CN118619893A - PGI2 receptor agonist compounds, pharmaceutical compositions and applications - Google Patents

Abstract

The present invention belongs to the technical field of chemical medicines, and provides a PGI2 receptor agonist compound shown in general formula I or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof. In the formula, _R1 and _R2 are independently selected from H, _C1 - _C3 alkyl or halogen atom; Z is selected from _CR7 or N atom, wherein _R7 is selected from H or _C1 - _C3 alkyl, halogen atom or halogenated _C1 - _C3 alkyl; _R3 is selected from _C1 - _C3 alkyl, C3 _{- C6} monocyclic cycloalkyl; _R4 is selected from H, _C1 - _C3 alkoxy or _3-6 heteroalicyclic group; _R5 represents OH, _OR6 , or _NHSO2R6 , wherein _R6 represents _C1-4 alkyl or _C1-4 alkyl substituted by halogen; It represents a single bond or a double bond. The compound has good affinity for PGI2 receptors and very low adverse reactions, avoiding the shortcomings of current PGI2 analogs and selexipag.

Description

PGI2 receptor agonist compounds, pharmaceutical compositions and applications

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of Chinese patent application CN202311824286.0, with an application date of December 27, 2023, and this application cites the full text of the above-mentioned Chinese patent application.

Technical Field

The present invention belongs to the technical field of chemical medicines, and in particular relates to a PGI2 receptor agonist compound, a pharmaceutical composition and an application thereof.

Background Art

Pulmonary arterial hypertension (PAH) is a disease characterized by vasospasm, intimal hyperplasia and remodeling of pulmonary arterioles. Vascular hyperplasia and remodeling of pulmonary arterioles lead to a progressive increase in pulmonary vascular resistance, eventually causing right heart failure and death. PAH has been ranked the third most common cardiovascular disease, second only to hypertension and coronary heart disease in prevalence. It has become a public health care issue that seriously threatens human physical and mental health, and has been included in the world's major chronic disease detection range by the World Health Organization.

PGI2 is a substance produced in vivo from arachidonic acid via prostaglandin H2 (PGH2). PGI2 deficiency can cause pulmonary hypertension. Currently, the PGI2 receptor agonists that have been marketed include epoprostenol, beraprost, iloprost, etc., all of which are PGI2 analogs. However, since the biological half-life of PGI2 is very short and its selectivity for the target is poor, it is difficult to separate the intended effect from other effects, so adverse reactions are prone to occur. Selexipag is currently the only PGI2 agonist that does not have a PGI2 skeleton but has good selectivity for PGI2 receptors and definite efficacy. It has been approved for marketing in many countries for the treatment of adult pulmonary hypertension. Its specific therapeutic effect is stronger and longer-acting than other drugs with similar mechanisms, but its high price undoubtedly adds a huge economic burden to patients with pulmonary hypertension who need long-term treatment. In addition, although selexipag has relatively good specificity, it still has obvious adverse reactions, such as headache, facial flushing, nausea, vomiting, etc. For patients who need long-term medication, the cumulative damage caused by the drug to the body will reduce their health level and quality of life to varying degrees.

Summary of the invention

In view of the above problems, the present invention provides a PGI2 receptor agonist compound, a pharmaceutical composition and an application. The compound has good affinity for the PGI2 receptor and very low adverse reactions, thus avoiding the shortcomings of the current PGI2 analogs and selexipag.

In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a PGI2 receptor agonist compound represented by general formula I or a pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof,

In the formula, R ₁ and R ₂ are independently selected from H, C ₁ -C ₃ alkyl or halogen atom;

Z is selected from CR ₇ or N atom, wherein R ₇ is selected from H or C ₁ -C ₃ alkyl, halogen atom or halogenated C ₁ -C ₃ alkyl;

R ₃ is selected from C ₂ -C ₄ alkyl, C _{3 -} C ₆ monocyclic cycloalkyl;

R ₄ is selected from H, C ₁ -C ₃ alkoxy or 3-6 membered heteroalicyclic group;

R ₅ represents OH, OR ₆ or NHSO ₂ R ₆ , wherein R ₆ represents C _1-4 alkyl or C _1-4 alkyl substituted by halogen; the carboxyl group forms an acid, ester or sulfonamide with R 5;

Indicates a single bond or a double bond.

One embodiment of the present invention relates to the above-mentioned compound of general formula I or its pharmaceutically acceptable salts, stable isotope derivatives, isomers and mixtures thereof, wherein Z is a N atom; or Z is a C atom and R ₇ is selected from H or CH ₃ .

In one embodiment, Represents a double bond, _R4 is H, and accordingly, its structural formula is shown in Formula II:

In formula II, _R1 and _R2 are selected from H, methyl or F; Z is a N atom, or Z is _CR7 , _R7 is selected from H or _CH3 , more preferably a H atom; _R3 is selected from isopropyl, ethyl or _cyclopropane , more preferably isopropyl; _R5 is selected from OH or _NHSO2R6 , _R6 is selected from _C1-4 alkyl or _C1-4 alkyl substituted by halogen, more preferably _methyl .

One embodiment of the present invention relates to the above-mentioned compound of general formula I or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, wherein Represents a single bond, and its structural formula is shown in Formula III:

In formula III, R ₄ is selected from H, C ₁ -C ₃ alkoxy or 3-6 membered heteroalicyclic group, preferably R ₄ is H, methoxy, ethoxy or More preferably, _R4 is H, methoxy or ethoxy.

One embodiment of the present invention relates to the above-mentioned compound of formula I or III or its pharmaceutically acceptable salts, stable isotope derivatives, isomers and mixtures thereof, wherein _R1 and _R2 are selected from H, methane or F; _R3 is selected from _isopropyl , ethyl or cyclopropane, preferably _R3 is isopropyl; _R5 is preferably OH or _NHSO2CH3 .

One embodiment of the present invention relates to a compound represented by the above general formula (I), wherein the compound is selected from but not limited to:

Or the above structural formula prodrug, stable isotope derivative, pharmaceutically acceptable salt, isomer and mixture thereof.

The compound of the present invention does not have a PGI2 skeleton, has strong selectivity and affinity for PGI2 receptors, and has good target selectivity compared with PGI2 analogs. Experimental verification shows that the compound has better in vivo efficacy than selexipag, and its toxicity is less than selexipag. Therefore, the compound can be used as a pulmonary arterial hypertension therapeutic drug with greater clinical value and development prospects.

The second aspect of the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and the above-mentioned compound or its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer and a mixture thereof as an active ingredient.

The above-mentioned pharmaceutical composition can be in any dosage form, optionally, conventional dosage forms such as tablets, granules, capsules, powders or injections.

Another aspect of the present invention relates to the use of the above-mentioned compound or its pharmaceutically acceptable salts, stable isotope derivatives, isomers and mixtures thereof, and the above-mentioned pharmaceutical composition in the preparation of drugs for treating pulmonary arterial hypertension.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the effects of intragastric administration of the compound in the example and Selexipag on pulmonary artery systolic pressure (*** indicates P≤0.001 compared with the model control group, ** indicates P≤0.01 compared with the model control group).

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

the term

In the compound preparation method of the present application, "eq" means equivalent, which means equivalent in chemistry. This concept is very critical in stoichiometry and chemical reactions, especially when it comes to the molar ratio of substances. For example, if 0.3 mol (1 eq) of A is used in a reaction, and the amount of B used is 6 times that of A, that is, 6 eq., then the amount of B used is 1.8 mol. This method of expression helps to make it more convenient and accurate when calculating the molar ratio of substances in chemical reactions. The "eq" of this application is calculated in molar ratio.

The notation "C _xy " used herein indicates a range of carbon number, wherein x and y are both integers, for example, C _3-6 cycloalkyl indicates a cycloalkyl group having 3 to 6 carbon atoms.

"Alkyl" means a saturated straight or branched hydrocarbon group having a certain number of carbon atoms, such as 1-6 carbon atoms or 1-4 carbon atoms. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl and 2-ethylbutyl, etc.

The heteroalicyclic group represents a monocyclic or condensed ring containing one or more heteroatoms of N, O or S. It is typically a 3-8-membered heterocyclic group containing one or more heteroatoms of N, O or S, preferably a 3-6-membered heterocyclic group containing one or more heteroatoms of N, O or S, such as propylene oxide, butylene oxide, piperazinyl, morpholino, piperidino and derivatives thereof.

For example, a morpholino group refers to a group having the following chemical structure: Piperidino refers to a group with the chemical structure:

"Halogen" refers to fluorine, chlorine, bromine or iodine.

As defined herein, "isomers" refer to compounds having the same molecular formula but differing in the nature or order of their atomic bonding or in the spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers". Stereoisomers include optical isomers, geometric isomers, and conformational isomers.

The compounds of the present invention may exist as optical isomers. Depending on the configuration of the substituents around the chiral carbon atom, these optical isomers are in the "R" or "S" configuration. Optical isomers include enantiomers and diastereomers. Methods for preparing and separating optical isomers are known in the art.

The compounds of the present invention may also exist as geometric isomers. The present invention contemplates various geometric isomers and mixtures thereof generated by the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyl or heterocyclic groups. Substituents around carbon-carbon double bonds or carbon-nitrogen bonds are designated as Z or E configurations, and substituents around cycloalkyl or heterocyclic rings are designated as cis or trans configurations.

"Isotope" includes all isotopes of atoms that occur in the compounds of the present invention. Isotopes include those atoms with the same atomic number but different mass numbers. Examples of isotopes suitable for incorporation into the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as but not limited to ^2H , ^3H , ^13C , ^14C , ^15N , ^18O , ^17O , ^35S , ^18F and ^36Cl , respectively. Isotope-labeled compounds of the present invention can be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the attached examples using suitable isotope-labeled reagents instead of non-isotope-labeled reagents. Such compounds have various potential uses, such as as standards and reagents in determining biological activity. In the case of stable isotopes, such compounds have the potential to advantageously change biological, pharmacological or pharmacokinetic properties.

"Prodrug" means that the compounds of the present invention can be administered in the form of prodrugs. Prodrugs refer to derivatives that are converted into the biologically active compounds of the present invention under physiological conditions in vivo, such as by oxidation, reduction, hydrolysis, etc. (each of which is carried out using an enzyme or without the participation of an enzyme). Examples of prodrugs are compounds in which the amino group in the compounds of the present invention is acylated, alkylated or phosphorylated, such as methylacylamino, alanylamino, pivaloyloxymethylamino, or in which the hydroxyl group is acylated, alkylated, phosphorylated or converted into a borate, such as acetoxy, fumaryloxy, alanyloxy, or in which the carboxyl group is esterified or amidated. These compounds can be prepared by the compounds of the present invention according to known methods.

"Pharmaceutically acceptable salts" or "pharmaceutically acceptable salts" refer to salts made from pharmaceutically acceptable bases or acids, including inorganic bases or acids and organic bases or acids. In the case where the compounds of the present invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically acceptable salts. Therefore, the compounds of the present invention containing acidic groups can exist in salt form and can be used according to the present invention, for example as alkali metal salts, alkaline earth metal salts or as ammonium salts, exemplified by sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines, such as ethylamine, ethanolamine, triethanolamine or amino acids. The compounds of the present invention containing basic groups can exist in salt form and can be used according to the present invention in the form of addition salts thereof with inorganic or organic acids. The example of suitable acid comprises hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acid well known to persons skilled in the art. If the compound of the present invention contains acidic and basic groups in the molecule simultaneously, the present invention also comprises inner salt or betaine except the salt form mentioned. Each salt can be obtained by conventional methods well known to persons skilled in the art, for example by making these contact with organic or inorganic acid or base in solvent or dispersant or by with other salt anion exchange or cation exchange.

"Pharmaceutical composition" refers to a composition containing one or more compounds described herein or their pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers and mixtures thereof, as well as other components such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

Therefore, when referring to "a compound", "a compound of the present invention" or "the compound described in the present invention" in this application, all forms of the compound, such as pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers and mixtures thereof, are included.

The present invention also provides a method for preparing the compound. The preparation of the compound described in the general formula (I) of the present invention can be completed by the following exemplary methods and examples, but these methods and examples should not be considered in any way to limit the scope of the present invention. The compound described in the present invention can also be synthesized by synthetic techniques known to those skilled in the art, or a combination of methods known in the art and the method described in the present invention. The product obtained by each step of the reaction is obtained by separation techniques known in the art, including but not limited to extraction, filtration, distillation, crystallization, chromatographic separation, etc. The starting materials and chemical reagents required for the synthesis can be synthesized or purchased according to the literature.

Reference Example 1

Preparation of 6-isopropylamino-3-ethoxy-hexanoic acid ethyl ester (denoted as SM1), the preparation route is as follows:

Preparation of SM1-B: Add SM1-A (4-isopropylamino-1-n-butanol, 200 g, 1 eq), Boc anhydride (349.3 g, 1.05 eq), and tetrahydrofuran (1200 ml) into the reaction bottle in sequence, react at room temperature for 2 to 2.5 hours, and concentrate to obtain 353 g of compound SM1-B.

Preparation of SM1-C: Compound SM1-B (300 g, 1 eq) and dichloromethane (1500 ml) were added to the reaction flask in sequence, cooled in an ice bath, and Dess-Martin reagent (660 g, 1.2 eq) was added in batches. The reaction was kept warm for 2 to 3 hours. The reaction solution was washed with saturated sodium bicarbonate solution, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue was purified by silica gel column to obtain 172 g of compound SM1-C.

SM1-D: Add triethyl phosphoacetate (199 g, 1.2 eq) and tetrahydrofuran (1300 ml) to the reaction bottle in sequence, cool in an ice bath, add sodium hydride (35.6 g, 1.2 eq) in batches, stir for 1-2 h, add compound SM1-C (170 g, 1 eq) in batches at 0-5°C, warm to room temperature and react for 2-3 h, add water dropwise to the reaction solution to quench, add ethyl acetate to extract, wash the organic phase with saturated sodium chloride aqueous solution and then dry over anhydrous sodium sulfate, concentrate to dryness, and purify the residue by silica gel column to obtain 135 g of compound SM1-D.

SM1-E: Compound SM1-D (30 g, 1 eq) and anhydrous ethanol (150 ml) were added to the reaction bottle in sequence, cooled in an ice bath, sodium hydride (6 g, 1.5 eq) was added, the temperature was raised to room temperature for reaction for 2-3 h, the temperature was lowered to 0-10°C, quenched with 100 ml of water, concentrated to remove most of the ethanol, extracted with ethyl acetate, dried the organic phase over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 26.3 g of compound SM1-E.

Preparation of SM1: SM1-E (25 g, 1 eq) and dichloromethane were added to a reaction flask, and hydrochloric acid/ethanol solution (1.1 eq) was added dropwise at room temperature. After the addition was completed, the mixture was concentrated to dryness to obtain 19.4 g of SM1.

Example 1

Preparation of 6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}-2-hexenoic acid, the compound is denoted as SYN-001, corresponding to the structural formula (1).

The preparation route is as follows:

STEP 1: Add compound 1A (4,4'-dimethylbenzil 300g, 1eq), 2-aminoacetamide hydrochloride (209g, 1.5eq), and methanol (2.0L) to the reaction bottle in sequence, raise the temperature to reflux, add sodium hydroxide aqueous solution (sodium hydroxide 101g, 2eq, purified water 200ml) dropwise, keep warm and stir for 4-5h, cool to room temperature, adjust the pH to 6-7 with hydrochloric acid, filter, rinse the filter cake with 300ml of methanol, and vacuum dry to obtain 320g of compound 1B.

STEP2: Add compound 1B (100g, 1eq) and phosphorus oxychloride (277.4g, 5eq) to the reaction flask in sequence, heat to 100-105°C, keep stirring for 3-5h, concentrate under reduced pressure to remove residual phosphorus oxychloride, add 200mL toluene, continue to concentrate to remove residual phosphorus oxychloride. Add 100ml of dichloromethane to the reactant and stir to dissolve, add the reaction solution dropwise to 1000ml of isopropanol, stir at 0-5°C for 1-3h, filter, and vacuum dry to obtain 90.7g of compound 1C.

STEP 3: Add compound 1C (100 g, 1 eq) and 4-(isopropylamino)butanol (245 g, 5.5 eq) to the reaction bottle in sequence, raise the temperature to 160-170°C, keep the reaction at this temperature for 20-30 h, lower the temperature to 60-80°C, pour the reaction solution into water (1.0 L), add ethyl acetate for extraction, wash the organic phase with saturated aqueous ammonium chloride solution, dry over anhydrous sodium sulfate, concentrate to dryness, and purify the residue by silica gel column to obtain 74 g of compound 1D.

STEP 4: Compound 1D (70 g, 1 eq) and dichloromethane (700 ml) were added to the reaction flask in sequence, cooled in an ice bath, and Dess-Martin reagent (152 g, 2 eq) was added in batches. The reaction mixture was kept warm for 3 to 5 hours. The reaction solution was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue was purified by silica gel column to obtain 21.5 g of compound 1E.

STEP 5: Add triethyl phosphoacetate (9 g, 1.2 eq) and tetrahydrofuran (130 ml) to the reaction bottle in sequence, cool in an ice bath, add sodium hydride (1.34 g, 1 eq) in batches, stir for 1 h, add compound 1E (13 g, 1 eq) in batches at 0-5°C, warm to room temperature and react for 2-3 h, add 50 ml of water dropwise to the reaction solution to quench, add ethyl acetate to extract, wash the organic phase with saturated sodium chloride aqueous solution, dry over anhydrous sodium sulfate, and concentrate to dryness. The residue is purified by silica gel column to obtain 13 g of compound 1F.

STEP 6: Compound 1F (3 g, 1 eq), tetrahydrofuran (30 ml), 3 ml of purified water were added to the reaction bottle in sequence, and sodium hydroxide (1.05 g, 4 eq) was added. The temperature was raised to reflux, and the reaction was carried out for 12 to 15 hours. The mixture was cooled to room temperature, and 50 ml of water was added. The pH was adjusted to 5 to 6 with hydrochloric acid, and ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 1.3 g of compound SYN-001.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.019 (s, 1H), 7.366～7.350 (m, 2H), 7.282 (s, 2H), 7.158～7.057 (m, 5H), 5.925～5.894 (d, 1H), 4.714～4.688(m,1H), 3.460～3.429(m,2H), 2.371～2.332(m,8H), 1.918～1.857(m, 2H), 1.291～1.277(m, 6H)ppm.

Example 2

Preparation of 6-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]-2-hexenoic acid, the compound is denoted as SYN-002, corresponding to the structural formula (2).

The preparation route is as follows:

The preparation method of 6-[N-(5,6-diphenylpyrazine-2-yl)-N-isopropylamino]-2-hexenoic acid is the same as the preparation method of 6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}-2-hexenoic acid (No. SYN-001) in Example 1, except that the starting material is benzil.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.050 (s, 1H), 7.449～7.430 (m, 2H), 7.366～7.347 (m, 2H), 7.285～7.247 (m, 6H), 7.232～7.211 ( m, 1H), 5.920 ~ 5.889 (m, 1H), 4.741 ~ 4.715 (m, 1H), 3.454 ~ 3.422 (m, 2H), 2.354 ~ 2.340 (m, 2H), 1.898 ~ 1.867 (m, 2H), 1.285～1.272(m,6H)ppm.

Example 3

Prepare 6-{N-[5,6-di(4-methylphenyl)-1,2,4-triazine]-2-yl-N-isopropylamino}hexanoic acid, the compound is denoted as SYN-003, corresponding to the structural formula (3).

The preparation route is as follows:

STEP1: Add glacial acetic acid (2.5L), 4,4'-dimethylbenzil (compound 6A; 500g, 1eq), semicarbazide hydrochloride (351g, 1.5eq), purified water (1L) to the reaction bottle in sequence, stir evenly, and heat to 100-110°C. Keep the reaction warm for 2-3h, cool to 30-40°C, add purified water, stir at room temperature for 0.5-1.5h, filter, add the filter cake to 3L ethyl acetate, heat to reflux and stir for 2-3h, cool to room temperature, filter, and vacuum dry to obtain 523g of intermediate 6B.

STEP2: Add phosphorus oxychloride (2L) and 6B (500g, 1eq) to the reaction flask in sequence, heat to 80-85°C, and stir to dissolve. Keep warm for 1-1.5h, concentrate to remove phosphorus oxychloride, add 1L toluene, and continue to concentrate to remove residual phosphorus oxychloride. Add tertiary methyl ether to the residue, stir and disperse for 1h, filter, and vacuum dry to obtain 320g of intermediate 6C.

STEP 3: Add intermediate 6C (100 g, 1 eq) and 4-(isopropylamino)butanol (155.3 g, 3.5 eq) to the reaction flask in sequence, raise the temperature to 140-160°C, keep the reaction for 14-20 h, cool to room temperature, pour the reaction solution into water, add ethyl acetate for extraction, wash the organic phase with saturated sodium chloride aqueous solution, dry over anhydrous sodium sulfate, and concentrate to dryness. The residue is purified by silica gel column to obtain 71 g of intermediate 6D.

STEP4: Add intermediate 6D (70 g, 1 eq) and dichloromethane (700 ml) to the reaction flask, cool to 0-10°C, add Dess-Martin reagent (152 g, 2 eq) in batches, and keep at 0-10°C for 3-5 hours. Stop the reaction, wash the reaction solution with saturated sodium bicarbonate solution twice, then with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, concentrate to dryness, and purify the residue with silica gel column to obtain 41.3 g of intermediate 6E.

STEP5: Add triethyl phosphoacetate (27.7 g, 1.2 eq) and tetrahydrofuran (400 ml) to the reaction bottle, cool to 0-10°C, add sodium hydride (4.1 g, 1 eq), stir for 1-2 h, add intermediate 6E (40 g, 1 eq) at 0-5°C, warm to room temperature and react for 2-3 h, add 100 ml of water to the reaction solution to quench the reaction, concentrate to remove most (more than 80%) of tetrahydrofuran, add 200 ml of ethyl acetate to the residue for extraction, wash the organic phase with saturated sodium chloride aqueous solution, dry over anhydrous sodium sulfate, and concentrate to dryness. The residue is purified by silica gel column to obtain 21.3 g of intermediate 6F.

STEP 6: Add 21 g of intermediate 6F, 2.1 g of 10% palladium carbon, and anhydrous ethanol (210 ml) into the reaction bottle, replace with hydrogen, react at room temperature for 5 to 7 hours, filter out the palladium carbon, and concentrate the filtrate to dryness to obtain 20.2 g of intermediate 6G.

STEP 7: Add intermediate 6G (20 g, 1 eq), tetrahydrofuran (200 ml), purified water (20 ml), and sodium hydroxide (6.9 g, 4 eq) to the reaction flask in sequence, raise the temperature to reflux, react for 2 to 3 hours, concentrate to remove tetrahydrofuran, add purified water (100 ml) and ethyl acetate (100 ml), stir evenly, stand to separate the liquids, retain the aqueous phase, add hydrochloric acid to the aqueous phase to adjust the pH to 3 to 5, add methyl tert-butyl ether for extraction, dry the organic phase over anhydrous sodium sulfate, and then concentrate the organic phase to dryness under reduced pressure to obtain 16.8 g of compound SYN-003.

1H NMR (500MHz, CDCl3): δ: 7.455～7.439(m,2H), 7.396～7.380(m,2H), 7.146～7.129(m,4H), 5.137(m, 1H), 3.599(m, 2H) , 2.462～2.378(m,8H), 1.795～1.735(m,4H), 1.527～1.454(m,2H), 1.365～1.238(m,6H)ppm.

Example 4

Preparation of 3-ethoxy-6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}hexanoic acid, the compound is denoted as SYN-004, and the corresponding structural formula is numbered as (4).

Compound 1F, named 6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}-2-hexenoic acid ethyl ester, (the preparation method of this compound is the same as that of "Compound 1F" in Example 1) (3 g, 1 eq), anhydrous ethanol (30 ml), cooled in an ice bath, sodium hydride (0.79 g, 3 eq) was added to the reaction bottle in sequence, and the reaction was refluxed for 12 to 15 hours, cooled to room temperature, and the pH was adjusted to 5 to 6 with hydrochloric acid. 30 ml of water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 1.2 g of compound SYN-004. ¹ H NMR (500MHz, CDCl ₃ ): δ: 8.013 (s, 1H), 7.378～7.366 (m, 2H), 7.350 (s, 1H), 7.090～7.065 (m, 5H), 4.803～4.776 (m, 1H), 3.814～3.792 (m, 1H), 3.594～3. 536(m,2H),3.449～3.398(m,2H),2.628～2.461(m,2H), 2.348～2.337(m,6H), 1.802～1.642(m,4H), 1.281～1.268(m,6H), 1.212～1.185(t,3H)ppm.

Example 5

Prepare 3-ethoxy-6-{N-[5,6-di(4-methylphenyl)-1,2,4-triazine]-2-yl-N-isopropylamino}hexanoic acid, the compound is denoted as SYN-005, corresponding to the structural formula (5).

The preparation route is as follows:

STEP 1: Add compound 6C (30 g, 1 eq, the preparation method is the same as compound 6C in Example 3, which is named 5,6-di(4-methylphenyl)-3-chloro-1,2,4-triazine), SM1 (37.2 g, 1.3 eq, the preparation method is shown in the reference example), potassium carbonate (42 g, 3 eq), and DMF (1.5 L) to the reaction bottle in sequence, raise the temperature to 80-85°C, keep the reaction warm for 2-3 h, add water, extract with ethyl acetate, and concentrate to obtain 43.9 g of compound 7D.

STEP 2: Compound 7D (10 g, 1 eq), tetrahydrofuran (100 ml), and 10 ml of purified water were added to the reaction bottle in sequence, and sodium hydroxide (2.4 g, 3 eq) was added. The temperature was raised to reflux, and the reaction was carried out for 12 to 15 h. The temperature was lowered to room temperature, and the pH was adjusted to 5 to 6 with hydrochloric acid. 100 ml of water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 4.8 g of compound SYN-005.

¹ H NMR (500MHz, CDCl ₃ ): δ: 7.451～7.425 (m, 2H), 7.381～7.365 (m, 2H), 7.133～7.117 (m, 4H), 3.835～3.811 (m, 1H), 3.605～ 3.654(m,4H), 2.601～2.573(m,2H), 2.384～2.365(m,7H), 1.699～1.688(m,4H), 1.328～1.315(m,6H), 1.210～1.182(t,3H) )ppm.

Example 6

Prepare 3-methoxy-6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}hexanoic acid, the compound is denoted as SYN-006, corresponding to the structural formula (6).

STEP 1: Add compound 1F (3 g, 1 eq, see compound 1F in Example 1 for preparation method) and anhydrous methanol (30 ml) into the reaction bottle in sequence, cool in an ice bath, add sodium hydride (0.79 g, 3 eq), warm to room temperature and react for 3 to 5 hours, add 50 ml of water to quench, add ethyl acetate to extract, dry the organic phase with anhydrous sodium sulfate, concentrate to dryness, and purify on a silica gel column to obtain 2.1 g of compound 4G.

STEP 2: Compound 4G (2 g, 1 eq), tetrahydrofuran (30 ml), and 3 ml of purified water were added to the reaction bottle in sequence, and sodium hydroxide (0.65 g, 4 eq) was added. The temperature was raised to reflux, and the reaction was carried out for 12 to 15 h. The mixture was cooled to room temperature, and the pH was adjusted to 5 to 6 with hydrochloric acid. 30 ml of water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 0.9 g of compound SYN-006.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.021 (s, 1H), 7.368～7.352 (m, 2H), 7.278～7.258 (m, 2H), 7.091～7.067 (m, 4H), 4.812 (m, 1H), 3.740～3.716(m,1H), 3.428～3.413(m,5H), 2.642～2.487(m,2H), 2.349～2.338(m,6H), 1.784～1.671(m, 4H), 1.293～ 1.269(m,6H)ppm.

Example 7

Prepare 6-[N-(5,6-diphenyl-1,2,4-triazine)-2-yl-N-isopropylamino]hexanoic acid, the compound is denoted as SYN-007, corresponding to the structural formula (7).

The preparation method of 6-[N-(5,6-diphenyl-1,2,4-triazine)-2-yl-N-isopropylamino]hexanoic acid is the same as the preparation method of 6-{N-[5,6-di(4-methylphenyl)-1,2,4-triazine]-2-yl-N-isopropylamino}hexanoic acid (No. SYN-003) in Example 3, except that the starting material is benzil.

¹ H NMR (500MHz, CDCl ₃ ): δ: 7.510～7.493 (m, 2H), 7.475～7.448 (m, 2H), 7.396～7.364 (m, 1H), 7.317～7.269 (m, 5H), 5.141～ 5.070 (m, 1H), 3.592 (m, 2H), 2.407 ~ 2.362 (m, 2H), 1.779 ~ 1.707 (m, 4H), 1.506 ~ 1.445 (m, 2H), 1.322 ~ 1.308 (m, 6H)ppm .

Example 8

Prepare 3-ethoxy-6-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]hexanoic acid, the compound is recorded as SYN-008, corresponding to the structural formula (8).

Compound 8F (3 g, 1 eq, compound 8F in preparation method example 2) and anhydrous ethanol (30 ml) were added to the reaction bottle in sequence, cooled in an ice bath, sodium hydride (0.84 g, 3 eq) was added, and the reaction was refluxed for 12 to 15 h, cooled to room temperature, and the pH was adjusted to 5 to 6 with hydrochloric acid. 100 ml of water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 1.2 g of compound SYN-008.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.037 (s, 1H), 7.443～7.226 (m, 10H), 4.785 (m, 1H), 3.790 (m, 1H), 3.545～3.414 (m, 4H) ,2.585～2.507(m,2H), 1.773～1.661(m,4H), 1.278～1.265(m,6H), 1.175(t,3H)ppm.

Embodiment 9

Preparation of 6-{N-[5,6-di(4-fluorophenyl)pyrazine]-2-yl-N-isopropylamino}-2-hexenoic acid, the compound is denoted as SYN-013, corresponding to the structural formula (9).

The preparation route is as follows:

The preparation method of 6-{N-[5,6-di(4-fluorophenyl)pyrazine]-2-yl-N-isopropylamino}-2-hexenoic acid is the same as the preparation method of 6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}-2-hexenoic acid (No. SYN-001) in Example 1, except that the starting material is 1,2-bis(4-fluorophenyl)ethane-1,2-dione.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.062 (s, 1H), 7.413～7.385 (m, 2H), 7.326～7.298 (m, 2H), 7.143～7.084 (m, 1H), 6.981～6.933 ( m, 4H), 5.919 ~ 5.888 (d, 1H), 4.709 ~ 4.683 (m, 1H), 3.455 ~ 3.424 (m, 2H), 2.361 ~ 2.319 (m, 2H), 1.886 ~ 1.840 (m, 2H), 1.297～1.274(m,6H)ppm.

Example 10

Prepare 6-{N-[5,6-di(4-fluorophenyl)pyrazine]-2-yl-N-isopropylamino}-hexanoic acid, the compound is denoted as SYN-010, corresponding to the structural formula (10).

STEP 1: Add compound 12F (6 g, 1 eq, for the preparation method, see Example 9 for compound 12F), 60 ml of anhydrous ethanol, and 0.6 g of 10% wet palladium carbon to the reaction bottle in sequence, replace H2, react at room temperature for 3 to 4 hours, filter out the palladium carbon, and concentrate the filtrate to obtain 5.8 g of compound 13G.

STEP 2: Compound 13G (5 g, 1 eq), tetrahydrofuran (50 ml), and 5 ml of purified water were added to the reaction bottle in sequence, and sodium hydroxide (1.3 g, 3 eq) was added. The temperature was raised to reflux, and the reaction was carried out for 12 to 15 h. The temperature was lowered to room temperature, and the pH was adjusted to 5 to 6 with hydrochloric acid. 100 ml of water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 2.3 g of compound SYN-010.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.125 (s, 1H), 7.423～7.388 (m, 2H), 7.333～7.298 (m, 2H), 7.194～7.100 (m, 4H), 4.768～4.735 ( m, 1H), 3.424 (m, 2H), 2.258 ~ 2.220 (m, 2H), 1.658 ~ 1.554 (m, 4H), 1.425 ~ 1.351 (m, 2H), 1.239 ~ 1.164 (m, 6H) ppm.

Embodiment 11

Preparation of 3-morpholinyl-6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}hexanoic acid, the compound is denoted as SYN-017, corresponding to the structural formula (11).

STEP 1: Add compound 1F (3 g, 1 eq, prepared by the same method as compound 1F in Example 1) and 6 ml of morpholine into a pressure bottle, heat to 100-105°C and react for 12-15 h, cool to room temperature, add aqueous ammonium chloride solution to quench, add ethyl acetate to extract, dry the organic phase over anhydrous sodium sulfate, concentrate to dryness, and purify on a silica gel column to obtain 2.1 g of compound 5G.

STEP 2: Compound 5G (2 g, 1 eq), tetrahydrofuran (30 ml), and 3 ml of purified water were added to the reaction flask in sequence, and sodium hydroxide (0.44 g, 3 eq) was added. The temperature was raised to reflux, and the reaction was carried out for 12 to 15 h. The mixture was cooled to room temperature, and the pH was adjusted to 5 to 6 with hydrochloric acid. 60 ml of water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 1.2 g of compound SYN-017.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.013 (s, 1H), 7.339～7.323 (m, 2H), 7.282 (m, 2H), 7.086～7.052 (m, 4H), 4.580～4.554 (m, 1H), 3.748(m,4H), 3.460～3.431(m,2H), 2.889～2.869(m,1H), 2.782～2.759(m,2H), 2.555～2.495(m,3H), 2.387～2.323( m,7H), 1.825～1.605(m,3H), 1.332～1.253(m,7H)ppm.

Example 12

Prepare 3-ethoxy-6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}-N-(p-methylsulfonyl)hexanamide, the compound is recorded as SYN-044, corresponding to the structural formula (12).

Compound SYN-004 (1 g, 1 eq), dichloromethane (20 ml), methanesulfonamide (300 mg, 1.5 eq), DMAP (308 mg, 1.2 eq), and EDCI (484 mg, 1.2 eq) were added to the reaction flask in sequence, and refluxed for 2 to 3 h. The mixture was cooled to room temperature, and the reaction solution was washed with purified water. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column to obtain 650 mg of compound SYN-044.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.013 (s, 1H), 7.292～7.272 (m, 2H), 7.208～7.188 (m, 2H), 7.129～7.066 (m, 4H), 4.769～4.737 ( m,1H), 3.771～3.741(m,1H), 3.493～3.459(m,2H), 3.183(s,3H), 2.434～2.290(m,10H), 1.694～1.525(m,4H), 1.246～ 1.229 (m, 6H), 1.092 ~ 1.057 (t, 3H) ppm.

Embodiment 13

Prepare 6-{N-[5,6-di(4-fluorophenyl)pyrazine]-2-yl-N-isopropylamino}-N-(p-methylsulfonyl)hexanamide, the compound is denoted as SYN-046, corresponding to the structural formula (13).

Compound SYN-010 (1.1 g, 1 eq), dichloromethane (20 ml), methanesulfonamide (357 mg, 1.5 eq), DMAP (367 mg, 1.2 eq), and EDCI (574 mg, 1.2 eq) were added to the reaction flask in sequence, and the mixture was refluxed for 2 to 3 h. The mixture was cooled to room temperature, and the reaction solution was washed with purified water. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 0.6 g of compound SYN-046.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.131 (s, 1H), 7.428～7.392 (m, 2H), 7.337～7.302 (m, 2H), 7.204～7.106 (m, 4H), 4.765 (m, 1H), 3.424 (m, 2H), 3.229 (s, 3H), 2.322 ~ 2.285 (m, 2H), 1.632 ~ 1.596 (m, 4H), 1.397 ~ 1.361 (m, 2H), 1.246 ~ 1.229 (m, 6H) ppm.

Embodiment 14

Preparation of {N-[5,6-di(4-methylphenyl)-1,2,4-triazine]-2-yl-N-isopropylamino}-2-hexenoic acid, the compound is denoted as SYN-018, corresponding to the structural formula (14).

Compound 6F (5 g, 1 eq, preparation method see Example 3), tetrahydrofuran (50 ml), 5 ml of purified water were added to the reaction flask in sequence, sodium hydroxide (0.87 g, 2 eq) was added, the temperature was raised to reflux, the reaction was carried out for 12 to 15 h, the temperature was lowered to room temperature, the pH was adjusted to 5 to 6 with hydrochloric acid, 100 ml of water was added, the mixture was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column to obtain 3.5 g of compound SYN-018.

¹ H NMR (500MHz, CDCl ₃ ): δ: 8.062 (s, 1H), 7.368～7.351

(m,2H),7.283～7.268(m,1H),7.143～7.050(m,5H),5.981～5.899(d,1H),4.719～4.690(m,1H),3.453～3.422(m,2H) ,2.371～2.330(m,8H), 1.916～1.855

(m, 2H), 1.293~1.278 (m, 6H)ppm.

Experimental example

1. Target Selectivity

Experimental procedure: HEK cells stably expressing prostacyclin receptors were used to study the functional activities of compounds numbered SYN-001, SYN-002, SYN-003, SYN-004, SYN-005, SYN-006, SYN-007, SYN-008, SYN-010, SYN-013, SYN-017, SYN-044, SYN-046 and SYN-018 against the following eight targets IP, EP1, EP2, EP3, EP4, DP, FP and TP using the HTRF cAMP and HTRF IP1 methods, and Selexipag (commercially available product Selexipag) was used as a comparison.

The experimental results are shown in Table 1.

Table 1 Target selectivity

The above results show that the compounds numbered SYN-001, SYN-002, SYN-003, SYN-004, SYN-005, SYN-006, SYN-007, SYN-008, SYN-010, SYN-013, SYN-017, SYN-044, SYN-046 and SYN-018 all have good agonist activity on IP receptors, and their effects are comparable to those of Selexipag. Except for SYN-001, SYN-002 and SYN-003, which have slight agonist activity on EP1, EP2, EP3, EP4, DP, FP and TP targets, the other compounds have no agonist activity on receptors other than IP receptors, and have high selectivity.

Among them, the chemical structural formula (Formula IV) of the active ingredient of Selexipag is shown below.

Its chemical name is: 2-{4-[(5,6-diphenylpyrazin-2-yl)(2-propyl)amino]butoxy}-N-(methylsulfonyl)acetamide.

ACT-333679 is a metabolite of selexipag, and its chemical structure is shown below.

Its chemical name is: 2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)acetic acid.

The structure of the compound in the embodiment of the present application is different from the butoxyacetamide structure in the branch chain of the active component of selexipag. The main structure of the branch chain in the embodiment of the present application is hexanoic acid or hexenoic acid, or hexanoic acid sulfonamide. Among them, SYN-001, SYN-002, SYN-003, SYN-004, SYN-005, SYN-006, SYN-007, SYN-008, SYN-010, SYN-013, SYN-017, SYN-044, SYN-046 and SYN-018 have good agonist activity on IP receptors. It has application prospects in the preparation of drugs for the treatment of pulmonary artery cancer.

2. Drug efficacy test in rats with pulmonary hypertension

Experimental process: The model was prepared by subcutaneous injection of monocrotaline 40 mg/kg, and the normal control group animals were subcutaneously injected with sodium chloride injection. The drugs were started about 2 hours after the animals were given the modeling agent, twice a day, for 19 consecutive days.

Specifically, the compounds numbered SYN-003, SYN-004, SYN-006, SYN-007, SYN-008, SYN-010, SYN-013, and SYN-017 were suspended in 10wt% DMSO + 90wt% NaCl to prepare a suspension, which was intragastrically administered at a dose of 3 mg/kg body weight of the active ingredient in a volume of 5 mL/kg.

After 19 days of continuous administration, the mean pulmonary artery pressure of rats was measured by right cardiac catheterization. The experimental results are shown in Figure 1, in which the Model group was given 10wt% DMSO + 90wt% NaCl, 5ml/kg body weight by intragastric administration, and the Control group was given Selexipag, 3mg/kg body weight by intragastric administration.

The results showed that the compounds numbered SYN-003, SYN-004, SYN-006, SYN-007, SYN-008, SYN-010, SYN-013, and SYN-017 all significantly reduced the pulmonary artery pressure of rats, among which SYN-004, SYN-007, and SYN-010 had the most obvious antihypertensive effect.

3. Survival rate test of rats with pulmonary hypertension

Experimental process: Monocrotaline 40mg/kg was injected subcutaneously for model preparation, and the normal control group animals were injected subcutaneously with sodium chloride injection. The animals were given the modeling agent and started to be administered about 2 hours after the administration, twice a day, for 45 consecutive days. The administration method was as follows: the compounds numbered SYN-004, SYN-007 and SYN-010 were suspended in 10wt% DMSO + 90wt% NaCl to prepare a suspension, and the active ingredient was administered by gavage at 1mg/kg body weight. Selexipag was used as the positive drug group and was administered by gavage at 1mg/kg body weight of the active ingredient. The model group was gavaged with normal saline.

The survival status of the rats was recorded. The experimental results are shown in Table 2.

Table 2 Effects of SYN-004, SYN-007 and SYN-010 on the survival rate of rats with pulmonary hypertension

Grouping Number of animals (D0) Number of remaining animals (D45) Survival rate (%) Normal control group 10 10 100 Model Group 10 3 30 Selexipag Group 10 7 70 SYN-004 Group 10 7 70 SYN-007 Group 10 8 80 SYN-010 Group 10 7 70

The results showed that Selexipag, SYN-004, SYN-007 and SYN-010 could significantly improve the survival rate of rats with pulmonary hypertension.

4. Toxicity comparison

Acute toxicity experiment process: Rats were randomly divided into groups according to body weight, 10 rats/group, and given 250, 500, 1000, and 2000 mg/kg of the compound by single gavage, with the administration volume of 10 ml/kg. The rats were observed for 7 days after administration.

The experimental results are shown in Table 3.

Table 3 Acute toxicity of SYN-003, SYN-004, SYN-005, SYN-007 and SYN-010 in rats

The results showed that the oral lethal dose of Selexipag in rats was about 500 mg/kg, and the non-lethal dose was 250 mg/kg. No death was observed at 1000 mg/kg for SYN-003, SYN-004, SYN-005, SYN-007, and SYN-010, but death occurred at 2000 mg/kg, indicating that the toxicity was less than that of Selexipag.

Genetic toxicity test process: The test samples SYN-004, SYN-007, SYN-010 and ACT-333679 were diluted to 8 concentrations with a final concentration of 1000 μg/well. The 6-well plate incorporation method was used, 2 wells were treated in parallel, and negative (DMSO) and positive controls were set up at the same time. Parallel tests were performed with or without metabolic activation system (±S9). After 48 to 72 hours of incubation, the test samples were observed for precipitation and the growth of background plaques, and the number of reverse mutant colonies in each well was counted. The test results are shown in Tables 4 to 11.

Positive result determination

A result is considered positive if it meets 1 or 2 of the following criteria:

1) In at least one strain, the number of revertant colonies increased in a dose-dependent manner with or without metabolic activation, and the number of revertant colonies was 2 times or more than that of the negative control group.

2) With or without metabolic activation, the number of reverse mutant colonies in one or more dose groups increases significantly and reproducibly, and the number of reverse mutant colonies is 2 times or more than that in the negative control group.

After the test sample is tested with two test strains, as long as one of the test strains is positive, regardless of whether S9 mixed solution is added or not, the test sample can be determined to be a mutagen.

Negative result judgment

If the test results show that there is no dose-dependent increase in the number of reverted colonies of each test strain, and the peak value of the number of reverted mutant colonies in each dose group of all strains does not exceed twice that of the negative control group, the test article can be determined to be a non-mutagenic agent.

The results showed that under ±S9 conditions, non-interfering precipitation was observed for TA98 and TA100 strains when the final concentration of ACT-333679 was ≥250μg/well; non-interfering precipitation was observed only when the final concentration of SYN-004, SYN-007 and SYN-010 was 1000μg/well. Under ±S9 conditions, for TA98 and TA100 strains, background plaque reduction was observed when the final concentration of ACT-333679 was ≥250μg/well; background plaque reduction was observed when the final concentration of SYN-010 was 1000μg/well; no abnormal background plaque was observed at any concentration of SYN-004 and SYN-007. For strains TA98 and TA100, under ±S9 conditions, the number of reverse mutant colonies in each concentration group of SYN-004, SYN-007, SYN-010 and ACT-333679 did not reach twice that of the negative control group, and there was no concentration-effect relationship, so the test results were considered negative.

Therefore, SYN-004, SYN-007, SYN-010 and ACT-333679 are not genotoxic.

Table 4 Results of SYN-004 bacterial reverse mutation 6-well plate initial screening test (TA98 strain)

Remark:

Background plaque: T0: normal;

Solubility of test/reference substance: P0: normal/no precipitation, P1: non-interfering precipitation under the microscope;

*: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

Table 5 Results of SYN-004 bacterial reverse mutation 6-well plate initial screening test (TA100 strain)

Remark:

Background plaque: T0: normal;

Solubility of test/reference substance: P0: normal/no precipitation, P1: non-interfering precipitation under the microscope;

*: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

Table 6 Results of SYN-007 bacterial reverse mutation 6-well plate initial screening test (TA98 strain)

Remark:

Background plaque: T0: normal;

Solubility of test/reference substance: P0: normal/no precipitation, P1: non-interfering precipitation under the microscope;

*: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

Table 7 Results of SYN-007 bacterial reverse mutation 6-well plate initial screening test (TA100 strain)

Remark:

Background plaque: T0: normal;

Solubility of test/reference substance: P0: normal/no precipitation, P1: non-interfering precipitation under the microscope;

*: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

Table 8 Results of SYN-010 bacterial reverse mutation 6-well plate initial screening test (TA98 strain)

Remark:

Background plaque: T0: normal, T1: background plaque slightly reduced;

Solubility of test/reference substance: P0: normal/no precipitation, P1: non-interfering precipitation under the microscope;

*: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

Table 9 Results of SYN-010 bacterial reverse mutation 6-well plate initial screening test (TA100 strain)

Remark:

Background plaque: T0: normal, T1: background plaque slightly reduced;

Solubility of test/reference substance: P0: normal/no precipitation, P1: non-interfering precipitation under the microscope;

*: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

Table 10 Results of 6-well plate screening test of ACT-333679 bacterial reverse mutation (TA98 strain)

Remark:

NA: not counted;

Background plaque: T0: normal, T1: background plaque slightly reduced, T3: background plaque severely reduced, T4: background plaque disappeared;

Solubility of test/reference substance: P0: normal/no precipitation, P1: non-interfering precipitation under the microscope;

^a : The positive drugs without and with S9 were sodium azide (0.4 μg/well) and 2-aminoanthracene (0.6 μg/well);

*: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

Table 11 Results of 6-well plate screening test of ACT-333679 bacterial reverse mutation (TA100 strain)

Remark:

NA: not counted;

Background plaque: T0: normal, T1: background plaque slightly reduced, T3: background plaque severely reduced, T4: background plaque disappeared;

Solubility of test/reference substance: P0: normal/no precipitation, P1: non-interfering precipitation under the microscope;

^a : The positive drugs without and with S9 were sodium azide (0.4 μg/well) and 2-aminoanthracene (0.6 μg/well);

*: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

In summary, the present application synthesized a series of new structural compounds, which have good affinity for PGI2 receptors and very low adverse reactions, and have broad application prospects in the preparation of drugs for the treatment of pulmonary arterial hypertension.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (14)

1. A PGI2 receptor agonist compound represented by general formula I or a pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, In the formula, R ₁ and R ₂ are independently selected from H, C ₁ -C ₃ alkyl or halogen atom; Z is selected from CR ₇ or N atom, wherein R ₇ is selected from H or C ₁ -C ₃ alkyl, halogen atom or halogenated C ₁ -C ₃ alkyl; R ₃ is selected from C ₂ -C ₄ alkyl, C _{3 -} C ₆ monocyclic cycloalkyl; R ₄ is selected from H, C ₁ -C ₃ alkoxy or 3-6 membered heteroalicyclic group; R ₅ represents OH, OR ₆ or NHSO ₂ R ₆ , wherein R ₆ represents C _1-4 alkyl or C _1-4 alkyl substituted by halogen; Indicates a single bond or a double bond. 2. The PGI2 receptor agonist compound or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof according to claim 1, wherein Z is a N atom; or Z is _CR7 , wherein _R7 is selected from H or _CH3 . 3. The PGI2 receptor agonist compound according to claim 2 or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, wherein: represents a double bond, _R4 is H. 4. The PGI2 receptor agonist compound according to claim 2 or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, wherein: represents a single bond, and R ₄ is selected from H, C ₁ -C ₃ alkoxy or 3-6 membered heteroalicyclic group. 5. The PGI2 receptor agonist compound according to claim 4 or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, wherein _R4 is selected from H, methoxy, ethoxy, 6. The PGI2 receptor agonist compound according to any one of claims 2 to 5, or a pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, wherein _R1 and _R2 are selected from H, methane or F. 7. The compound according to claim 6 or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, characterized in that _R3 is selected from isopropyl, ethyl or cyclopropane. 8. The compound according to claim 7 or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, characterized in that _R3 is selected from isopropyl. 9. The compound or pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof according to claim 6, wherein _R5 is selected from OH or _{NHSO2CH3 .} 10. The compound according to claim 1 or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, characterized in that the compound is selected from: 11. A pharmaceutical composition, characterized in that it comprises a pharmaceutically acceptable carrier, excipient or diluent, and as an active ingredient, the compound according to any one of claims 1 to 10 or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof. 12. The pharmaceutical composition according to claim 11, characterized in that the dosage form of the pharmaceutical composition is a pharmaceutically acceptable dosage form. 13. The pharmaceutical composition according to claim 12, characterized in that the dosage form comprises tablets, granules, capsules, powders or injections. 14. Use of the compound according to any one of claims 1 to 10 or its pharmaceutically acceptable salts, stable isotope derivatives, isomers and mixtures thereof or the pharmaceutical composition according to any one of claims 11 to 13 in the preparation of a drug for treating pulmonary arterial hypertension.