A type of nitrogen-containing cyclic compound and its preparation method and application

Abstract

The present invention discloses a class of nitrogen-containing cyclic compounds and their preparation methods and applications. Specifically, the present invention discloses a compound as shown in formula (I), its stereoisomers, tautomers, crystal forms, pharmaceutically acceptable salts, hydrates, solvates or prodrugs. The compound of the present invention is an orexin receptor antagonist, which can be used to prepare drugs for treating and / or preventing diseases related to the activity or expression of orexin receptor 2.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a class of nitrogen-containing and cyclic compounds and a preparation method and application thereof. Background Art

[0002] Orexin is a neuropeptide discovered in 1998. It is synthesized and secreted by the lateral hypothalamic orexin neurons. There are two types, OrexinA and OrexinB. Orexin A contains 33 amino acids and Orexin B contains 28 amino acids. They act on the G-protein-coupled orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) receptor subtypes, respectively.

[0003] OX1R and OX2R are widely expressed in the central nervous system and have complex relationships with other neuropeptides that affect feeding. Rodents injected with orexin intraperitoneally have prolonged wakefulness (Piper et al. J. Neurosci. 2000 12.726-730), OX2R damage or mutation leads to long-term sleep in dogs (lin et al. Cell 1999, 365-376), and insufficient orexin signals in cerebrospinal fluid will cause long-term narcolepsy in humans. All of these indicate that wakefulness and sleep can be regulated by regulating the two receptors. OX1R and OX2R play different roles in controlling non-rapid eye movement sleep (NREM, deep sleep) and rapid eye movement sleep (REM, brain activity is the same as when awake). OX2R is a key receptor for regulating wakefulness and NREM sleep, while REM sleep is controlled by two receptor subtypes. OX2R plays a dominant role in regulating sleep, while OX1R alone contributes little to sleep induction, but works synergistically with OX2R to regulate REM sleep.

[0004] Orexin receptor antagonists are divided into selective OX1R antagonists, selective OX2R antagonists and dual OX1 / 2R antagonists according to their binding affinity. Dual OX1 / 2R antagonists from multiple companies have been launched on the market, including Suvorexant from Merck & Co, Lemborexant from Eisai, and daridorexant from Johnson & Johnson. However, acting on OX1R will change the normal physiological structure of REM and NREM, that is, sacrificing NREM time, prolonging REM time, and thus increasing the risk of sleepiness.

[0005] Currently, the only selective antagonist is Johnson & Johnson's Seltorexant in phase III clinical trials, with indications for major depression, insomnia, etc. Although it shows certain selectivity for OX1R and OX2R, its selectivity still needs to be improved.

[0006] Therefore, there is an urgent need in the art for an OX2R antagonist with high selectivity and safety. Summary of the invention

[0007] In order to solve the above problems, the present invention provides an OX2R antagonist with high antagonist selectivity, high efficacy and high safety.

[0008] In the first aspect of the present invention, there is provided a compound as represented by formula (I), its stereoisomers, tautomers, crystal forms, pharmaceutically acceptable salts, hydrates, solvates or prodrugs, .

[0009] In a preferred embodiment, the method comprises the following steps:

[0010] (1) In the presence of condensing agent HATU, compound 01 and compound 1 undergo condensation reaction to obtain compound 02: (2) In the presence of TFA, compound 02 is subjected to a deprotection reaction to obtain compound 03: (3) In the presence of a base, compound 03 and compound 2 undergo a substitution reaction to obtain compound 04, i.e., a compound of formula (I): .

[0011] In a preferred embodiment, the step (1) is carried out in the presence of HATU and DIEA.

[0012] In another preferred embodiment, the step (1) is carried out in DMF solvent.

[0013] In another preferred embodiment, in the step (1), the reaction temperature of the condensation reaction is 20-30°C; preferably 22-28°C.

[0014] In another preferred embodiment, the reaction time of the condensation reaction is 10 to 20 h; preferably 12 to 18 h; more preferably 14 to 18 h.

[0015] In another preferred embodiment, in the step (1), the molar ratio of compound 01 to compound 1 is (0.1-5):1; preferably (0.5-2):1; more preferably (0.5-1.5):1.

[0016] In another preferred embodiment, in the step (1), the molar ratio of the compound 01 to HATU is 1: (1-5); preferably 1: (1-3); more preferably 1: (1-1.5).

[0017] In another preferred embodiment, in the step (1), the molar ratio of compound 01 to DIEA is 1: (1-10); preferably 1: (2-8); more preferably 1: (3-7).

[0018] In a preferred embodiment, the step (2) is carried out in the presence of TFA and DCM.

[0019] In another preferred embodiment, in the step (2), the reaction temperature of the deprotection reaction is 20-30°C; preferably 22-28°C.

[0020] In another preferred embodiment, the reaction time of the deprotection reaction is 1 to 5 h; preferably 1 to 3 h.

[0021] In a preferred embodiment, the step (3) is carried out in the presence of a base and DMF.

[0022] In a preferred example, the base is selected from the following group: Cs2CO3, K2CO3, or a combination thereof.

[0023] In another preferred embodiment, in the step (3), the reaction temperature of the substitution reaction is 20-30°C; preferably 22-28°C.

[0024] In another preferred embodiment, in the step (3), the reaction time of the substitution reaction is 1 to 3 h.

[0025] In another preferred embodiment, in the step (3), the molar ratio of compound 03 to compound 2 is (0.1-5):1; preferably (0.5-2):1; more preferably (0.5-1.5):1.

[0026] In another preferred embodiment, in the step (3), the molar ratio of the compound 03 to the base is 1: (1-10); preferably 1: (1-5); more preferably 1: (2-4).

[0027] The second aspect of the present invention provides a pharmaceutical composition, comprising:

[0028] (i) the compound according to the first aspect of the present invention, its stereoisomer, tautomer, crystalline form, pharmaceutically acceptable salt, hydrate, solvate or prodrug; and

[0029] (ii) a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0030] The third aspect of the present invention provides a use of the compound as described in the first aspect of the present invention, its stereoisomers, tautomers, crystal forms, pharmaceutically acceptable salts, hydrates, solvates or prodrugs, or the pharmaceutical composition as described in the second aspect of the present invention for preparing a drug for treating and / or preventing diseases related to the activity or expression of orexin receptor 2.

[0031] In a preferred embodiment, the disease associated with the activity or expression of orexin receptor 2 is selected from the following group: sleep-wake cycle disorder, insomnia, restless legs syndrome, jet lag syndrome, sleep disturbance, depression, Alzheimer's disease, sleep disorders secondary to the following diseases: neurotic disorders, mania, depression, bipolar disorder, schizophrenia, pain syndrome, fibromyalgia, neuropathic pain, tension, Parkinson's disease, Tourette's syndrome, anxiety, delirium, dementia, overweight or obesity, and diseases related to overweight or obesity, insulin resistance, type II diabetes, hyperlipidemia, gallstones, angina pectoris, hypertension, dyspnea, tachycardia, infertility, sleep apnea, back and joint pain, varicose veins, osteoarthritis, hypertension, tachycardia, arrhythmia, angina pectoris, acute heart failure, ulcer, irritable bowel syndrome, diarrhea, gastroesophageal reflux.

[0032] In a preferred embodiment, the disease related to the activity or expression level of orexin receptor 2 is selected from the following group: insomnia, depression, and Alzheimer's disease.

[0033] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. DETAILED DESCRIPTION

[0034] After long-term and in-depth research and extensive screening, the inventors have developed for the first time an OX2R antagonist with high antagonist selectivity, high efficacy and high safety. Based on this, the inventors have completed the present invention.

[0035] Abbreviations

[0036]

[0037] Active ingredients

[0038] As used herein, "compound of the present invention" refers to the compound represented by formula (I), and also includes its stereoisomers, optical isomers, pharmaceutically acceptable salts, crystal forms, isotopic derivatives, prodrugs, metabolites, solvates or hydrates thereof.

[0039] Unless otherwise specified, the structural formulas described in the present invention are intended to include all stereoisomers (such as cis-trans isomers, enantiomers, diastereomers and conformational isomers): R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, cis-trans isomers of cycloalkanes, etc. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers or conformational isomers are all within the scope of the present invention.

[0040] The compounds of the present invention may contain cis-trans isomers, one or more chiral carbon atoms, and thus may produce stereoisomeric forms of cis-trans isomers, chiral isomers, enantiomers, diastereomers and other combinations. Cis-trans isomerism refers to the diastereoisomerism phenomenon that occurs due to the restriction of free rotation in the compound molecule, which causes the different arrangements of various groups in space. This restriction is generally caused by the presence of functional groups that cannot rotate freely in the structure of organic compounds, such as C=C double bonds, C=N double bonds, C=S double bonds, N=N double bonds, heterocycles or cycloalkanes. Organic molecules containing such isomers, such as olefins, azo compounds, cycloalkanes, etc., are considered cis-trans isomers. The cis form refers to the same ligands in adjacent positions, generally represented by "cis" or "cis-"; the trans form refers to the same ligands in diagonal positions, generally represented by "trans" or "trans-". Each chiral carbon atom can be defined as (R)- or (S)- based on stereochemistry. The present invention is intended to include all possible isomers, as well as racemates and optically pure forms thereof. The compounds of the present invention may be prepared using racemates, cis-trans isomers, chiral isomers, diastereomers or enantiomers as starting materials or intermediates. Optically active isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

[0041] Conventional techniques for preparing / isolating individual optical isomers (ie, cis-trans and chiral isomers) include chiral synthesis from appropriate cis-trans or optically pure precursors, or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography.

[0042] If a synthesis of a specific stereoisomer of the compound of the present invention is to be designed, it can be prepared by asymmetric synthesis, or derivatized with a chiral auxiliary, the resulting stereo mixture can be separated, and then the chiral auxiliary can be removed to obtain a pure cis-trans monomer, a chiral monomer or a mixed stereo monomer. If the molecule contains a cis-trans isomeric center, it can be purified by column chromatography (normal phase silica gel column or reverse phase high performance liquid preparation) to obtain a pure cis product or trans product; in addition, if the molecule contains a basic functional group, such as an amino acid, or an acidic functional group, such as a carboxyl group, it can be used with a suitable optically active acid or base to form a diastereomeric salt, and then separated by conventional means such as fractional crystallization or chromatography, and then a pure enantiomer is obtained.

[0043] The present invention also includes isotopically labeled compounds (i.e., isotopic derivatives) that are equivalent to the original compounds disclosed herein. However, in practice, it is common for one or more atoms to be replaced by atoms having a different atomic mass or mass number than the original atoms. Examples of isotopes in the isotopic derivatives of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes, such as 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F and 36 Cl. The isotopic derivatives of the compounds of the present invention are all within the protection scope of the present invention. 3 H-labeled compounds and 14 C-labeled compounds are useful in drug and substrate tissue distribution experiments. 3 H) and carbon-14 (i.e. 14 C) labeled compounds are easier to prepare and detect, and are the first choice among isotopes. In addition, heavier isotope substitutions such as deuterium, i.e. 2 H, due to its good metabolic stability in certain therapies has advantages, such as increasing half-life or reducing dosage in vivo, therefore, in some cases, it can be preferred. Isotope-labeled compounds can be prepared by general methods by replacing readily available isotope-labeled reagents with non-isotopic reagents using the schemes disclosed in the examples.

[0044] As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0045] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, naphthalene disulfonates, and the like. These salts may be prepared by methods known in the art.

[0046] "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. The salt derived from organic base includes but is not limited to the following salt: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the profession.

[0047] The metabolites of the compound represented by formula (I) and its pharmaceutically acceptable salts, and the prodrugs that can be converted into the compound represented by formula (I) and its pharmaceutically acceptable salts in vivo are also included in the protection scope of the present invention.

[0048] As used herein, the term "solvate" refers to a complex formed by coordination of the compound represented by formula (I) with solvent molecules in a specific ratio.

[0049] As used herein, the term "hydrate" refers to a complex formed by the compound represented by formula (I) and water molecules coordinated to form a specific ratio.

[0050] As described herein, the compounds of the present invention can be substituted with any number of substituents or functional groups to expand their scope. In general, the term "substituted" refers to replacing hydrogen radicals with specified structural substituents. When multiple positions in a specific structure are substituted with multiple specific substituents, the substituents can be the same or different at each position. The term "substituted" used herein includes all allowed organic group substitutions. In a broad sense, allowed substituents include non-cyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic groups. Herein, heteroatom nitrogen can have hydrogen substituents or any allowed organic groups described above to supplement its valence. In addition, the present invention is not intended to limit the allowed substitution of organic groups in any way. The present invention believes that the combination of substituents and variable groups is good in the treatment of diseases in the form of stable compounds. The term "stable" here refers to a stable compound that is tested for a long enough time to maintain the integrity of the compound structure, preferably for a long enough time, and is used for the above purposes herein.

[0051] Pharmaceutical compositions and methods of administration

[0052] Since the compounds of the present invention can selectively inhibit orexin receptor 2 (OX2R), and are used to treat diseases such as sleep-wake cycle disorders, insomnia, restless legs syndrome, jet lag syndrome, depression, Alzheimer's disease, etc. Therefore, the compounds of the present invention and their stereoisomers, optical isomers, pharmaceutically acceptable salts, crystal forms, isotope derivatives, prodrugs, metabolites, solvates or hydrates thereof, and pharmaceutical compositions containing the compounds of the present invention as main active ingredients can be used to prevent and / or treat (stabilize, alleviate or cure) diseases related to the activity or expression of orexin receptor 2 (sleep-wake cycle disorders, insomnia, restless legs syndrome, jet lag syndrome, depression, Alzheimer's disease, etc.).

[0053] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0054] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0055] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include, but are not limited to, oral administration and parenteral administration (intravenous, intramuscular or subcutaneous administration).

[0056] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0057] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0058] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0059] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0060] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0061] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0062] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0063] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0064] When administered in combination, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds can be used simultaneously, separately or sequentially with the compound of the present invention to prevent and / or treat diseases related to the activity or expression of orexin receptor 2.

[0065] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1-2000 mg, preferably 20-500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0066] The main advantage of the present invention is that the OX2R antagonist of the present invention has high selectivity for OX2R and thus can have higher safety than marketed drugs.

[0067] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually carried out under normal conditions or under the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0068] Example 1. Preparation of Compound 04: (3aS,6aS)-1-(4,6-dimethylpyrimidin-2-yl)-hexahydroxy-pyrrolyl[3,4-b]pyrrol-5(1H)-yl)(2-fluoro-6-(2H-1,2,3-triazol-2-yl)phenyl)methanone

[0069]

[0070] first step

[0071]

[0072] At 25 °C, compound 01 (102.5 mg, 482.71 µmol, 1.0 eq), compound 1 (100.0 mg, 482.71 µmol, 1.0 eq), HATU (220.3 mg, 579.25 µmol, 1.2 eq), and DIEA (311.9 mg, 2.41 mmol, 5.0 eq) were added to 2 mL of DMF, and the reaction system was stirred for 16 h. The reaction process was monitored by liquid quality control. After the reaction was completed, about 20 mL of water was added to the reaction solution, and then extracted with ethyl acetate (20 mL) three times, the organic phases were combined and washed once with about 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography using a 0%-50% ethyl acetate / petroleum ether mobile phase gradient elution, and the obtained fractions were concentrated under reduced pressure to give compound 02 (colorless oil, 140.8 mg, yield 78.0%).

[0073] Step 2

[0074]

[0075] At 25 °C, compound 02 (140.8 mg, 367.19 µmol, 1.0 eq) was dissolved in 2 mL of DCM, and 1 mL of TFA was added. The mixture was stirred for about 2 h, and the reaction process was monitored by TLC. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and compound 03 (145.4 mg) was directly used for the next step.

[0076] Step 3

[0077]

[0078] At 25 °C, 2 mL of DMF was added to compound 03 (145.4 mg, 482.53 µmol, 1.0 eq), compound 2 (68.8 mg, 482.53 µmol, 1.0 eq) and Cs2CO3 (471.7 mg, 1.45 mmol, 3.0 eq), and the reaction system was placed in a 120 °C oil bath and stirred for about 2 h. The reaction process was monitored by liquid chromatography-mass spectrometry. After the reaction was completed and cooled to room temperature, about 20 mL of water was added to the reaction solution, and then extracted with ethyl acetate (20 mL) three times. The organic phases were combined and washed once with about 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was separated by high pressure preparation to obtain a white solid product, namely compound 04 (54.6 mg), MS (ESI, m / z): 408.26 [M+H] + .

[0079] Example 2. Determination of the inhibitory activity of compounds on OX1 & PFSK-1 (OX2) receptors

[0080] 1. Cell Information

[0081]

[0082] 1.1 In this study, stably transfected cells expressing OX1 receptor and PFSK-1 (OX2) cells were incubated with different concentrations of test compounds, and the inhibition of the compounds on OX1 & OX2 receptors was determined using the FLIPR CALCIUM 6 ASSAY KIT kit.

[0083] 1.2. OX1-CHO cell line was cultured in F12 medium containing 10% fetal bovine serum and 0.2 mg / mL Hygromycin B, and PFSK-1 (OX2) was cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% carbon dioxide concentration.

[0084] 1.3. Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of TrypLE™ Express solution and incubate at 37℃ for about 2 min. When the cells detach from the bottom of the dish, add about 5 mL of complete culture medium preheated at 37℃. Gently pipette the cell suspension to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min.

[0085] 1.4. In order to maintain the physiological activity of cells, the cell confluence degree was controlled at about 80%.

[0086] 2. Experimental Procedure

[0087] 2.1 Cell plating: OX1&PFSK-1(OX2)-CHO cells were digested and collected, resuspended and counted, and then inoculated into 384-well cell plates at a density of 1.2×10 4 cells / 25 μL / well. Then place the cell plate in a 37°C, 5% CO2 incubator for about 16-20 hours;

[0088] 2.2 Day 2: Prepare the Assay Buffer according to the instructions of the FLIPR Calcium 6 Assay Kit. Freeze and thaw 20× Compound A to room temperature, dilute it with Assay buffer to 1× loading buffer, and keep it at room temperature for later use;

[0089] 2.3 Remove the culture medium from the cell plate, quickly add 35 μL 1× loading buffer to each well, centrifuge and incubate the cell plate at 37°C in the dark for 120 min;

[0090] 2.4 Prepare the working solution of positive compound and test compound, transfer 5 μL to the corresponding cell wells, and incubate at 37°C in the dark for 30 min;

[0091] 2.5 Prepare agonist and transfer 20 μL / well to a 384-well compound source plate;

[0092] 2.6 Place the cell plate, source plate and pipette tip in the corresponding position of the FLIPR instrument. Use the FLIPR Tetra to add 10 μL of the diluted compound in step 5 to each experimental well and collect data at a wavelength of 515 nm-575 nm.

[0093] 2.7 Plot the signal value against the compound concentration and use the GraphPad Prism software nonlinear regression method to fit the IC curve 50 calculate.

[0094] 3. Data Analysis

[0095] 1) Z' factor = 1-3*(SD Max+SD Min) / (AVG Max- AVG Min);

[0096] 2) CV Max = (SD Max / AVG Max)*100%;

[0097] 3) CV Min = (SD Min / AVG Min)*100%;

[0098] 4) S / B = Signal / Background;

[0099] 5) Calculate compound IC using GraphPad nonlinear fitting formula 50 :

[0100] Y=Bottom + (Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0101] 6) % Inhibition rate formula:

[0102]

[0103] Mean values ​​of positive controls.

[0104] Mean values ​​of negative controls (DMSO).

[0105] 4. Test results:

[0106] Among them, the positive compound is Seltorexant, and its structure is shown below:

[0107]

[0108]

[0109] Note: Selection ratio = molecule's inhibitory activity on OX1R (nM) / molecule's inhibitory activity on OX2R (nM). The experimental data were completed in the same test batch.

[0110] 5. Experimental Conclusion

[0111] As can be seen from the above table, the inhibitory selectivity of the compounds invented in the present application on OX2 / OX1 is higher than that of the positive control.

[0112] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing compound 04, characterized in that: The method comprises the following steps: (1) At 25°C, 102.5 mg of compound 01, 100.0 mg of compound 1, 220.3 mg of HATU, and 311.9 mg of DIEA were added to 2 mL of DMF, and the reaction system was stirred for 16 h. The reaction process was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, about 20 mL of water was added to the reaction solution, and then extracted three times with 20 mL of ethyl acetate, the organic phases were combined and washed once with about 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product; The crude product was purified by silica gel column chromatography using a 0%-50% ethyl acetate / petroleum ether mobile phase gradient elution, and the obtained fractions were concentrated under reduced pressure to obtain compound 02; (2) At 25°C, 140.8 mg of compound 02 was dissolved in 2 mL of DCM, and 1 mL of TFA was added. The mixture was stirred for about 2 h, and the reaction process was monitored by TLC. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and compound 03 was directly used for the next reaction; (3) At 25°C, 2 mL of DMF was added to 145.4 mg of compound 03, 68.8 mg of compound 2, and 471.7 mg of Cs2CO3. The reaction system was placed in an oil bath at 120°C and stirred for about 2 h. The reaction process was monitored by liquid chromatography-mass spectrometry. After the reaction was completed and cooled to room temperature, about 20 mL of water was added to the reaction solution, and then extracted three times with 20 mL of ethyl acetate. The organic phases were combined and washed once with about 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was separated by high pressure preparation to obtain a white solid product, namely compound 04.

Images