloropyridin-2-amine (purchased from Shanghai Bide Pharmatech Ltd.) as raw material, the key Intermediate U was synthesized in accordance with the synthesis processes of steps 41.1 to 41.4 in Example 41, and then 1-(2-aminothiazo[4,5-b]pyridin-6-yl)-3-(4-bromo-3-fluorophenyl)-1-[2-(4-morpholinyl)ethyl]urea was synthesized in accordance with the synthesis process of step 41.5 in Example 41. Then 1-(2-aminothiazo[4,5-b]pyridin-6-yl)-3-(4-bromo-3-fluorophenyl)-1-[2-(4-morpholinyl)ethyl]urea and phenyl benzylcarbamate (0.4 g, 0.7 mmol) were added to 1,4-dioxane (4 mL), and triethylamine (0.3 g, 2.3 mmol) was dropwise added to the reaction solution. The system was heated to 60° C. and reacted for 3 h. After the reaction was completed, the solution was cooled to room temperature, and the solvent was concentrated under reduced pressure. To the residue water (10 mL) was added. After adjusting the pH to 10 with 2.5% NaOH solution, the solution was filtered by suction, and dried, to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=20:1), to obtain 0.25 g of white solid. Yield: 40%.
The structures as well as 1H-NMR and MS data for the compounds of Examples are shown in Table 1.
1H-NMR, MS m/z data
1H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 7.83 (d, J = 1.9 Hz, 1H), 7.24 (s, 2H), 7.19 (d, J = 8.6 Hz, 1H), 7.06 (dd, J = 8.6, 2.0 Hz, 1H), 5.78 (s, 2H). MS (ESI) m/z: 209.5 [M + H]+
1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.99 (s, 1H), 7.88 (d, J = 1.6 Hz, 1H), 7.48 (d, J = 7.9 Hz, 2H), 7.29 (d, J = 1.9 Hz, 2H), 7.26 (s, 1H), 7.24 (d, J = 3.6 Hz, 1H), 7.19 (dd, J = 8.6, 1.7 Hz, 1H), 6.94 (t, J = 7.3 Hz, 1H). MS (ESI) m/z:
1H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.23 (s, 2H), 7.18 (d, J = 8.6 Hz, 1H), 7.06 (dd, J = 8.6, 2.2 Hz, 1H), 2.09 (s, 1H), 1.97 (s, 2H), 1.62 (d, J = 14.8 Hz, 2H), 1.57 (s, 3H), 1.53 (s, 2H), 1.50 (s, 1H), 1.48 (d, J =
1H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 7.83 (s, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.24 (s, 2H), 7.19 (d, J = 8.6 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 6.83 (s, 1H), 4.27 (d, J = 5.8 Hz, 2H). MS (ESI) m/z: 333.5 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 9.66 (s, 2H), 8.19 (d, J = 9.3 Hz, 2H), 7.91 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 9.2 Hz, 2H), 7.31 (d, J = 8.6 Hz, 1H), 7.24 (dd, J = 8.6, 2.1 Hz, 1H), 3.61-3.54 (m, 4H), 3.47 (t, J = 6.6 Hz, 2H), 2.53 (t, J = 6.6 Hz, 2H), 2.42 (s, 4H). MS (ESI) m/z: 443.3 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 10.25 (s, 1H), 9.90 (s, 1H), 7.90 (s, 2H), 7.89 (s, 2H), 7.62 (s, 1H), 7.60 (s, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.22 (dd, J = 8.6, 2.0 Hz, 1H), 3.58 (t, J = 4.5 Hz, 4H), 3.47 (dd, J = 12.2, 6.4 Hz, 2H), 2.53 (d, J = 6.6 Hz, 2H), 2.51 (s, 3H), 2.42 (s, 4H). MS (ESI) m/z: 440.2 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 9.77 (s, 1H), 9.30 (d, J = 5.4 Hz, 2H), 7.83 (d, J = 2.1 Hz, 1H), 7.72 (t, J = 5.4 Hz, 1H), 7.39 (d, J = 8.9 Hz, 2H), 7.32 (d, J = 8.9 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 7.13 (dd, J = 8.6, 2.1 Hz, 1H), 3.51 (t, J = 4.5 Hz, 4H), 3.39 (dd, J = 12.2, 6.4 Hz, 2H), 2.47-2.44 (m, 2H), 2.35 (s, 4H), 1.94 (s, 3H). MS (ESI) m/z: 455.3 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 9.62 (s, 1H), 9.53 (s, 1H), 7.99 (s, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.5 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 3.86 (s, 4H), 3.80 (s, 2H), 3.42 (d, J = 5.2 Hz, 2H), 3.38 (s, 4H). MS (ESI) m/z: 432.3 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 7.63 (s, 1H), 7.37 (d, J = 8.6 Hz, 2H), 7.12 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.2 Hz, 1H), 6.63 (d, J = 5.0 Hz, 1H), 3.59 (t, J = 4.6 Hz, 4H), 2.53-2.52 (m, 2H), 2.47-2.44 (m, 2H), 2.39 (s, 4H). MS (ESI) m/z: 466.3 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 10.58 (s, 1H), 9.45 (s, 1H), 8.28 (dd, J = 5.0, 1.1 Hz, 1H), 7.95 (d, J = 2.1 Hz, 1H), 7.74 (ddd, J = 8.9, 7.4, 1.9 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 8.6 Hz, 1H), 7.25 (dd, J = 8.6, 2.2 Hz, 1H), 7.02- 6.98 (m, 1H), 3.58 (t, J = 4.6 Hz, 4H), 3.47 (t, J = 6.5 Hz, 2H), 2.53 (t, J = 6.6 Hz, 2H), 2.42 (s, 4H). MS (ESI) m/z: 399.6 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 8.25 (dd, J = 5.7, 3.8 Hz, 1H), 8.00 (d, J = 8.6 Hz, 2H), 7.88-7.84 (m, 1H), 7.52 (d, J = 7.7 Hz, 2H), 7.50-7.47 (m, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 8.7 Hz, 2H), 3.60-3.57 (m, 4H), 3.46 (t, J = 6.6 Hz, 4H), 2.53 (d, J = 6.6 Hz, 2H), 2.42 (s, 2H). MS (ESI) m/z: 448.3 [M + H]+
1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 2H), 7.95 (dd, J = 6.3, 2.6 Hz, 1H), 7.88 (d, J = 2.1 Hz, 1H), 7.37 (dd, J = 8.9, 4.2, 2.7 Hz, 1H), 7.28 (dt, J = 8.8, 4.5 Hz, 2H), 7.19 (dd, J = 8.6, 2.1 Hz, 1H), 3.60-3.56 (m, 4H), 3.46 (t, J = 6.6 Hz, 2H), 2.53 (d, J = 6.6 Hz, 2H), 2.42 (s, 4H). MS (ESI) m/z: 494.5 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 9.48 (s, 1H), 9.21 (s, 1H), 7.87 (d, J = 40.0 Hz, 2H), 7.57 (s, 1H), 7.28 (s, 4H), 3.55 (s, 6H), 2.41 (s, 6H). MS (ESI) m/z: 482.6 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 8.59 (s, 2H), 7.88 (d, J = 2.0 Hz, 1H), 7.79 (s, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.6 Hz, 1H), 7.16 (dd, J = 8.6, 2.1 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H), 3.58 (t, J = 4.4 Hz, 4H), 3.48-3.45 (m, 2H), 2.54 (t, J = 6.2 Hz, 2H), 2.51 (dd, J = 5.9, 2.2 Hz, 2H), 2.42 (s, 4H), 1.15 (t, J = 7.6 Hz, 3H). MS (ESI) m/z: 426.3 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.90 (s, 1H), 7.88 (s, 1H), 7.77 (s, 1H), 7.35 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.5 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 8.6 Hz, 2H), 4.53-4.47 (m, 1H), 3.58 (s, 4H), 3.46 (d, J = 5.1 Hz, 2H), 3.33 (s, 2H), 2.42 (s, 4H), 1.24 (s, 3H), 1.23 (s, 3H). MS (ESI) m/z: 456.1 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 7.89 (d, J = 1.8 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.6 Hz, 1H), 7.19 (dd, J = 8.6, 1.7 Hz, 1H), 7.12 (d, J = 8.5 Hz, 2H), 3.59-3.57 (m, 4H), 3.45 (t, J = 6.6 Hz, 2H), 2.85-2.79 (m, 1H), 2.53 (d, J = 6.7 Hz, 2H), 2.42 (s, 4H), 1.19 (s, 3H), 1.17 (s, 3H). MS (ESI) m/z: 440.2 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.64 (s, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.76 (t, J = 5.5 Hz, 1H), 7.48 (d, J = 8.9 Hz, 2H), 7.31 (d, J = 8.9 Hz, 2H), 7.28 (d, J = 8.6 Hz, 1H), 7.16 (dd, J = 8.6, 2.2 Hz, 1H), 3.39 (dd, J = 12.8, 6.3 Hz, 2H), 2.61 (t, J = 6.8 Hz, 2H), 2.53 (d, J = 7.1 Hz, 2H), 2.50-2.49 (m, 2H), 0.97 (t, J = 7.1 Hz, 6H). MS (ESI) m/z: 418.5 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 9.76 (s, 1H), 9.58 (s, 1H), 7.89 (s, 1H), 7.75 (s, 1H), 7.54 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H), 7.26 (s, 1H), 7.23 (d, J = 9.8 Hz, 1H), 3.43 (d, J = 4.9 Hz, 2H), 3.34 (s, 4H), 2.82 (dd, J = 22.5, 11.2 Hz, 4H), 1.93 (t, J = 10.7 Hz, 3H), 0.87 (t, J = 6.7 Hz, 3H). MS (ESI) m/z: 444.3 [M + H]+
1H NMR (400 MHz, DMSO-d6) δ 7.93 (s, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.33-7.19 (m, 2H), 3.44 (t, J = 6.5 Hz, 2H), 2.51 (s, 4H), 2.43 (s, 2H), 2.32 (d, J = 5.2 Hz, 4H), 2.14 (s, 3H). MS (ESI) m/z: 479.2 [M + H]+
1H NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 9.39 (s, 1H), 7.88 (s, 1H), 7.83 (d, J = 5.3 Hz, 1H), 7.53 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 8.6 Hz, 2H), 7.22 (d, J = 8.5 Hz, 1H), 3.45 (d, J = 5.2 Hz, 2H), 2.63 (t, J = 6.6 Hz, 2H), 1.69 (s, 4H), 1.23 (s, 4H). MS (ESI) m/z: 416.5 [M + H]+
1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.95 (s, 1H), 7.80 (d, J = 1.9 Hz, 1H), 7.73 (t, J = 5.2 Hz, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H), 7.12 (dd, J = 8.4, 1.9 Hz, 1H), 2.39 (t, J = 6.5 Hz, 2H), 2.12 (s, 6H), 2.11 (s, 2H). MS (ESI) m/z: 390.6 [M + H]+
1H NMR (600 MHz, DMSO-d6) δ 7.85 (s, 1H), 7.47 (d, J = 8.6 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H), 7.17 (d, J = 8.5 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 3.56 (s, 4H), 3.32 (t, J = 6.8 Hz, 2H), 2.35 (d, J = 7.0 Hz, 2H), 2.33 (s, 4H), 1.75-1.71 (m, 2H). MS (ESI) m/z: 446.3 [M + H]+
The compounds of the present invention were studied for the in vitro enzyme activity of soluble epoxide hydrolase (sEH), and the results were as follows.
The benzothiazole skeleton compounds (concentration: 0.1 μM) according to the above formula I of the present invention were tested for the in vitro sEH enzyme activity by fluorescence analysis. The reference t-AUCB was home-made by the research group (Hwang S H, Wecksler A T, Zhang G, et al. Synthesis and biological evaluation of sorafenib- and regorafenib-like sEH inhibitors. Bioorganic & Medicinal Chemistry Letters, 2013, 23(13): 3732-3737.).
12 μL of sEH enzyme (human recombinant, 0.5 mg/mL, Cayman Chemical) was diluted with 588 μL of buffer (25 mM bis-Tris, pH 7.0). The fluorescent substrate was PHOME (cyano-(6-methoxy-naphthalen-2-yl)-methyl (3-phenyl-oxiranyl)-acetate) in DMSO (10 μM, Cayman Chemical). The compound to be tested was dissolved in 100% DMSO at a concentration of 4 μM. 185 μL of buffer (25 mM bis-Tris, pH 7.0), 5 μL of compound solution, and 5 μL of diluted sEH enzyme were added to the wells of a 96-well plate, and finally 5 μL of substrate solution was added to initiate the reaction. After incubating at 37° C. for 15 min, the fluorescence signal was measured with a microplate reader (excitation wavelength 330 nm, emission wavelength 465 nm). The background group and the full-active group were set at the same time, wherein the background group excluded the compound and the enzyme (replaced by DMSO and buffer, respectively), and the full-active group excluded the compound (replaced by DMSO). The inhibition was calculated from the data of the full-active group (F0) and the compound group (F1) after deducting the fluorescence signal of the background group:
Inhibition (%)=(F0−F1)/(F0)×100.
The in vitro sEH enzyme activity test results of the compounds of Examples were shown in Table 2.
Some compounds of the present invention were studied for the in vitro IC50 of the soluble epoxide hydrolase (sEH), and the results were as follows.
The benzothiazole skeleton compounds according to the above formula I of the present invention were tested for the in vitro sEH enzyme activity by fluorescence analysis. The reference t-AUCB was home-made by the research group.
12 μL of sEH enzyme (human recombinant, 0.5 mg/mL, Cayman Chemical) was diluted with 588 μL of buffer (25 mM bis-Tris, pH 7.0). The fluorescent substrate was PHOME (cyano-(6-methoxy-naphthalen-2-yl)-methyl (3-phenyl-oxiranyl)-acetate) in DMSO (10 μM, Cayman Chemical). The compound to be tested was dissolved in 100% DMSO at a concentration of 4 μM, and successively diluted into solutions with different concentrations (2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.04 μM, 0.02 μM, 0.004 μM, and 0.0004 μM). The inhibitions at different concentrations for the compound were respectively measured in accordance with the inhibition assay, to calculate IC50.
The sEH enzyme IC50 activity data for some Examples were shown in Table 3.
The preliminary in vitro sEH enzyme activity test results showed that, the compounds of general formula I as claimed in the present invention had a good inhibitory activity on sEH, and some compounds were equivalent to or better than the positive control drug t-AUCB.
Male C57BL/6J mice (6 to 8 weeks old) (SPF (Beijing) Biotechnology Co., Ltd.) were divided into 3 groups: sham group, unilateral ureteral obstruction (UUO) group, and UUO+Ex. 16 compound group. After the mice were anesthetized, the left ureter was ligated and cut to create a model. In the sham group, the ureter was only isolated without ligation. The experimental animals were administrated 72 hours before modeling, and the aqueous solution of Ex. 16 compound (10% PEG400, 10% Tween 80, concentration: 9 mg/mL) was intraperitoneally injected at a dose of 30 mg·kg−1·d−1; and the sham group and UUO group were intraperitoneally injected with an equal volume of blank solvent. 7 days after modeling, the animals were sacrificed, and samples were taken for testing. The results showed that, the compound of Example 16 could maintain the level of EETs in mouse kidney tissue (Kim J, Imig J D, Yang J, et al. Inhibition of soluble epoxide hydrolase prevents renal interstitial fibrosis and inflammation. Am J Physiol Renal Physiol, 2014, 307(8): F971-F980.), reduce the collagen deposition, and decrease the fibrotic areas. The results were shown in
Male KM mice (6 to 8 weeks old) (SPF (Beijing) Biotechnology Co., Ltd.) were subcutaneously with 50 μL of 1% λ-carrageenan saline solution at planta to prepare the model of inflammation and swelling (Posadas I, Bucci M, Roviezzo F, et al. Carrageenan-induced mouse paw oedema is biphasic, age-weight dependent and displays differential nitric oxide cyclooxygenase-2 expression. British Journal of Pharmacology, 2004, 142(2):331-338.). 1 hour in advance, the mice were intraperitoneally injected with the solutions of the compounds of Examples 12, 18, 27, and 40 (10% PEG 400, 10% Tween 80) or the blank solvent, respectively. Taking the initial paw thickness of the mice before modeling as 100%, the changes in the paw thickness of the mice were measured. The results showed that, the compounds could significantly inhibit the carrageenan-induced paw swelling in mice, and the effects were better than that of celecoxib. The results were shown in
45 male SD rats (6 to 8 weeks old) (SPF (Beijing) Biotechnology Co., Ltd.) were divided into the sham group, blank solvent group, and dosing group. 72 hours after intraperitoneally injecting the compound of Example 12 or blank solvent, a traumatic brain injury model was established using the modified Feeney method (Marmarou A, Foda M A, van den Brink W, et al., A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics. Journal of Neurosurgry, 1994, 80(2):291 to 300.). 23 hours after model establishment, three mice in each group were injected with Evans blue solution through the tail vein, and after 1 hour of circulation, the mice were decapitated and the brain was taken out to determine the content of Evans blue (EB), so as to study the permeability. Another three rats were taken to measure the water content in brain. The results showed that, the compound of Example 12 could reduce the water content and permeability in brain. The results were shown in Table 4.
Male Balb/c mice (6 to 8 weeks old) (SPF (Beijing) Biotechnology Co., Ltd.) were intraperitoneally injected with streptozotocin STZ (40 mg/kg) once a day for 5 consecutive days, to prepare a diabetic model (Like A A, Rossini A A. Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus. Science, 1976, 193(4251):415-7). The mice after modeling were intraperitoneally injected with the solution of compound of Example 12 once a day. After two weeks, the blood glucose of the dosing group was significantly lower than that of the model group, and the glucose tolerance was higher than that of the model group. The results were shown in
Male Wistar rats (180 to 200 g) (Experimental Animal Center, Hebei Medical University) were intraperitoneally injected with streptozotocin STZ (60 mg/kg) to prepare a diabetic model, and were fed for 3 months to induce a retinopathy model (Miyamoto K, Khosrof S, Bursell S E, et al. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc Natl Acad Sci USA, 1999, 96(19): 10836-10841). The compound of Example 40 was dissolved in 10% hydroxypropyl-p-cyclodextrin solution, to be prepared into eye drops with a content of 0.1% for the compound of Example 40. The rats were topically administrated in eyes every day. After 6 months, the rats developed non-proliferative diabetic retinopathy symptoms of increased retinal capillary free pericytes and increased vascular leakage, while in Example 40 group these symptoms were significantly alleviated. The results were shown in
Male SD rats (6 to 8 weeks old) (SPF (Beijing) Biotechnology Co., Ltd.) were subcutaneously injected with 50 μL of 1% λ-carrageenan saline solution at planta to prepare an inflammatory pain model (Rose T E, Morisseau C, Liu J Y, et al. 1-Aryl-3-(1-acylpiperidin-4-yl)urea inhibitors of human and murine soluble epoxide hydrolase: structure-activity relationships, pharmacokinetics, and reduction of inflammatory pain. J Med Chem, 2010, 53(19): 7067-7075). The compounds of Examples 12 and 40 were added to 0.5% sodium carboxymethylcellulose solution and ground to prepare a suspension with a concentration of 15 mg/mL. 1 hour before modeling, the rats were intragastrically administrated. The changes in the paw withdrawal time of rats were measured with a hot plate dolorimeter. The results showed that, the compounds of Examples 12 and 40 could significantly prolong the paw withdrawal time of rats in the hot plate dolorimeter, increase the pain threshold and relieve the pain. The results were shown in
Male KM mice (6 to 8 weeks old) (SPF (Beijing) Biotechnology Co., Ltd.) swam in advance for 10 min, and were injected with lipopolysaccharide (0.5 mg/kg). After 23 hours, the mice were subcutaneously injected with the solutions of the compounds of Examples 12 and 40 or the blank solvent. After 1 hour, the mice were subjected to the forced swimming test for 6 min, and the immobility time (s) in later 4 minute for the mice in each group was recorded. The results showed that, the compounds of Examples 12 and 40 could significantly reduce the immobility time of mice in forced swimming. The results were shown in
Those skilled in the art should understand that, although the invention is described in details with reference to the above Examples, the invention is not limited to these specific Examples. Based on the methods and technical solutions taught by the invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the invention, and the equivalent embodiments thus obtained are all within the scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010511064.3 | Jun 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/097571 | 6/1/2021 | WO |
