Patent Application
20250145564
This application claims the priority of the Chinese patent application No. 202110935522.0 filed on Aug. 16, 2021, the disclosure of which is incorporated herein by reference in its entirety.
The present invention belongs to the field of pharmaceutical chemistry and chemical synthesis. Specifically, the present invention relates to a sulfonylurea compound, and preparation method and use thereof.
Heart failure is a condition in which disorders of the contraction and/or relaxation function of the heart results in blood stasis in the venous system and insufficient blood perfusion in the arterial system, thereby causing circulatory disorders in the heart. Myocardium is closely related to the contraction and relaxation function of the heart. Myocardial damage caused by various reasons may lead to heart failure, among which inflammation is one of the important causes for myocardial damage.
Epoxyeicosatrienoic acids (EETs) are a class of functional small molecules generated by arachidonic acid under the action of CYP450. Arachidonic acid is one of the most widely distributed and abundant polyunsaturated essential fatty acid in living organisms. It can generate through the CYP450 pathway four different EETs isomers, namely 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, which are collectively known as EETs. EETs have various regulatory effects in the human body, such as increasing sodium-hydrogen reverse transport activity, decreasing cyclooxygenase (COX) activity, and promoting the release of calcium from intracellular storage.
EETs are unstable and can be hydrolyzed by soluble epoxide hydrolase (sEH) to weakly bioactive dihydroxyeicosaterionic acid (DHET), which can be quickly excreted from the body. The CYP-EETs-sEH metabolic pathway plays a regulatory role in multiple key pathways in the human body. Research has shown that sEH inhibitors can reverse endothelial dysfunction, regulate blood pressure, reduce the occurrence of stroke and epilepsy, enhance insulin action, and reduce myocardial damage, and can be anti-inflammatory and analgesic. sEH inhibitors for the treatment of hypertension and diabetes is extensively studied.
In view of this, the present invention is proposed.
The primary object of the present invention is to provide a sulfonylurea compound of formula I, or a stereoisomer or pharmaceutically acceptable salt thereof.
The second object of the present invention is to provide a method for preparing the sulfonylurea compound.
The third object of the present invention is to provide use of the sulfonylurea compound as a sEH inhibitor in preparation of a medicament for prevention and/or treatment of heart failure.
To achieve the above objects, the following technical solution is adopted.
The present invention provides a sulfonylurea compound of formula I, or a stereoisomer or pharmaceutically acceptable salt thereof,
Furthermore, the sulfonylurea compound of formula I of the present invention is selected from the compounds of formulas IA and IB:
The present invention also provides a method for preparing the compound of formula IA, which is one of the following two methods:
The present invention also provides a method for preparing the compound of formula IB, as follows:
The present invention also provides a pharmaceutical composition comprising one or more selected from the group consisting of the aforementioned sulfonylurea compound and the stereoisomer or pharmaceutically acceptable salt thereof.
The present invention also provides a soluble epoxide hydrolase inhibitor, comprising one or more selected from the group consisting of the aforementioned sulfonylurea compound and the stereoisomer, pharmaceutically acceptable salt thereof, or comprising the aforementioned pharmaceutical composition.
The present invention also provides use of the aforementioned sulfonylurea compound, or the stereoisomer or pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition, wherein the use is selected from the group consisting of: use in preparation of a medicament for inhibiting the activity of soluble epoxide hydrolase; use in preparation of a medicament for increasing the level of epoxyeicosatrienoic acids (EETs), use in preparation of a medicament for reducing inflammatory response, and use in preparation of a medicament for preventing and/or treating heart failure.
The present invention also provides use of a sulfonylurea drug in preparation of a medicament for prevention and/or treatment of heart failure, wherein the sulfonylurea drug is selected from the group consisting of glimepiride, gliclazide, gliquidone, glibenclamide, glipizide, tolazamide, torasemide and acetohexamide.
The technical solution of the present invention has at least the following technical effects:
The compound of the present invention has soluble epoxide hydrolase (sEH) inhibitory activity, which can increase the level of epoxyeicosatrienoic acids (EETs), thereby reducing inflammatory response, and can be used for prevention and treatment of heart failure.
The preparation method of the present invention has the technical advantages of simple steps, high yield, and easily available raw materials.
In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details.
The term “C1-C6 alkyl” refers to a straight or branched alkyl having 1 to 6 carbon atoms. For example, it includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, amyl, isopentyl, hexyl, etc.
The term “C3-C7 cycloalkyl” refers to a cyclic alkyl having 3 to 7 carbon atoms, including, but not limited to, cyclopropyl, methylcyclopropyl, ethylcyclopropyl, dimethylcyclopropyl, cyclobutyl, methylcyclobutyl, ethylcyclobutyl, cyclopentyl, cyclohexyl, etc.
The term “C4-C8 N-heterocyclyl” refers to a saturated or unsaturated non-aromatic heterocyclyl having 4 to 8 carbon atoms and at least one nitrogen atom on the ring, including, but not limited to,
The term “C3-C6 cycloalkyl-fused pyrrolidinyl” includes, but is not limited to,
The term “C2-C6 alkenyl” refers to a straight or branched alkenyl having 2 to 6 carbon atoms, for example, including, but not limited to, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl, pentenyl, isopentenyl, hexenyl, etc.
The term “C1-C2 alkyl substituted by C2-C6 alkenyl” refers to a C1-C2 alkyl substituted with the aforementioned “C2-C6 alkenyl”, for example, including, but not limited to, allyl, etc.
The term “C1-C6 acyl” refers to an acyl having 1 to 6 carbon atoms, for example, including, but not limited to, formyl, acetyl, propionyl, isopropyl, butyryl, isobutyryl, tert-butyryl, valeryl, isovaleryl, neovaleryl, hexyl, tert-hexyl, etc.
The term “C1-C6 alkoxy” refers to a straight, branched or cyclic alkoxy having 1 to 6 carbon atoms. For example, it includes, but is not limited to, methoxy, ethoxy, propioxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, isopentoxy, cyclopentoxy, hexoxy, cyclohexoxy, etc.
The term “C1-C6 alkylene” refers to an alkylene having 1 to 6 carbon atoms, such as, but not limited to, methylene, methyl-substituted methylene, ethyl-substituted methylene, propyl-substituted methylene, ethylidene, methyl-substituted ethylene, ethyl-substituted ethylene, propylene, methyl-substituted propylene, ethyl-substituted propylene, pentene, hexylene, etc.
The term “aryl” refers to a substituent with aromatic ring structural properties, preferably is “C6-C14 aryl”, which represents an aryl having 6 to 14 carbon atoms, for example, including, but not limited to, phenyl, substituted phenyl, naphthyl, substituted naphthyl, anthryl, etc.
The term “heteroaryl” refers to a monocyclic or polycyclic group (each ring having 4 to 6 atoms) having 5 to 14 ring atoms, wherein one or more atoms are heteroatoms selected from N, O, or S, and the rest is carbon. The “heteroaryl” has certain aromatic properties. The preferred heteroaryl herein is “C2-C9 heteroaryl”, which represents a heteroaryl having 2 to 9 carbon atoms, including, such as, but not limited to, furyl, substituted furyl, benzofuranyl, substituted benzofuranyl, thienyl, substituted thienyl, benzothienyl, substituted benzothienyl, indolyl, substituted indolyl, isoindolyl, substituted isoindolyl, pyrrolyl, substituted pyrrolyl, thiazolyl, substituted thiazolyl, oxazolyl, substituted oxazolyl, pyrazolyl, substituted pyrazolyl, imidazolyl, substituted imidazolyl, pyranyl, substituted pyranyl, pyridazinyl, substituted pyridaziny, pyrazinyl, substituted pyrazinyl, pyrimidinyl, substituted pyrimidinyl, pyridyl, substituted pyridyl, quinolyl, substituted quinolyl, isoquinolyl, carbazolyl, substituted carbazolyl, etc.
The term “halogen” is selected from fluorine, chlorine, bromine, and iodine.
The term “halogenation” includes monohalogenation, polyhalogenation, or perhalogenation, that is, one or more, or all hydrogen atoms are replaced by halogen.
The term “substitution” means that one or more hydrogen atoms on a group are substituted by one or more substituents.
The term “5-6 membered lactam ring” refers to a saturated or unsaturated non-aromatic heterocyclic group having 5 to 6 ring atoms and containing an amide moiety on the ring, including, but not limited to,
where the hydrogen atom on the ring can be replaced by a substituent.
In one aspect, the present invention relates to a sulfonylurea compound of formula I, or a stereoisomer or pharmaceutically acceptable salt thereof:
wherein Rei is a C1-C6 alkylene, Rb2 is a C3-C7 cycloalkyl;
wherein Rd and Re are each independently selected from H, C1-C6 alkyl, C3-C7 cycloalkyl, substituted or unsubstituted C6-C14 aryl; or Rd and Re, together with the N atom connected thereto, form a substituted or unsubstituted pyrrolidinyl, piperidyl or piperazinyl; wherein the substituent for the “substituted” is selected from C1-C6 alkyl, halogenated C1-C6 alkyl, —OH, halogen, C1-C6 alkoxy, halogenated C1-C6 alkoxy and —CN;
wherein the substituent for the “substituted” is selected from C1-C4 alkyl, —C(═O)XRh, and —(CH2)mORi;
wherein Rb1 is a C1-C6 alkylene; Rb2 is a C3-C7 cycloalkyl;
wherein Rp1, Rp2, Rp3 and Rp4 are each independently a C1-C6 alkyl;
The sulfonylurea compound of the present invention can be used as a sEH inhibitor with strong inhibitory activity on sEH enzymes, and some compounds have an IC50 lower than 1 μM.
In one embodiment, the sulfonylurea compound of formula I of the present invention may be selected from the compounds of formulas IA and IB below.
In some embodiments, Rb is a C1-C3 alkyl, a C1-C3 acyl, or
In some embodiments, Rc is a C1-C3 alkyl, a C3-C6 cycloalkyl, a C1-C3 acyl, —CO(CH2)nNRdRe,
wherein Rd, Re and n are defined as above.
In some embodiments, Rd and Re are each independently selected from H, C1-C3 alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted phenyl, or Rd and Re, together with the N atom connected thereto, form a substituted or unsubstituted pyrrolidinyl, piperidyl or piperazinyl; wherein the substituent for the “substituted” is selected from C1-C3 alkyl, halogenated C1-C3 alkyl, —OH, halogen, C1-C3 alkoxy, halogenated C1-C3 alkoxy and —CN.
or a substituted cyclohexyl; wherein the substituent for the “substituted” is selected from C1-C4 alkyl, —C(═O)XRh, and —(CH2)mORi;
In some embodiments, Rh is a C1-C3 alkyl, a C1-C3 acyl, or
In some embodiments, Ri is a C1-C3 alkyl, a C3-C6 cycloalkyl, a C1-C3 acyl, —CO(CH2)nNRdRe,
wherein Rd, Re and n are defined as above.
In some embodiments, Rd and Re are each independently selected from H, C1-C3 alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted phenyl, or Rd and Re, together with the N atom connected thereto, form a substituted or unsubstituted pyrrolidinyl, piperidyl or piperazinyl; wherein the substituent for the “substituted” is selected from C1-C3 alkyl, halogenated C1-C3 alkyl, —OH, halogen, C1-C3 alkoxy, halogenated C1-C3 alkoxy and —CN.
In some embodiments, the compound of formula IB is selected from the compounds of formulas IB1-IB7.
In some embodiments, the sulfonylurea compound of formula I of the present invention is selected from the following compounds:
In some embodiments, the sulfonylurea compound, or the stereoisomer or pharmaceutically acceptable salt thereof of the present invention may exist in the form of a crystalline hydrate or solvate. These crystalline hydrate or solvate are also included within the scope of the present invention.
On the basis of knowing the structure of the compound of the present invention, those skilled in the art may design and synthesize the compound of the present invention using a reaction known in the art. Therefore, there is no special limitation on the specific preparation method for synthesizing the compound of the present invention, as long as the compound of the present invention can be obtained.
In some embodiments, the compound of formula IA of the present invention can be prepared using one of the following two methods:
the compound of formula V reacts with the compound of formula VI through nucleophilic substitution to obtain the compound of formula IA;
the compound of formula VII reacts with the compound of formula V through nucleophilic substitution to obtain the compound of formula VIII, which is then subjected to esterification, acylation, etherification, or phosphorylation to obtain the compound of Formula IA,
wherein, R13A is —COOH or —(CH2)mOH; and R11, R13 and m are defined as above.
In some embodiments, the compound of formula V is prepared by the method below:
the compound of formula IV is subjected to acylation to prepare the sulfonylcarbamate of formula V;
wherein, R11 is defined as above.
In some embodiments, the sulfonylcarbamate of formula V may be prepared by acylating the compound of formula IV with ethyl chloroformate in an organic solvent; wherein the preferred reaction temperature is the ice bath temperature, and the preferred organic solvent is dichloromethane.
In some embodiments, the compound of formula V and the compound of formula VI may be subjected to nucleophilic substitution in an organic solvent under protection of an inert gas (nitrogen or argon); wherein the preferred organic solvent is toluene, and the preferred reaction temperature is reflux temperature.
In some embodiments, the compound of formula IB of the present invention may be prepared by the following method:
the compound of formula IX reacts with R21—I through alkylation to obtain the compound of formula IB,
wherein, R21, R22 and R23 are defined as above.
In some embodiments, the compound of formula IX is prepared by one of the following methods:
the compound of formula X is subjected to acylation to prepare the sulfonylcarbamate of formula XI, which then reacts with the compound of formula XII through nucleophilic substitution to obtain the compound of formula IX.
the compound of formula X is subjected to acylation to prepare the sulfonylcarbamate of formula XI, which then reacts with the compound of formula XIV through nucleophilic substitution to obtain the compound of formula XV; and the compound of formula XV is subjected to esterification, acylation, etherification, or phosphorylation to obtain the compound of formula IX;
wherein, R23A is a substituted cyclohexyl, wherein the substituent for the “substituted” is selected from —COOH and —(CH2)mOH; and R22, R23 and m are defined as above.
In some embodiments, the alkylation may be carried out using R21—I and the compound of formula IX in the presence of potassium carbonate in an organic solvent as the solvent. R21—I is added dropwise to an organic solvent dissolving with IX under an ice bath. After the dropwise addition is complete, the system is raised to room temperature. After the TLC detection indicates that the reaction is complete, water and ethyl acetate are added. After layered, the organic phase is washed with water, concentrated and purified by silica gel column chromatography. The preferred organic solvent is DMF.
In another respect, the present invention provides a pharmaceutical composition comprising one or more selected from the group consisting of the sulfonylurea compound and the stereoisomer and pharmaceutically acceptable salt thereof. The pharmaceutical composition may also include one or more pharmaceutically acceptable excipients, diluents, carriers, excipients, or adjuvants.
It has been experimentally confirmed that the compound of the present invention has inhibitory activity on soluble epoxide hydrolase (sEH), can increase the level of epoxyeicosatrienoic acids (EETs), and can thereby reduce inflammatory response and can be used for prevention and/or treatment of heart failure.
Therefore, in still another respect, the present invention further provides a soluble epoxide hydrolase inhibitor, comprising one or more selected from the group consisting of the sulfonylurea compound and the stereoisomer and pharmaceutically acceptable salt thereof, or comprising the pharmaceutical composition.
The present invention also provides a use of the sulfonylurea compound or the stereoisomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, wherein the use is selected from the group consisting of: use in preparation of a medicament for inhibiting the activity of soluble epoxide hydrolase; use in preparation of a medicament for increasing the level of epoxyeicosatrienoic acids (EETs); use in preparation of a medicament for reducing inflammatory response; and use in preparation of a medicament for preventing and/or treating heart failure.
The present invention also provides the sulfonylurea compound or the stereoisomer or pharmaceutically acceptable salt thereof or the pharmaceutical composition for inhibition of the activity of soluble epoxide hydrolase, for increasing the level of epoxyeicosatetraenoic acids (EETs), for reducing inflammatory response, or for preventing and/or treating heart failure.
The present invention also provides a method for suppressing the activity of soluble epoxide hydrolase, a method for increasing the level of epoxyeicosatetraenoic acids (EETs), a method for reducing inflammatory response, or a method for preventing and/or treating heart failure, comprising: administering to a subject in need thereof an effective amount of the sulfonylurea compound or the stereoisomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition.
The present invention also provides use of a sulfonylurea drug in preparation of a medicament for prevention and/or treatment of heart failure, wherein the sulfonylurea drug is selected from the group consisting of glimepiride, gliclazide, gliquidone, glibenclamide, glipizide, tolazamide, torasemide and acetohexamide.
The present invention also provides a method for prevention and/or treatment of heart failure, comprising: administering to a subject in need thereof an effective amount of a sulfonylurea drug selected from the group consisting of glimepiride, gliclazide, gliquidone, glibenclamide, glipizide, tolazamide, torasemide and acetohexamide.
In order to clarify the purpose, technical solution, and advantages of the examples of the present invention, the following will provide a clear and complete description of the technical solution in the examples of the present invention. Obviously, the described examples are partial examples of the present invention, rather than the entire examples. Based on the examples in the present invention, all other examples, which are obtained by those skilled in the art without creative labor, fall within the scope of protection of the present invention. In some examples, the raw materials, components, methods, means, etc. familiar to those skilled in the art are not described in detail to highlight the main idea of the present invention.
The following examples further explain the synthesis method of the compounds and intermediates of the present invention, without limiting the scope of the present invention.
1H NMR was performed on Bruker-400 or Bruker-500. Low-resolution mass spectrometry was performed on a Thermo Fisher FINNIGAN LTQ mass spectrometer. The reagents and raw materials used in the present invention are all commercially available raw materials unless otherwise specified.
200 mg of I-1-a, 56 mg of potassium carbonate and 2 ml of N,N-dimethylformamide (DMF) were added into a 25 ml of three necked flask, and 55 mg of methyl iodide was dropped into the reaction solution at 0° C. The reaction solution was warmed to 25° C. and reacted for 1 h. 2 ml of water and 10 ml of ethyl acetate were added to the reaction solution and layered. The organic phase was washed three times with water (2 ml×3), concentrated and purified by column chromatography to obtain 105 mg of I-1 as a white solid with a yield of 51.0%.1H NMR (DMSO-d6, 500 MHz): 8.37 (t, 1H), 7.79 (d, 2H), 7.50 (d, 2H), 7.23 (d, 1H), 4.17 (s, 2H), 3.51 (q, 2H), 3.05 (s, 3H), 2.92 (t, 2H), 2.19 (q, 2H), 2.02 (s, 3H), 1.73 (d, 2H), 1.64 (d, 2H), 1.28 (m, 2H), 0.98 (t, 3H), 0.92 (m, 2H), 0.85 (d, 3H). ESI-MS m/z 505.3 (M+H)+.
I-2 was prepared with a yield of 65.3% following the same method as that in Example 1, except that I-1-a was replaced with I-2-a. 1H NMR (CDCl3, 500 MHz): 7.75 (m, 2H), 7.69 (d, 1H), 7.49 (m, 2H), 7.37 (d, 1H), 7.22 (m, 2H), 4.27 (m, 2H), 3.91 (s, 3H), 3.12 (s, 3H), 3.05 (m, 2H), 1.91 (m, 1H), 1.74 (dt, 2H), 1.65 (s, 3H), 1.63 (m, 1H), 1.55 (s, 6H), 1.42-1.24 (m, 6H). ESI-MS m/z 542.2 (M+H)+.
I-3 was prepared with a yield of 72.3% following the same method as that in Example 1, except that I-1-a was replaced with I-3-a. 1H NMR (CDCl3, 500 MHz): 8.19 (d, 1H), 7.83 (s, 1H), 7.79 (d, 2H), 7.45 (d, 2H), 7.41 (dd, 1H), 7.23 (d, 1H), 6.90 (d, 1H), 3.81 (s, 3H), 3.77 (q, 2H), 3.14 (s, 3H), 3.05 (t, 2H), 1.78 (m, 3H), 1.30 (m, 6H). ESI-MS m/z 508.2 (M+H)+.
I-4 was prepared with a yield of 61.6% following the same method as that in Example 1, except that I-1-a was replaced with I-4-a. 1H NMR (CDCl3, 500 MHz): 7.96 (s, 1H), 7.72 (d, 2H), 7.36 (d, 2H), 3.23 (d, 2H), 3.13 (s, 2H), 2.66 (d, 2H), 2.46 (m, 5H), 1.68-1.50 (m, 6H).
ESI-MS m/z 338.4 (M+H)+.
I-5 was prepared with a yield of 58% following the same method as that in Example 1, except that I-1-a was replaced with I-5-a. ESI-MS m/z 339.1 (M+H)+.
I-6 was prepared with a yield of 43% following the same method as that in Example 1, except that iodomethane was replaced with allyl bromide. ESI-MS m/z 531.3 (M+H)+.
I-7 was prepared with a yield of 53% following the same method as that in Example 1, except that I-1-a was replaced with I-7-a and iodomethane was replaced with isopropyl bromide. ESI-MS m/z 366.2 (M+H)+.
I-8 was prepared with a yield of 52% following the same method as that in Example 1, except that iodomethane was replaced with cyclopropyl bromide. ESI-MS m/z 531.2 (M+H)+.
I-9-a (1.1 g, 2.18 mmol) was dissolved in 20 ml of DMF, and imidazole (0.75 g, 10.92 mmol) was added. tert-Butylchlorodiphenylsilane (TBDPSCl) (0.6 ml, 2.29 mmol) was dropped therein, and stirred overnight. TLC showed that the raw material was completely converted, and the reaction mixture was diluted with ethyl acetate. The organic phase was washed with water and saline, dried with anhydrous sodium sulfate, filtered and concentrated to obtain I-9-b, which was directly used for the next step without further purification.
I-9-c was prepared following the same method as that in Example 1, except that I-1-a was replaced with I-9-b.
I-9-c (0.2 g, 0.26 mmol) was dissolved in tetrahydrofuran, added dropwisely with 1.5 mL of concentrated hydrochloric acid, and stirred at room temperature for 5 hours. TLC showed that the raw material was completely converted, and the reaction mixture was diluted with dichloromethane. The organic phase was wash with saline, dried with anhydrous sodium sulfate, filtered, concentrated and purified to obtain I-9 with a yield of 54%. ESI-MS m/z 521.2 (M+H)+.
Step 1: I-10-a (143 mg, 0.62 mmol) was suspended in dichloromethane (DCM), added with 3 eq of triethylamine, followed by dropwise addition of 2.5 eq of ethyl isocyanate at room temperature. The reaction mixture was reacted overnight at room temperature (10-18° C.), and TLC showed that the raw material was completely converted. The reaction mixture was diluted with ethyl acetate (EA). The organic phase was washed with diluted hydrochloric acid (14 mL of 1N hydrochloric acid was diluted to 80 mL) and saline, dried, filtered and concentrated to obtain I-10-b (180 mg, 0.6 mmol) as a white solid.
Step 2: I-10-b (180 mg, 0.6 mmol) was dissolved in DCM, added dropwisely with 10 eq of trifluoroacetic acid at room temperature, and stirred at room temperature for 3 hours after adding. The reaction mixture was concentrated to remove most of the trifluoroacetic acid, and the residue was diluted with DCM and then added with aqueous sodium bicarbonate to adjust the pH to be weakly basic. The organic phase was separated, and the aqueous phase was repeatedly extracted with DCM. The combined organic phase was dried, filtered and concentrated to obtain I-10-c.
Step 3: I-10-d (2 g, 5.70 mmol) was suspended in 30 mL of dichloromethane, and added with triethylamine (1.98 mL, 14.2 mmol), followed by dropwise addition of ethyl chloroformate (810 μL, 8.54 mmol). After the addition, the temperature was slowly raised to room temperature. After 7 hours, TLC showed that the raw material was completely converted. The reaction mixture was diluted with 100 mL of dichloromethane, and the organic phase was washed with diluted hydrochloric acid (14 mL of 1N hydrochloric acid was diluted to 80 mL) and saline, dried with anhydrous sodium sulfate, concentrated to obtain a crude, which was added with 8 mL of acetone, stirred for 2 hours under reflux and then for 2 hours at room temperature, filtered, dried to obtain I-10-e as a white solid.
Step 4: I-10-c (50 mg, 0.25 mmol) and I-10-e (105 mg, 0.25 mmol) were mixed in a small amount of toluene, purged with nitrogen, and refluxed for 5 hours. The reaction mixture was concentrated, and slurred in acetone to obtain a total of 95 mg of I-10. 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 8.37 (t, J=5.8 Hz, 1H), 7.87-7.73 (m, 2H), 7.46 (d, J=8.2 Hz, 2H), 7.02 (d, J=6.1 Hz, 1H), 6.31 (d, J=7.7 Hz, 1H), 4.17 (s, 2H), 3.71 (d, J=6.5 Hz, 2H), 3.50 (q, J=6.8 Hz, 2H), 3.19 (dd, J=7.7, 3.8 Hz, OH), 2.97 (qd, J=7.2, 5.6 Hz, 2H), 2.90 (t, J=7.2 Hz, 2H), 2.18 (q, J=7.5 Hz, 2H), 2.01 (s, 3H), 1.69 (dd, J=28.0, 12.3 Hz, 4H), 1.45 (s, 1H), 1.16-1.04 (m, 2H), 0.98 (td, J=7.4, 2.8 Hz, 7H). ESI-MS m/z 578.3 (M+H)+.
I-11 was prepared with a yield of 57% following the same method as that in Example 1, except that I-1-a was replaced with I-10. ESI-MS m/z 592.3 (M+H)+.
I-12 was prepared with a yield of 52% following the same method as that in Example 10, except that ethyl isocyanate was replaced with isopropyl isocyanate in Step 1. ESI-MS m/z 590.3 (M+H)+.
I-13 was prepared with a yield of 62% in the final step following the same method as that in Example 10, except that ethyl isocyanate was replaced with 4-trifluoromethoxyphenyl isocyanate in Step 1. The final step yield was 62%. 1H NMR (500 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.82 (s, 1H), 8.37 (t, J=5.8 Hz, 1H), 7.81 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.9 Hz, 2H), 7.46 (d, J=8.4 Hz, 2H), 7.28 (d, J=8.6 Hz, 2H), 6.34 (d, J=7.7 Hz, 1H), 4.17 (s, 2H), 3.89 (d, J=6.5 Hz, 2H), 3.53-3.46 (m, 2H), 3.27-3.16 (m, 1H), 2.90 (t, J=7.2 Hz, 2H), 2.18 (q, J=7.5 Hz, 2H), 2.00 (s, 3H), 1.74 (td, J=14.7, 3.7 Hz, 4H), 1.60-1.49 (m, 1H), 1.12 (qd, J=12.7, 3.7 Hz, 2H), 1.07-0.88 (m, 5H). ESI-MS m/z 710.2 (M+H)+.
1.1 eq of N,N-dimethylglycine, 1.2 eq of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) and a catalytic amount of 4-dimethylaminopyridine (DMAP) were mixed in a small amount of DMF, stirred for 5 minutes, added with I-9-a (50 mg, 1 eq) at once, stirred overnight at room temperature, and diluted with EA. The organic phase was washed with aqueous ammonium chloride and saline, dried, concentrated and purified with silica gel column chromatography to obtain I-14 with a yield of 67%. 1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 10.21 (s, 1H), 8.38 (t, J=5.8 Hz, 1H), 7.81 (d, J=8.1 Hz, 2H), 7.45 (d, J=8.1 Hz, 2H), 6.63 (d, J=7.7 Hz, 1H), 4.20 (s, 2H), 4.17 (s, 2H), 3.99 (d, J=6.3 Hz, 2H), 3.49 (q, J=6.8 Hz, 2H), 3.22 (d, J=10.4 Hz, 1H), 2.90 (t, J=7.2 Hz, 2H), 2.82 (s, 6H), 2.19 (q, J=7.5 Hz, 2H), 2.01 (s, 3H), 1.78-1.64 (m, 4H), 1.56 (s, 1H), 1.08 (ddd, J=31.6, 13.7, 6.9 Hz, 3H), 0.98 (t, J=7.5 Hz, 4H). ESI-MS m/z 592.5 (M+H)+.
I-15 was prepared with a yield of 57% following the same method as that in Example 1, except that I-1-a was replaced with I-14. ESI-MS m/z 606.3 (M+H)+.
I-16 was prepared with a yield of 49% following the same method as that in Example 14, except that N,N-dimethylglycine was replaced with 2-(piperidin-1-yl)acetic acid. ESI-MS m/z 632.5 (M+H)+.
I-17-b and 1.1 eq of I-17-a were dissolved in DMF, added with 2 eq of potassium carbonate, and reacted for 1 hour at room temperature. TLC showed that the reaction was complete. The system was added with water, and extracted several times with EA. The EA phase was washed with water and saturated saline, dried with anhydrous sodium sulfate, filtered and rotary evaporated to dryness, and the residue was purified with column chromatography to obtain I-17 with a yield of 32%. ESI-MS m/z 691.3 (M+H)+.
I-10-e (100 mg, 0.2 mmol, 1 eq) was dissolved in DCM (2 mL). After dropwise addition of 2,6-dimethylpyridine (46.6 μL, 0.4 mmol, 2.0 eq), the reaction solution was cooled to −30° C., added with I-18-a (1.5 eq) dissolved in DCM, and then warmed to room temperature. After TLC showed that the reaction was complete, the reaction solution was poured into water (10 mL), extracted with dichloromethane (15 mL×3), and layered. The organic phase was washed with 0.5 M diluted hydrochloric acid (10 mL), saturated sodium bicarbonate (10 mL) and saturated saline (10 mL) successively, dried with anhydrous sodium sulfate, filtered, and rotary evaporated to dryness. The residue was purified with column chromatography to obtain I-18 with a yield of 20% as a white solid. ESI-MS m/z 819.3 (M+H)+.
I-19 was prepared with a yield of 19% following the same method as that in Example 18, except that I-18-a was replaced with I-19-a. ESI-MS m/z 776.3 (M+H)+.
I-20 was prepared with a yield of 28% following the same method as that in Example 1, except that I-1-a was replaced with 1-17. ESI-MS m/z 705.4 (M+H)+.
I-21 was prepared with a yield of 22% following the same method as that in Example 1, except that I-1-a was replaced with I-18. ESI-MS m/z 847.5 (M+H)+.
I-22 was prepared with a yield of 19% following the same method as that in Example 1, except that I-1-a was replaced with I-19. ESI-MS m/z 790.5 (M+H)+.
I-9-a (0.14 g, 0.27 mmol) was suspended in dichloromethane, added with N,N-diisopropylethylamine (96 μL, 0.55 mmol) and DMAP (4 mg, 0.03 mmol) at room temperature, added dropwisely with acetic anhydride (39 μL, 0.41 mmol), and stirred overnight. TLC showed that the raw material was completely converted, the reaction mixture was diluted with dichloromethane, and the organic phase was washed with dilute hydrochloric acid and saline, dried with anhydrous sodium sulfate, filtered and concentrated to obtain the crude, which was recrystallized in an appropriate amount of acetone, filtered and dried to obtain I-23 as a white solid with a yield of 57%. 1H NMR (500 MHz, DMSO-d6) δ 10.35 (s, 1H), 8.37 (t, J=5.8 Hz, 1H), 7.81 (d, J=8.0 Hz, 2H), 7.46 (d, J=8.0 Hz, 2H), 6.33 (d, J=7.8 Hz, 1H), 4.17 (s, 2H), 3.79 (d, J=6.6 Hz, 2H), 3.50 (q, J=6.7 Hz, 2H), 3.25-3.16 (m, 1H), 2.90 (t, J=7.2 Hz, 2H), 2.18 (q, J=7.5 Hz, 2H), 2.01 (s, 3H), 1.99 (s, 3H), 1.77-1.70 (m, 2H), 1.66 (d, J=13.0 Hz, 2H), 1.50 (s, OH), 1.16-1.05 (m, 2H), 0.97 (t, J=7.4 Hz, 4H). ESI-MS m/z 571.3 (M+Na)+.
I-24 was prepared with a yield of 48% following the same method as that in Example 1, except that I-1-a was replaced with I-23. ESI-MS m/z 585.3 (M+Na)+.
I-25 was prepared with a yield of 78% following the same method as that in Example 10, except that ethyl isocyanate was replaced with iodomethane to obtain I-25-a in Step 1. ESI-MS m/z 521.2 (M+Na)+.
Step 1: The raw material I-26-a (2.5 g, 13.92 mmol) was suspended in 20 mL of methanol, added dropwisely with concentrated sulfuric acid (275 mg, 2.8 mmol), and reacted overnight at 60° C. The solvent was rotary evaporated off to form a large number of white solid, which was slurried in 15 mL of acetone, filtered, and dried to obtain I-26-b as a white solid with a yield of 86%.
Step 2: I-26 was prepared with a yield of 72% by reacting I-26-b with I-10-e following the same method as that in Step 4 of Example 10. 1H NMR (500 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.37 (t, J=5.8 Hz, 1H), 7.86-7.74 (m, 1H), 7.46 (d, J=8.5 Hz, 1H), 6.35 (d, J=7.7 Hz, 1H), 4.17 (s, 1H), 3.56 (s, 2H), 3.52-3.47 (m, 1H), 3.22 (dt, J=7.7, 3.9 Hz, OH), 2.90 (t, J=7.2 Hz, 1H), 2.27-2.13 (m, 2H), 2.08 (s, 1H), 2.01 (s, 1H), 1.89-1.81 (m, 1H), 1.73 (dd, J=12.8, 3.6 Hz, 1H), 1.31 (qd, J=13.2, 3.4 Hz, 1H), 1.20-1.08 (m, 1H), 0.97 (t, J=7.5 Hz, 2H). ESI-MS m/z 535.4 (M+H)+.
Step 1: I-27-a was prepared with a yield of 48% following the same method as that in Example 1, except that I-1-a was replaced with I-26.
Step 2: I-27-a (55 mg, 0.1 mmol) was suspended in 1 mL of tetrahydrofuran, added with lithium hydroxide monohydrate (0.12 mmol) dissolved in 0.1 mL of water, added with 0.1 mL of methanol, and refluxed for 3 hours. The solvent was rotary evaporated off, and water was added. The reaction mixture was adjusted pH with 1 M hydrochloric acid, and carefully added with 1.2 mL of 1 M hydrochloric acid to precipitate a large amount of solid, which was filtered to obtain I-27 with a yield of 48%. ESI-MS m/z 557.2 (M+Na)+.
I-28 was prepared with a yield of 65% following the same method as that of Example 26, except that methanol was replaced with isopropanol in Step 1. ESI-MS m/z 563.3 (M+H)+.
I-29 was prepared with a yield of 58% following the same method as that in Step 4 of Example 10, except that I-10-c was replaced with I-29-a. ESI-MS m/z 534.5 (M+H)+.
I-30 was prepared with a yield of 38% following the same method as that in Step 4 of Example 10, except that I-10-c was replaced with I-30-a and I-10-e was replaced with I-30-b. ESI-MS m/z 549.2 (M+H)+.
I-31 was prepared with a yield of 42% following the same method as that in Step 4 of Example 10, except that I-10-c was replaced with I-31-a. ESI-MS m/z 536.2 (M+H)+.
I-32 was prepared with a yield of 25% following the same method as that in Example 1, except that I-1-a was replaced with I-32-a. ESI-MS m/z 460.2 (M+H)+.
I-33 was prepared with a yield of 35% following the same method as that in Example 1, except that I-1-a was replaced with I-33-a. ESI-MS m/z 326.2 (M+H)+.
Step1: I-34-b was prepared following the same method as that in Step 4 of Example 10, except that I-10-c was replaced with I-34-a.
Step2: I-34 was prepared with a yield of 35% following the same method as that in Example 1, except that I-1-a was replaced with I-34-a. ESI-MS m/z 519.3 (M+H)+.
I-35 was prepared with a yield of 33% following the same method as that in Example 1, except that I-1-a was replaced with I-35-a. ESI-MS m/z 464.2 (M+H)+.
I-36 was prepared with a yield of 38% following the same method as that in Example 1, except that I-o-a was replaced with I-36-a. ES-MS m/z 381.1(M+H)+.
I-37 was prepared with a yield of 39% following the same method as that in Example 1, except that iodomethane was replaced with iodoethane. 1H NMR (400 MHz, DMSO-d6) δ 8.35 (t, J=5.8 Hz, 1H), 7.84-7.74 (m, 2H), 7.53-7.45 (m, 2H), 7.27 (d, J=7.7 Hz, 1H), 4.15 (s, 2H), 3.60 (q, J=7.0 Hz, 2H), 3.57-3.46 (m, 2H), 3.36 (dt, J=7.8, 3.9 Hz, OH), 2.91 (t, J=7.1 Hz, 2H), 2.18 (q, J=7.5 Hz, 2H), 2.01 (s, 3H), 1.73 (dd, J=13.0, 3.8 Hz, 2H), 1.67-1.59 (m, 2H), 1.25 (qd, J=12.7, 3.4 Hz, 3H), 1.05 (t, J=7.0 Hz, 3H), 0.97 (t, J=7.5 Hz, 3H), 0.92 (dd, J=12.0, 3.1 Hz, 1H), 0.84 (d, J=6.5 Hz, 3H). ESI-MS m/z 518.8 (M+H)+.
I-38 was prepared with a yield of 21.3% following the same method as that in Example 1, except that iodomethane was replaced with 2-iodopropane. ESI-MS m/z 533.5 (M+H)+.
I-39 was prepared with a yield of 42% following the same method as that in Example 23, except that acetic anhydride was replaced with propionic anhydride. ESI-MS m/z 563.2 (M+H)+.
I-40 was prepared with a yield of 38% following the same method as that in Example 1, except that I-1-a was replaced with I-39. ESI-MS m/z 599.3 (M+Na)+.
I-41 was prepared with a yield of 35% following the same method as that in Example 23, except that acetic anhydride was replaced with isobutyric anhydride. ESI-MS m/z 577.2 (M+H)+.
I-42 was prepared with a yield of 41% following the same method as that in Example 1, except that I-1-a was replaced with I-41. ESI-MS m/z 591.3 (M+H)+.
I-43 was prepared with a yield of 37% following the same method as that in Example 23, except that acetic anhydride was replaced with pivalic anhydride. ESI-MS m/z 591.3 (M+H)+.
I-44 was prepared with a yield of 45% following the same method as that in Example 1, except that I-1-a was replaced with I-43. ESI-MS m/z 605.3 (M+H)+.
sEH Assay Buffer was purchased from Cayman Chemical; sEH (human recombinant) was purchased from Cayman Chemical; sEH substrate was purchased from Cayman Chemical. The working solution of 20% DMSO for the compounds was prepared by mixing dimethyl sulfoxide with water in a volume ratio of 1:4, and shaking to mix well.
sEH Assay Buffer (10×), SEH (human recombinant) and sEH substrate were thawed on ice.
The sEH Assay Buffer (lux) was prepared into a 1×sEH Assay Buffer with deionized water. 12 μL of sEH was added with 588 L of 1× Assay Buffer to be diluted 50 times to obtain a sEH working solution. 180 L of 1× Assay Buffer was added into the corresponding wells; the sEH working solution was added to the corresponding wells at 5 μL/well; the working solution of 20% DMSO for the compounds was added into the corresponding wells at 5 μL/well, and blank wells and negative control wells added with an equal amount of DMSO were set. After centrifuged at 1000 rpm for 1 minute, the wells were incubated at 25° C. for 30 minutes. After incubation, the substrate was added into the 96-wells plate at 5 μL/well and the plate was centrifuged at 1000 rpm for 1 minute. After incubation at 25° C. for 15 minutes, fluorescence values were read at emission wavelengths of 320 nm and 460 nm.
The experimental results were shown in Table 1, wherein A indicates an IC50 less than 200 nM, B indicates an IC50 between 200 nM and 1 μM, C indicates an IC50 between 1 μM and 10 μM, and D indicates an IC50 between 10 μM to 100 μM.
The compounds I-1, I-4 and I-23 of the present invention and glimepiride were used for the assay, and 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA) was used as the positive control.
Biomaterial sources: Human cardiomyocyte cell line AC16 was purchased from ATCC, USA. Phenylephrine (PE) was purchased from Sigma Company. AUDA was purchased from Cayman Company.
The compounds were dissolved in DMSO. The working concentrations were 0.1, 1 and 10 μM for the compounds of the present invention, 0.1, 1 and 10 μM for glimepiride, and 1 μM for AUDA. The incubation time was 24 hours.
The ELISA detection kit (BIOTARGET 14,15-EET/DHET ELISA KIT) from Detroit R&D company was used, and the experiment was conducted according to the instructions of the kit.
After 24 hours of incubation, cardiomyocytes were collected to perform the above detection.
The experimental results are shown in
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PE was dissolved in disinfected deionized water with a working concentration of 100 μM. The compounds were dissolved in DMSO, and the working concentrations were 0.1, 0.5, 1 μM for the compounds of the present invention, 10 μM for glimepiride, and 1 μM for AUDA. The incubation time was 24 hours.
Ventricular Remodeling Biomarker mRNA Detection:
Total RNA was extracted using Invitrogen's Trizol reagent, then cDNA was obtained using TAKARA's reverse transcription kit, and finally the mRNA content of related genes was amplified with specific primers.
After 24 hours of incubation, cardiomyocytes were collected to perform the above detection.
The results are shown in
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12-week-old male C57BL/6 mice, which were purchased from Gempharmatech Co., Ltd, were raised in the SPF animal house of Tongji Medical College, Huazhong University of Science and Technology. After one week of acclimatization in the animal house, the mice were subjected to thoracic aortic constriction as the TAC group. Another group of mice were subjected to thoracotomy without ligation of thoracic aorta as Sham group. Meanwhile, AUDA was used as the positive control.
Cardiac ultrasound detection was performed on a Visual Sonics Vevo 2100 small animal ultrasound imager. The major measurement indicators include heart rate (HR), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal dimension-diastole (LVIDd) and left ventricular internal dimension-systole (LVIDs), and left ventricular posterior wall thickness (LVPWd and LVPWs).
The detection was performed on a Millar pressure-volume system from Millar Instr Ment PowerLab. After the animals were anesthetized to an appropriate extent, a midline incision was made in the neck, the right common carotid artery was separated, ligated at the distal end and clipped at the proximal end. A V-shaped incision was made on the artery with microscissors, and a microcatheter was inserted to the left ventricle. Signals were recorded through a conduction system to obtain hemodynamic data such as heart rate (HR), left ventricular end diastolic pressure (PED), left ventricular end systolic pressure (PES), maximum rate of decrease in left ventricular pressure (−dp/dtmin), and maximum rate of increase in left ventricular pressure (+dP/dtmax).
The myocardial tissue was placed in an embedding frame, soaked and fixed in neutral formaldehyde solution, dehydrated, and embedded in paraffin. The paraffin block was then sliced into sections with a thickness of 4 μm on a microtome. The general appearance of the myocardium was observed through HE staining, and the myocardial fibrosis was observed through Sirius red staining.
Ventricular Remodeling Biomarker mRNA Detection:
Total RNA was extracted using Invitrogen's Trizol reagent, then cDNA was obtained using TAKARA's reverse transcription kit, and finally the mRNA content of related genes was amplified with specific primers.
Cardiac ultrasound examination was performed two weeks after TAC surgery. 24 TAC mice with no significant difference in cardiac function were selected and randomly divided into a model group (TAC) and a treatment group (TAC+compound). The model group received no other treatment, while the treatment group received daily gavage (0.2 mg/kg and 0.4 mg/kg for I-1 and I-23 of the present application, and 1.2 mg/kg for glimepiride). Eight weeks later, i.e., at the chronic heart failure phase, the animals were sacrificed, and tissue specimens were collected to perform the above detections.
The results are shown in
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Cardiac ultrasound examination was performed two weeks after TAC surgery. 24 TAC mice with no significant difference in cardiac function were selected and randomly divided into a model group (TAC) and a treatment group (TAC+compound). The model group received no other treatment, while the treatment group received daily gavage (0.44 mg/kg for I-23 or 10 mg/kg for Empagliflozin). Eight weeks later, i.e., at the chronic heart failure phase, the animals were sacrificed, and tissue specimens were collected to perform the above detections.
This experiment was to compare the ameliorative effects of I-23 and Empagliflozin on chronic heart failure.
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The administration concentration of AUDA that was not specifically specified in the listed experimental examples was 1 μM.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110935522.0 | Aug 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/112383 | 8/15/2022 | WO |
