GHSR 1a agonist, pharmaceutical composition as well as preparation method and application of GHSR 1a agonist

Abstract

A compound represented by formula I, a racemate, a stereoisomer, a tautomer, a nitrogen oxide, a solvate, a polymorphic substance, a pharmaceutically acceptable salt or a prodrug thereof. The compound has a good GHSR1a agonist effect and can be used for diagnosing, treating or preventing diseases and diseases related to growth hormone deficiency or growth hormone dependence and preparing drugs for diagnosing, treating or preventing the diseases and diseases. # imgabs0 #

Description

GHSR 1a agonist, pharmaceutical composition, preparation method and application thereof

[0001] The present invention claims:

[0002] Priority to the prior application, patent application number 202310435486.0, filed with the State Intellectual Property Office of China on April 21, 2023, entitled “GHSR 1a agonists, pharmaceutical compositions, preparation methods and uses thereof”;

[0003] Priority to the prior application, patent application number 202311345870.8, filed with the State Intellectual Property Office of China on October 17, 2023, entitled “GHSR 1a agonists, pharmaceutical compositions, preparation methods and uses thereof”;

[0004] The entire contents of said prior application are incorporated herein by reference. Technical Field

[0005] The present invention belongs to the field of pharmaceutical compounds, and in particular relates to a GHSR 1a agonist, a pharmaceutical composition, and a preparation method and application thereof. Background Art

[0006] Ghrelin is an endogenous 28-amino acid growth hormone-releasing peptide and an endogenous ligand for the growth hormone secretagogue receptor type 1a (GHSR 1a). Both in vivo and in vitro studies have demonstrated that ghrelin significantly promotes growth hormone (GH) secretion. Clinical studies have also demonstrated that intravenous ghrelin can strongly stimulate GH release in a dose-dependent manner.

[0007] Human growth hormone is a peptide hormone secreted by the anterior pituitary gland. It is composed of 191 amino acids and acts directly or indirectly on peripheral organs by inducing the synthesis of insulin-like growth factor 1 (IGF-1) or epidermal growth factor (EGF). Its main physiological functions include promoting the linear growth of the body, promoting muscle and skin cell proliferation, and playing an important role in the regeneration of tissues after trauma.

[0008] The release of GH is believed to treat physiological or pathophysiological conditions characterized by a deficiency in growth hormone secretion, as well as conditions that are ameliorated by the anabolic effects of growth hormone. GH has been shown to be promising in treating conditions such as loss of muscle mass, accumulation of adipose tissue, bone demineralization, and reduced tissue regeneration after injury.

[0009] GH is synthesized and stored in the pituitary gland, but its release is controlled by hormones from the hypothalamus. Two hormones are known to be involved in GH release: growth hormone-releasing hormone (GHRH) and the inhibitory hormone somatostatin (SRIF). In most cases, GH deficiency involves GH release (hypothalamic defect) rather than GH synthesis (pituitary defect). Therefore, stimulating GH release from the pituitary using GHSR agonists may be a new therapeutic alternative to recombinant human growth hormone.

[0010] GHSR has two subtypes, 1a and 1b. Subtype 1a is the functional receptor subtype, while the function of subtype 1b awaits further investigation. Within the central nervous system, GHSR 1a is distributed in the hypothalamus and multiple regions beyond the hypothalamus, including the pituitary gland, the arcuate nucleus of the hypothalamus, and the ventromedial nucleus. In the periphery, GHSR is also expressed at low levels in the thyroid gland, pancreas, and myocardium. Therefore, ghrelin and its receptor, GHSR 1a, may be involved in regulating multiple functions in the body.

[0011] Studies have found that some clinical peptide or peptidomimetic compounds exhibit GHSR agonist activity and have the ability to induce GH release. Compounds currently in clinical development include examorelin, tabimorelin, pralmorelin, ibutamoren, tesamorelin, anamorelin, and macimorelin. Among these, the injectable peptide tesamorelin (used to reduce excess abdominal fat in HIV-infected individuals) and the small molecule peptidomimetic macimorelin have been approved for marketing by the FDA. Macimorelin is the only oral drug approved for the diagnosis of growth hormone deficiency in adults, but macimorelin also has drawbacks such as poor oral bioavailability and potential risk of cardiotoxicity.

[0012] Ghrelin has been shown to not only promote gastrointestinal motility through the vagus and pelvic nerves but also stimulate GH release. However, ghrelin's short half-life hinders its drugability, necessitating the development of GHSR agonists with enhanced pharmacokinetics to treat related diseases. Studies have shown that capromorelin exhibits a favorable safety profile and some GHSR agonist activity in clinical trials, but failed to achieve clinical endpoints after long-term administration. Therefore, the development of GHSR agonists based on capromorelin, while avoiding its potential drawbacks, would have promising clinical application prospects.

[0013] Summary of the Invention

[0014] To improve the above technical problems, the present invention provides a compound represented by Formula I, its racemate, stereoisomer, tautomer, nitrogen oxide, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof:

[0015] in,

[0016] X is selected from N or CR x ; R x is selected from oxo (=O) or phenyl;

[0017] Y is selected from NR y or O; R y Selected from H, C 1-6 Alkyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, halogenated C 1-6 Alkyl, C 3-6 Cycloalkyl-C 1-6 alkyl;

[0018] Z is selected from N or C-Ph;

[0019] W is selected from N or CH;

[0020] R1 and R2 are the same or different and are independently selected from H or C 1-6 Alkyl; or, R1, R2 together with the carbon atom to which they are attached form a 3-8 membered N-containing heterocyclic group;

[0021] R3 is selected from H or amino;

[0022] R4 is absent or selected from methylene substituted by R5; R5 is selected from unsubstituted or optionally substituted by 1, 2 or more R 51 Substituted phenyl or pyridyl; each R 51 are the same or different and are independently selected from halogen or CN;

[0023] Each R a The same or different, independently selected from halogen, CN, C 1-6 alkyl;

[0024] Each R b The same or different, independently selected from halogen, CN, C 1-6 Alkyl; or two R attached to the same carbon atom b Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; or two non-adjacent R groups attached to different carbon atoms b Connected by their end groups, together forming C 1-3 alkylene;

[0025] represents a single bond or a double bond; and when X is C(=O), is a single bond;

[0026] m is selected from 0, 1, 2 or 3; n is selected from 0, 1, 2 or 3.

[0027] According to some embodiments, X is selected from C(═O);

[0028] According to some embodiments, Y is selected from NR y ; R y Selected from C 3-6 Cycloalkyl or 3-6 membered O heterocyclic group;

[0029] According to some embodiments, Y is selected from NR y ; R y is selected from H, methyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 3-oxetanyl, cyclopropylmethyl, tert-butyl, difluoromethyl;

[0030] According to some embodiments, Y is selected from NR y ; R y is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or 3-oxetanyl;

[0031] According to some embodiments, Z is selected from N;

[0032] According to some embodiments, W is selected from CH;

[0033] According to some embodiments, R1 and R2 are selected from methyl groups, and R3 is selected from amino groups;

[0034] According to some embodiments, R4 is absent or selected from benzyl, Preferably, R4 is benzyl.

[0035] According to some embodiments, the compound represented by Formula I is selected from the structures shown below:

[0036] Among them, R y has the definitions set out herein.

[0037] According to some embodiments, the compound represented by formula I is selected from the following structures:

[0038] According to some embodiments, the compound represented by formula I is selected from the following structures:

[0039] According to some embodiments, the compound represented by formula I is selected from the following structures:

[0040] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of at least one of the compound of Formula I, its racemate, stereoisomer, tautomer, nitrogen oxide, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof.

[0041] According to an embodiment of the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0042] The excipients in the pharmaceutical compositions are "acceptable" in the sense that they are compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject being treated. One or more pharmaceutical excipients can be used to deliver the active compound.

[0043] According to some embodiments of the present invention, the pharmaceutical composition may further contain one or more additional therapeutic agents.

[0044] The present invention further provides use of the compound of formula I, its racemate, stereoisomer, tautomer, nitrogen oxide, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof, or the pharmaceutical composition in the preparation of a GHSR 1a agonist.

[0045] The present invention further provides the use of the compound of formula I, its racemate, stereoisomer, tautomer, nitrogen oxide, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof, or the pharmaceutical composition in the preparation of a medicament for diagnosing, preventing and / or treating diseases or conditions related to growth hormone deficiency or growth hormone dependence.

[0046] According to an embodiment of the present invention, the disease or condition is related to growth hormone deficiency or growth hormone dependence. Preferably, the compound can increase the level of growth hormone in the plasma of humans and animals after administration. This property can be used to diagnose and treat physiological or medical symptoms characterized by a lack of growth hormone secretion, such as the diagnosis of growth hormone deficiency patients, slow growth and short stature in children with growth hormone deficiency, and the treatment of other diseases that can be improved by the physiological effects of growth hormone.

[0047] The present invention also provides a method for diagnosing, preventing, and / or treating conditions requiring stimulation of growth hormone production or secretion, such as in humans with natural growth hormone deficiency or animals used for food production, where growth hormone stimulation would result in larger, more productive animals. The method comprises administering to a human or animal in need thereof at least one compound or pharmaceutical composition of the present invention to increase the level of growth hormone secretion.

[0048] In some embodiments, the compounds are useful as GHSR 1a agonists, including but not limited to: regulation of energy balance and food intake; treatment of adipogenesis, obesity, and weight loss; treatment of cachexia; improvement of gastrointestinal motility, treatment of gastroparesis and diabetic gastroparesis, and postoperative ileus; increase in muscle mass and skin thickness, reduction in fat mass, and slight increase in bone density in elderly patient populations; treatment of burns, AIDS, and cancer conditions, as well as wound and bone healing.

[0049] The compounds of the present invention may be used in combination with additional therapeutic agents.

[0050] According to some embodiments, the compound of formula I, its racemate, stereoisomer, tautomer, nitrogen oxide, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof can be prepared into a form suitable for administration by any appropriate route, and can be formulated using one or more pharmaceutically acceptable carriers by conventional methods. Thus, the compound of formula I, its racemate, stereoisomer, tautomer, nitrogen oxide, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof can be formulated into various dosage forms for oral administration, injection (e.g., intravenous, intramuscular or subcutaneous) administration, inhalation or insufflation administration; can also be formulated into sustained release dosage forms, such as tablets, hard or soft capsules, aqueous or oily suspensions, emulsions, injections, dispersible powders or granules, suppositories, lozenges or syrups. Beneficial effects

[0051] The compounds provided by the present invention exhibit excellent GHSR 1a agonist activity. Preferred compounds exhibit significantly stronger agonist activity against human and canine GHSR than capromorelin. Furthermore, preferred compounds exhibit slower clearance from human liver microsomes, superior pharmacokinetic properties, and enhanced pituitary tissue distribution. In vivo pharmacodynamics studies have shown that preferred compounds are significantly more effective than capromorelin in releasing growth hormone in vivo and exhibit good safety. In summary, the preferred compounds of the present invention possess enhanced drugability.

[0052] Definitions and Explanations of Terms

[0053] Unless otherwise indicated, the definitions of groups and terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The group definitions and compound structures resulting from such combinations and couplings should be understood to be within the scope of this specification and / or claims.

[0054] The term "optional" (or "optionally", "optionally") in the general formula definitions of this application means the situation of being substituted by zero, one or more substituents, for example, "optionally substituted by one, two or more R" means that it may not be substituted by R (unsubstituted) or may be optionally substituted by one, two or more R.

[0055] "More" means three or more, for example, 3, 4, 5, 6, 7, 8, 9 or 10.

[0056] Unless otherwise indicated, numerical ranges recited in this specification and claims are equivalent to reciting at least each specific integer value therein. For example, the numerical range "1-6" is equivalent to reciting each integer value in the numerical range "1-6", i.e., 1, 2, 3, 4, 5, and 6.

[0057] The term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0058] The term "C 1-6 The term "alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or the like or isomers thereof.

[0059] The term "C 3-8 "Cycloalkyl" is understood to mean a saturated monovalent monocyclic, bicyclic (such as bridged ring, spirocyclic) hydrocarbon ring having 3, 4, 5, 6, 7, 8 carbon atoms. 3-8 The cycloalkyl group may be a monocyclic hydrocarbon group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon group such as borneol, indolyl, hexahydroindolyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl, 2,7-diazaspiro[3,5]nonyl, 2,6-diazaspiro[3,4]octyl.

[0060] The term "3-8 membered heterocyclyl" means a saturated or unsaturated non-aromatic ring or ring system and contains at least one heteroatom selected from O, S and N. The heterocyclyl group may be attached to the rest of the molecule through any of the carbon atoms or the nitrogen atom (if present).

[0061] The term "nitrogen oxide" refers to a compound formed by oxidation of a nitrogen atom in a tertiary amine or nitrogen-containing (aromatic) heterocyclic compound structure.

[0062] The term "C-Ph" refers to a C atom with a benzene ring substituent attached to it.

[0063] Unless otherwise specified, a heterocyclic group, heteroaryl group, or heteroarylene group includes all possible isomeric forms thereof, such as positional isomers thereof. Thus, for some illustrative non-limiting examples, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-positions, etc. (if present) may include 1, 2, or more substituted or bonded forms thereof, including pyridin-2-yl, pyridin-2-ylene, pyridin-3-yl, pyridin-3-ylene, pyridin-4-ylene, and pyridin-4-ylene; thienyl or thienylene group includes thien-2-yl, thien-2-ylene, thien-3-ylene, and thien-3-ylene; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl, and pyrazol-5-yl.

[0064] The term "oxo" refers to a substituent in which a carbon atom, a nitrogen atom, or a sulfur atom is oxidized to form an oxy group (=O).

[0065] "Haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0066] In the chemical structure of the compound of the present invention, the bond Indicates that the configuration is not specified. or Indicates the absolute configuration, that is, if there are stereoisomers in the chemical structure, the bond Can be or or include both and Two configurations.

[0067] In the present invention, the compounds referred to also include isotopically labeled compounds, which are the same as those shown in Formula I, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of H, C, N, O, S, F, and Cl, such as 2 H.3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 32 P. 35 S. 18 F and 36 Cl. Compounds of the invention, prodrugs thereof, or pharmaceutically acceptable salts of said compounds or prodrugs containing the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the invention. Certain isotopically labeled compounds of the invention, for example, those incorporating radioactive isotopes (such as 3 H and 14 C) compounds can be used in drug and / or substrate tissue distribution assays. 3 H) and carbon 14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. 2 Substitution with hydrogen (H or D) may provide certain therapeutic advantages (e.g., increased in vivo half-life or reduced dosage requirements) derived from greater metabolic stability and may therefore be preferred in certain circumstances. The compounds of the present invention as claimed in the claims may be specifically limited to substitution with deuterium or tritium. Furthermore, the absence of separate listing of the term deuterium or tritium for hydrogen present in a substituent does not exclude deuterium or tritium, but rather may also include deuterium or tritium.

[0068] It will be appreciated by those skilled in the art that the compounds of formula (I) may exist in the form of various pharmaceutically acceptable salts. If these compounds have a basic center, they may form acid addition salts; if these compounds have an acidic center, they may form base addition salts; if these compounds contain both an acidic center (e.g., a carboxyl group) and a basic center (e.g., an amino group), they may also form internal salts.

[0069] The compounds of the present invention may exist in the form of solvates (e.g., hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0070] Depending on their molecular structure, the compounds of the present invention may be chiral and therefore may exist in various enantiomeric forms. Thus, these compounds may exist in racemic or optically active forms. The compounds of the present invention encompass isomers or mixtures thereof, racemates, in which each chiral carbon is in the R or S configuration. The compounds of the present invention or their intermediates can be separated into enantiomeric compounds by chemical or physical methods well known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers are prepared from the mixture by reaction with an optically active resolving agent. Examples of suitable resolving agents are optically active acids, such as R and S forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, appropriate N-protected amino acids (e.g., N-benzoylproline or N-phenylsulfonylproline) or various optically active camphorsulfonic acids. Chromatographic enantiomer resolution can also be advantageously performed with the aid of optically active resolving agents (e.g., dinitrobenzoylphenylglycine, cellulose triacetate or other carbohydrate derivatives or chirally derivatized methacrylate polymers immobilized on silica gel). Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, e.g., hexane / isopropanol / acetonitrile.

[0071] The corresponding stable isomers can be separated according to known methods, for example by extraction, filtration or column chromatography.

[0072] The term "patient" refers to any animal including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and most preferably humans.

[0073] The term "therapeutically effective amount" refers to that amount of an active compound or drug that will elicit the biological or medical response that a researcher, veterinarian, physician, or other clinician is seeking in a tissue, system, animal, individual, or human, and includes one or more of the following: (1) prevents disease, e.g., prevents a disease, disorder, or condition in an individual who is susceptible to the disease, disorder, or condition but who is not yet experiencing or developing the pathology or symptoms of the disease. (2) inhibits disease, e.g., inhibits the disease, disorder, or condition (i.e., prevents further development of the pathology and / or symptoms) in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder, or condition. (3) alleviates disease, e.g., alleviates the disease, disorder, or condition (i.e., reverses the pathology and / or symptoms) in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder, or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG1 shows the effect of compound G1a on the level of circulating GH in SD rats. DETAILED DESCRIPTION

[0075] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0076] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0077] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm).

[0078] Unless otherwise specified, all reactions of the present invention are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, with dry solvents and reaction temperatures in degrees Celsius.

[0079] Example 1

[0080] Step 1: 2,5-dioxopyrrolidin-1-yl 2-[(tert-butoxycarbonyl)amino]-2-methylpropanoate 1b

[0081] To a solution of 2-[(tert-butoxycarbonyl)amino]-2-methylpropanoic acid 1a (10 g, 49.203 mmol, 1 equiv) in tetrahydrofuran (100 mL) at room temperature under nitrogen was added N-hydroxysuccinimide (5.66 g, 49.203 mmol, 1 equiv) and N,N′-dicyclohexylcarbodiimide (15.23 g, 73.805 mmol, 1.5 equiv). The mixture was stirred overnight and the reaction was complete after liquid chromatography-mass spectrometry. The mixture was filtered, the filter cake washed with tetrahydrofuran (3 x 100 mL), and the filtrate concentrated under reduced pressure to yield compound 1b (11 g, 74.44%).

[0082] LC-MS: (ESI, m / z)=318.15[M+H2O] +

[0083] Step 2: 3-(Benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid 1c

[0084] Under nitrogen, O-benzyl-DL-serine (3.25 g, 16.649 mmol, 1 equiv) and triethylamine (3.37 g, 33.298 mmol, 2 equiv) were added to a solution of compound 1b (5 g, 16.649 mmol, 1 equiv) in tetrahydrofuran (50 mL) and water (25 mL) at room temperature. The reaction was stirred for 4 hours, and the reaction was complete by liquid chromatography-mass spectrometry. The reaction mixture was acidified to pH 5 with hydrochloric acid. The reaction mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic phases were backwashed with saturated brine (1 x 300 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography using the following conditions: a C18 column, mobile phase: water and acetonitrile, gradient from 0% to 50% over 20 minutes, and UV detection at 254 nm. Compound 1c (4 g, 63.15%) was obtained.

[0085] LC-MS: (ESI, m / z)=381.20[M+H] +

[0086] Step 3: tert-Butyl N-{1-[(1-{3a-benzyl-2-cyclopropyl-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl}-3-(benzyloxy)-1-oxopropan-2-yl)carbamoyl]-1-methylethyl}carbamate 1d

[0087] To a solution of 3a-benzyl-2-cyclopropyl-4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-one (300 mg, 1.114 mmol, 1 equiv) in dichloromethane (5 mL) was added compound 1c (635.60 mg, 1.671 mmol, 1.5 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (320.27 mg, 1.671 mmol, 1.5 equiv), N-hydroxy-7-azabenzotriazole (227.41 mg, 1.671 mmol, 1.5 equiv), and triethylamine (169.06 mg, 1.671 mmol, 1.5 equiv) in an ice bath under nitrogen. The mixture was removed from the ice bath, warmed to room temperature, and stirred for 1 hour. The reaction was complete as monitored by liquid chromatography-mass spectrometry. The reaction mixture was extracted with ethyl acetate (3 x 5 mL). The organic phases were combined, backwashed with saturated brine (1 x 20 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography using a C18 column, mobile phase: water and acetonitrile, gradient from 0% to 50% over 20 minutes, and UV detection at 254 nm. Compound 1d (200 mg, 28.42%) was obtained.

[0088] LC-MS: (ESI, m / z)=632.45[M+H] +

[0089] Step 4: N-(1-{3a-benzyl-2-cyclopropyl-3-oxo-4H, 6H, 7H-pyrazolo[4,3-c]pyridin-5-yl}-3-(benzyloxy)-1-oxopropan-2-yl)-2-amino-2-methylpropionamide G1

[0090] Under nitrogen, a 4 M solution of hydrogen chloride in 1,4-dioxane (2 mL) was added to a solution of compound 1d (200 mg, 0.317 mmol, 1 equiv) in 1,4-dioxane (2 mL) at room temperature and stirred for 1 hour. The desired product was found in the liquid phase. The reaction mixture was concentrated in vacuo. The resulting residue was purified by HPLC to yield compound G1 (100 mg, 59.42%). The conditions were as follows: Column: Kinetex 5μm EVO C18, 30 mm x 150 mm; Mobile phase A: Water (10 mmol / L NH4HCO3), Mobile phase B: Acetonitrile; Flow rate: 60 mL / min; Gradient: 20% B to 48% B over 8 min; Wavelength: 254 nm / 220 nm; Retention time: 7.63 min.

[0091] LC-MS: (ESI, m / z)=532.35[M+H] +

[0092] Step 5

[0093] Compound G1 (100 mg) was chirally resolved at room temperature to obtain four optically pure isomer compounds, namely compound G1a (18.00 mg), compound G1b (20.21 mg), compound G1c (7.74 mg) and compound G1d (7.95 mg).

[0094] Compound G1a:

[0095] LC-MS: (ESI, m / z)=532.00[M+H] +

[0096] 1H NMR (400MHz, DMSO-d6) δ8.42 (s, 1H), 7.33 (m, 4H), 7.18 (m, 4H), 7.02 (m, 2H), 5.08 (d, 1H), 4.88-4.60 (m, 1H), 4.57-4.36 (m, 3H), 3.71 (m, 1H), 3.63(m, 1H), 3.08-2.84(m, 3H), 2.84-2.66(m, 3H), 2.45(s, 1H), 1.22( s, 3H), 1.16 (s, 3H), 0.64 (m, 2H), 0.47-0.28 (m, 1H), 0.25-0.05 (m, 1H).

[0097] Retention time (RT) = 5.9 min. (Chromatographic column: CHIRAL Cellulose SB 4.6*100 mm, 3 μm; Mobile phase A: HEX (0.1% DEA), Mobile phase B: ETOH: DCM = 4:1; Flow rate: 1 mL / min; Wavelength: 254 / 220 nm)

[0098] Compound G1b:

[0099] LC-MS: (ESI, m / z)=532.00[M+H] +

[0100] 1 H NMR (400MHz, DMSO-d6) δ8.41 (s, 1H), 7.33 (m, 4H), 7.18 (m, 4H), 7.08-6.81 (m, 2H ), 5.08 (d, 1H), 4.77-4.59 (m, 1H), 4.58-4.38 (m, 3H), 3.71 (m, 1H), 3.63 (dd, J=9. 7, 6.1Hz, 1H), 3.08-2.85 (m, 3H), 2.84-2.65 (m, 3H), 2.45 (s, 1H), 2.23 (s, 2H), 1. 22(s, 3H), 1.16(s, 3H), 0.73-0.55(m, 2H), 0.45-0.35(m, 1H), 0.25-0.14(m, 1H).

[0101] Retention time (RT) = 4.6 min. (Chromatographic column: CHIRAL Cellulose SB 4.6*100 mm, 3 μm; Mobile phase A: HEX (0.1% DEA), Mobile phase B: ETOH: DCM = 4:1; Flow rate: 1 mL / min; Wavelength: 254 / 220 nm)

[0102] Compound G1c:

[0103] LC-MS: (ESI, m / z)=532.00[M+H] +

[0104] 1 H NMR (400MHz, DMSO-d6) δ8.33 (s, 1H), 7.40-7.23 (m, 5H), 7.14 (m, 3H), 7.02-6.70 (m, 2H), 5.16 (s, 1H), 4.86-4.38 (m, 4H), 3.79-3.58 (m, 2H) ), 3.03(m, 1H), 2.96-2.75(m, 2H), 2.74-2.65(m, 3H), 2.45(d, 1H), 1.19(d, 6H), 0.71-0.53(m, 2H), 0.35-0.24(m, 1H), 0.18-0.04(m, 1H).

[0105] Retention time (RT) = 2.9 min. (Chromatographic column: CHIRAL Cellulose SB 4.6*100 mm, 3 μm; Mobile phase A: HEX (0.1% DEA), Mobile phase B: ETOH:DCM = 4:1; Flow rate: 1 mL / min; Wavelength: 254 / 220 nm)

[0106] Compound G1d:

[0107] LC-MS: (ESI, m / z)=532.00[M+H] +

[0108] 1 H NMR (400MHz, DMSO-d6) δ8.32 (s, 1H), 7.38-7.27 (m, 5H), 7.14 (m, 3H), 7.0 0-6.70 (m, 2H), 5.15 (s, 1H), 4.72-4.41 (m, 4H), 3.69 (d, 2H), 3.05 (d, 1H), 2.90-2.76 (m, 2H), 2.71 (m, 3H), 2.43 (s, 1H), 1.58 (d, J=1.6Hz, 2H), 1.17 ( d, J=4.5Hz, 6H), 0.72-0.53(m, 2H), 0.38-0.24(m, 1H), 0.17-0.07(m, 1H).

[0109] Retention time (RT) = 5.3 min. (Chromatographic column: CHIRAL Cellulose SB 4.6*100 mm, 3 μm; Mobile phase A: HEX (0.1% DEA), Mobile phase B: ETOH:DCM = 4:1; Flow rate: 1 mL / min; Wavelength: 254 / 220 nm)

[0110] Example 2

[0111] Step 1 3a-Benzyl-2-(oxetan-3-yl)-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester 2b

[0112] To a solution of 2a (1 g, 3.036 mmol, 1 equiv) in N,N-dimethylformamide (10 mL) under nitrogen protection and ice-bath was added sodium hydride (0.15 g, 6.072 mmol, 2 equiv). The mixture was stirred at room temperature for 1 hour, followed by the addition of 3-iodooxetane (0.84 g, 4.554 mmol, 1.5 equiv). The temperature was raised to 60°C and the reaction was stirred for 3 hours. The reaction was complete after liquid chromatography-mass spectrometry. The reaction mixture was cooled to room temperature and quenched with water (20 mL). The aqueous phase was extracted with ethyl acetate (3×30 mL), and the resulting residue was concentrated under reduced pressure to afford compound 2b (480 mg, 41.02%).

[0113] LC-MS: (ESI, m / z)=386.15[M+H] +

[0114] Step 2 3a-Benzyl-2-(oxetan-3-yl)-4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-one 2c

[0115] To a solution of 2b (480 mg, 1.245 mmol, 1 equiv) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL). The mixture was stirred at room temperature for 2 hours. The reaction was complete by liquid chromatography-mass spectrometry. Saturated aqueous sodium bicarbonate (10 mL) was added to the reaction mixture, and the reaction mixture was extracted with dichloromethane (3 × 1 mL). The combined organic phases were backwashed with water (2 × 1 mL), dried over anhydrous sodium sulfate (10 g), and the resulting mixture was filtered. The filtrate was concentrated under reduced pressure to afford compound 2c (350 mg, 89.54%).

[0116] LC-MS: (ESI, m / z)=286.15[M+H] +

[0117] Step 3: Tert-butyl (1-(((2R)-1-(3-benzyl-2-(oxetan-3-yl)-3-oxo-2,3,3a,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)-3-(benzyloxy)-1-oxopropan-2-yl)amino)-2-methyl-1-oxopropan-2-yl)carbamate 2d

[0118] To a solution of 2c (100 mg, 0.154 mmol, 1 equiv), (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (400 mg, 1.051 mmol, 1.5 equiv), and N,N-diisopropylethylamine (272 mg, 2.103 mmol, 3 equiv) in dichloromethane (2 mL) was added 1-propylphosphonic anhydride (446 mg, 1.402 mmol, 2 equiv). The mixture was stirred at room temperature for 1 hour. The reaction was complete as monitored by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-25%) to afford compound 2d (150 mg, 28.74%).

[0119] LC-MS: (ESI, m / z)=648.45[M+H] +

[0120] Step 4: N-(1-([(2R)-1-[3a-benzyl-2-(oxetane-3-yl)-3-oxo-4H, 6H, 7H-pyrazolo[4, 3-c]pyridin-5-yl]-3-(benzyloxy)-1-oxopropan-2-yl]carbamoyl-1-methylethyl)carbamic acid tert-butyl ester G2

[0121] To a solution of 2d (100 mg, 0.154 mmol, 1 equiv) in dichloromethane (2 mL) was added trifluoroacetic acid (0.2 mL). The reaction was stirred at room temperature for 1 hour, and the reaction was complete by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in N,N-dimethylformamide (1 mL). The residue was filtered, and the filter cake was washed with N,N-dimethylformamide (2 mL). The filtrate was purified by HPLC using the following conditions: XBridge BEH OBD C18 column, 30 mm x 150 mm, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 25% B to 40% B over 10 min; wavelength: 254 nm / 220 nm; retention time: 9.74 min. Compound G2 (30.35 mg, 35.68%) was obtained.

[0122] Step 5

[0123] Compound G2 (30 mg) was chirally resolved at room temperature to obtain optically pure isomers. The two optically pure compounds were compound G2a (10.12 mg) and compound G2b (8.65 mg).

[0124] Compound G2a:

[0125] LC-MS: (ESI, m / z)=548.05[M+H] +

[0126] 1 H NMR (400MHz, DMSO-d6) δ8.43 (s, 1H), 7.44-6.87 (m, 10H), 5.11 (d, 2H), 4.79-4.61 (m, 5H), 4.4 8(m, 5H), 3.87(m, 1H), 3.77-3.69(m, 1H), 3.64(m, 1H), 3.01(m, 3H), 2.80(m, 2H), 1.20(d, 6H).

[0127] Retention time (RT) = 8.7 min. (Chromatographic column: CHIRAL Cellulose SB 4.6*100 mm, 3 μm; Mobile phase A: HEX (0.1% DEA), Mobile phase B: ETOH: DCM = 4:1; Flow rate: 1 mL / min; Wavelength: 254 / 220 nm)

[0128] Compound G2b

[0129] LC-MS: (ESI, m / z)=548.05[M+H] +

[0130] 1 H NMR (400MHz, DMSO-d6) δ8.32 (s, 1H), 7.41-7.25 (m, 5H), 7.19-7.06 (m, 3H), 6.79 (d, 2H), 5.17(s, 1H), 5.05(m, 1H), 4.75-4.55(m, 5H), 4.49(s, 1H), 4.38(t, 1H), 3.79(m 1H), 3.71(d2H), 3.08(m, 1H), 2.96-2.81(m, 2H), 2.77(d, 2H), 2.6l(s, 1H), 2.04(s, 2H), 1.18(d, 6H).

[0131] Retention time (RT) = 5.1 min. (Chromatographic column: CHIRAL Cellulose SB 4.6*100 mm, 3 μm; Mobile phase A: HEX (0.1% DEA), Mobile phase B: ETOH: DCM = 4:1; Flow rate: 1 mL / min; Wavelength: 254 / 220 nm)

[0132] Example 3

[0133] Step 1 8-(tert-butyl)-2-methyl-3-oxo-8-azabicyclo[3.2.1]octane-2,8-dicarboxylate 3b

[0134] Sodium hydride (3.5 g, 60%, 88.9 mmol, 2 equiv) was suspended in toluene (60 mL) and heated to 90°C. Under nitrogen, a solution of dimethyl carbonate (8 g, 88.9 mmol, 2 equiv) in toluene (20 mL) was added dropwise, followed by methanol (1 mL). Finally, a solution of 3a (10 g, 44.4 mmol, 1 equiv) in toluene (20 mL) was added dropwise. After the addition was complete, the mixture was allowed to react at 90°C for 3 h. The reaction solution was poured into ice water and extracted with ethyl acetate and methane (3 x 100 mL). The organic phases were combined, backwashed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The resulting residue was purified by column chromatography (ethyl acetate / petroleum ether = 0-20%) to afford compound 3b (10 g, 79.3%).

[0135] LC-MS: (ES, m / z)=228.05[M+H-56] +

[0136] Step 2 8-(tert-Butyl) 2-methyl 2-benzyl-3-oxo-8-azabicyclo[3.2.1]octane-2,8-dicarboxylate 3c

[0137] Under nitrogen, sodium hydride (2.8 g, 60%, 70.6 mmol, 2 equiv) was added to a solution of 3b (10 g, 35.3 mmol, 1 equiv) in N,N-dimethylformamide (70 mL) at 0°C. The mixture was stirred at room temperature for 1.5 h, then the temperature was lowered to 0°C and a solution of benzyl bromide (6.6 g, 38.8 mmol, 1.1 equiv) in N,N-dimethylformamide (10 mL) was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction mixture was poured into ice water and extracted with ethyl acetate and methane (3 x 100 mL). The organic phases were combined, backwashed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography (acetonitrile / water (0.05% ammonium bicarbonate) = 0-55%) to afford compound 3c (2.5 g, 19.1%).

[0138] LC-MS: (ES, m / z)=374.10[M+H] +

[0139] Step 3: 3a-Benzyl-3-oxo-2,3,3a,4,5,6,7,8-octahydro-4,7-epiaminocyclohepta[c]pyrazole-9-carboxylic acid tert-butyl ester 3d

[0140] 3c (1 g, 2.7 mmol, 1 equiv) and hydrazine hydrate (0.67 g, 13.5 mmol, 5 equiv) were dissolved in anhydrous ethanol (10 mL) and stirred at 85°C for 36 h. The reaction solution was concentrated under reduced pressure, toluene (10 mL) was added, and the mixture was stirred at 110°C for 36 h. The reaction solution was concentrated, and the resulting residue was purified by reverse phase column chromatography (acetonitrile / water (0.05% ammonium bicarbonate) = 0-50%) to afford compound 3d (650 mg, 68.4%).

[0141] LC-MS: (ES, m / z)=356.10[M+H] +

[0142] Step 4: 3a-Benzyl-2-methyl-3-oxo-2,3,3a,4,5,6,7,8-octahydro-4,7-epiaminocyclohepta[c]pyrazole-9-carboxylic acid tert-butyl ester 3e

[0143] Under nitrogen, sodium hydride (113 mg, 60%, 2.8 mmol, 2 equiv) was added to a solution of 3d (500 mg, 1.4 mmol, 1 equiv) in N,N-dimethylformamide (15 mL) at 0°C and stirred at room temperature for 1 h. Iodomethane (300 mL, 2.1 mmol, 1.5 equiv) was then added dropwise and stirred at room temperature for 2 h. The reaction solution was poured into ice water and extracted with ethyl acetate (3 x 30 mL). The organic phases were combined, backwashed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The resulting residue was purified by column chromatography (ethyl acetate / petroleum ether = 0-35%) to afford compound 3e (450 mg, 86.7%).

[0144] LC-MS(ES+): m / z=370.10[M+H] +

[0145] Step 5: 3a-Benzyl-2-methyl-3a,4,5,6,7,8-hexahydro-4,7-epiaminocyclohepta[c]pyrazol-3(2H)-one 3f

[0146] 3e (450 mg, 1.22 mmol) was dissolved in 1,4-dioxane hydrochloride solution and stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure, and the resulting residue was dissolved in water and adjusted to pH 8 with saturated sodium bicarbonate solution. The mixture was extracted with dichloromethane (3 x 30 mL). The organic phases were combined, backwashed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to afford compound 3f (300 mg, 91.4%).

[0147] LC-MS(ES+): m / z=270.10[M+H] +

[0148] Step 6: tert-Butyl (1-(((2R)-1-(3a-benzyl-2-methyl-3-oxo-2,3,3a,4,5,6,7,8-octahydro-4,7-epiaminocycloheptyl[c]pyrazol-9-yl)-3-(benzyloxy)-1-oxopropyl-2-yl)amino)-2-methyl-1-oxopropyl-2-yl)carbamate 3g

[0149] 3f (300 mg, 1.11 mmol, 1 equiv), (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (636 mg, 1.67 mmol, 1.5 equiv), and N,N-diisopropylethylamine (432 mg, 3.33 mmol, 3 equiv) were dissolved in dichloromethane (15 mL). 1-Propylphosphonic anhydride (1.42 g, 50% DMF solution, 2.22 mmol, 2 equiv) was added dropwise under ice-cooling. After the addition, the mixture was stirred at room temperature for 3 h. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reverse phase column chromatography (acetonitrile / water (0.05% ammonium bicarbonate) = 0-55%) to afford compound 3g (150 mg, 21.3%).

[0150] LC-MS(ES+): m / z=632.30[M+H] +

[0151] Step 7: 2-amino-N-((2R)-1-(3a-benzyl-2-methyl-3-oxo-2,3,3a,4,5,6,7,8-octahydro-4,7-epiaminocyclohepta[c]pyrazol-9-yl)-3-(benzyloxy)-1-oxopropan-2-yl)-2-methylpropanamide G3

[0152] 3g (150mg, 0.24mmol) was dissolved in dichloromethane (5mL), trifluoroacetic acid (0.5mL) was added, and the mixture was stirred at room temperature for 2h. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by HPLC using the following conditions: column: Kinetex 5m EVO C18, 30mm x 150mm; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60ml / min; gradient: 25% B to 40% B over 10min; wavelength: 254nm / 220nm; retention time: 9.25min. Compound G3 (38.6mg, 30.63%) was obtained.

[0153] Step 8

[0154] G3 (34 mg, 0.06 mmol) was subjected to chiral separation to obtain compound G3a and compound G3b.

[0155] G3a

[0156] LC-MS(ES+): m / z=532.45[M+H] +

[0157] 1H NMR (400MHz, DMSO-d6) δ8.37-8.3l (m, 1H), 7.36-6.83 (m, 10H), 4.96 (s, 2H), 4.78 -4.71 (m, 1H), 4.65-4.56 (m, 1H), 4.52-4.49 (m, 1H), 3.84-3.63 (m, 2H), 3.05-3.0 2(m, 1H), 2.92-2.84(m, 4H), 2.41-2.37(m, 1H), 2.35-2.28(m, 1H), 2.14-2.11(m, 2H), 1.93-1.68(m, 2H), 1.54-1.39(m, 1H), 1.25-1.03(m, 6H), 1.06-0.98(m, 1H).

[0158] Retention time (RT) = 4.547 min. (Chromatographic column: CHIRALPAK-IF 2*25 cm, 5 μm; mobile phase A: HEX (0.1% DEA), mobile phase B: ETOH:DCM = 1:1; flow rate: 20 mL / min; gradient: isocratic 50; wavelength: 254 / 220 nm; sample solvent: ETOH:DCM; injection volume: 1.0 mL; number of runs: 2)

[0159] G3b

[0160] LC-MS(ES+): m / z=532.45[M+H] +

[0161] 1H NMR(400MHz, DMSO-d6)δ 8.35 (s, 1H), 7.49-6.76 (m, 10H), 5.84 (d, J=4.6Hz, 1H), 4.97 (s, 1H), 4.83-4.78 (m, 1H), 4.62-4.44 (m, 2H), 3.74-3.55 (m, 2H), 3.46-3.42(m, 2H), 3.13(s, 3H), 2.61-2.56(m, 2H), 2.43-2.39(m, 2H), 1.93-1.63(m, 3H), 1.41-1.34(m, 1H), 1.30-1.04(m, 6H).

[0162] Retention time (RT) = 7.07 min. (Chromatographic column: CHIRALPAK-IF 2*25 cm, 5 μm; mobile phase A: HEX (0.1% DEA), mobile phase B: ETOH:DCM = 1:1; flow rate: 20 mL / min; gradient: isocratic 50; wavelength: 254 / 220 nm; sample solvent: ETOH:DCM; injection volume: 1.0 mL; number of runs: 2)

[0163] Example 4

[0164] Step 1: tert-Butyl 3a-benzyl-3-oxo-2,3,3a,4,5,6,7,8-octahydro-4,7-epiaminocyclohepta[c]pyrazole-9-carboxylate 4b

[0165] 3c (1 g, 2.7 mmol, 1 equiv) and hydrazine hydrate (0.4 g, 5.1 mmol, 3 equiv) were dissolved in anhydrous ethanol (10 mL) and reacted at 85°C for 36 hours. The reaction solution was concentrated under reduced pressure, and toluene (10 mL) was added, followed by reaction at 110°C for 36 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure and the resulting residue was purified by reverse-phase column chromatography (acetonitrile / water (0.05% ammonium bicarbonate) = 0-55%) to afford compound 4b (500 mg, 52.6%).

[0166] LC-MS: (ESI, m / z)=356.10[M+H] +

[0167] Step 2 3a-Benzyl-3a,4,5,6,7,8-hexahydro-4,7-epiaminocyclohepta[c]pyrazol-3(2H)-one 4c

[0168] 4b (500 mg, 1.4 mmol) was dissolved in a 4 M solution of hydrogen chloride in 1,4-dioxane (10 mL) and allowed to react at room temperature for 2 hours. After completion of the reaction, the reaction mixture was concentrated and the resulting residue was dissolved in water and adjusted to pH 8 with saturated sodium bicarbonate solution. The mixture was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, backwashed with saturated sodium chloride solution (50 mL), and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure to afford compound 4c (300 mg, crude product), which was used directly in the next step without further purification.

[0169] LC-MS: (ESI, m / z)=256.10[M+H] +

[0170] Step 3: Tert-Butyl (1-(((2R)-1-(3a-benzyl-3-oxo-2,3,3a,4,5,6,7,8-octahydro-4,7-epiaminocyclohepta[c]pyrazol-9-yl)-3-(benzyloxy)-1-oxopropan-2-yl)amino)-2-methyl-1-oxopropan-2-yl)carbamate 4d

[0171] 4d (100 mg, 0.4 mmol, 1 equiv), (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (224 mg, 0.6 mmol, 1.5 equiv), and N,N-diisopropylethylamine (152 mg, 1.2 mmol, 3 equiv) were dissolved in dichloromethane (10 mL), followed by the dropwise addition of 1-propylphosphonic anhydride (500 mg, 50% N,N-dimethylformamide solution, 0.8 mmol, 2 equiv). The reaction was allowed to react at room temperature for 3 hours. The reaction was monitored for completion by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reverse phase column chromatography (acetonitrile / water (0.05% ammonium bicarbonate) = 0-55%) to afford compound 4d (40 mg, 16.7%).

[0172] LC-MS: (ESI, m / z)=618.25[M+H] +

[0173] Step 4: 2-amino-N-((2R)-1-(3a-benzyl-3-oxo-2,3,3a,4,5,6,7,8-octahydro-4,7-epiaminocyclohepta[c]pyrazol-9-yl)-3-(benzyloxy)-1-oxopropan-2-yl)-2-methylpropanamide G4

[0174] 4d (40 mg, 0.06 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (0.5 mL) was added. The mixture was allowed to react at room temperature for 2 hours. After completion of the reaction, the reaction solution was concentrated and the resulting residue was purified by HPLC using the following conditions: column specifications: Kinetex 5m EVO C18, 30 mm x 150 mm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; elution gradient: 20% B to 50% B over 10 minutes; detection wavelength: UV 254 nm / 220 nm; retention time: 7.9 min. Compound G4 (7.2 mg, 21.5%) was obtained.

[0175] LC-MS(ESI+): m / z=518.10[M+H] +

[0176] 1H NMR(400MHz, DMSO-d6)δ 9.76 (s, 1H), 8.43-8.28 (m, 1H), 7.37-7.22 (m, 5H), 7.21-7.08 (m, 5H), 6.23-5.96 (m, 1H), 4.89 (d, 1H), 4.79 (s, 1H), 4.49 (d, 1H), 4. 08(s, 1H), 3.59(t, 1H), 3.58-3.39(m, 3H), 2.94-2.77(m, 1H), 2.05(s, 2H), 1.94-1.70(m, 2H), 1.55-1.38(m, 1H), 1.28-1.00(m, 6H).

[0177] Example 5

[0178] Step 1 5-(tert-butyl)7-methyl8-oxo-5-azaspiro[2.5]octane-5,7-dicarboxylate 5b

[0179] 5a (1 g, 4.4 mmol, 1 equiv) was dissolved in anhydrous tetrahydrofuran (15 mL) under nitrogen atmosphere. Sodium hydride (444 mg, 60%, 11 mmol, 2.5 equiv) was added under ice-cooling. After addition, the mixture was warmed to room temperature and stirred for 0.5 hours. Dimethyl carbonate (1 g, 11 mmol, 2.5 equiv) was slowly added dropwise under ice-cooling. After addition, the mixture was warmed to room temperature and stirred for 12 hours. Liquid chromatography-mass spectrometry was performed to monitor the reaction completion. The reaction solution was poured into water and extracted with ethyl acetate (3 × 30 mL). The product was backwashed with saturated sodium chloride solution (30 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography (ethyl acetate / petroleum ether = 0-15%) to afford compound 5b (1 g, 79.3%).

[0180] LC-MS: (ESI, m / z)=228.05[M+H-56] +

[0181] Step 2 5-(tert-Butyl)7-methyl7-benzyl-8-oxo-5-azaspiro[2.5]octane-5,7-dicarboxylate 5c

[0182] 5b (1 g, 3.5 mmol, 1 equiv) was dissolved in N,N-dimethylformamide (12 mL) and, under nitrogen, sodium hydride (0.28 g, 60%, 7 mmol, 2 equiv) was added under ice-cooling. The mixture was stirred at room temperature for 1 hour. A solution of benzyl bromide (0.6 g, 3.5 mmol, 1 equiv) in N,N-dimethylformamide (2 mL) was slowly added dropwise under ice-cooling. After the addition was complete, the mixture was stirred at room temperature for 4 hours. Liquid chromatography-mass spectrometry was performed to confirm the completion of the reaction. The reaction solution was poured into water, extracted with ethyl acetate (3 × 30 mL), backwashed with saturated sodium chloride solution (30 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (acetonitrile / water (0.1% ammonium bicarbonate) = 0-65%) to afford compound 5c (900 mg, 69.2%).

[0183] LC-MS: (ESI, m / z)=318.05[M+H-56] +

[0184] Step 3 3a′-Benzyl-2′-methyl-3′-oxo-2′, 3′, 3a′, 4′-tetrahydrospiro[cyclopropane-1, 7′-pyrazolo[4, 3-c]pyridine]-5′(6′H)-carboxylic acid tert-butyl ester 5d

[0185] To an aqueous solution (10 mL) of sodium hydroxide (193 mg, 4.8 mmol, 2 equiv) and methylhydrazine hydrochloride (299 mg, 3.6 mmol, 1.5 equiv) was slowly added dropwise a solution of 5c (900 mg, 2.4 mmol, 1 equiv) in methanol (5 mL) under ice-cooling. The mixture was stirred at 0°C for 2 hours, then warmed to room temperature and stirred for 3 hours. The reaction solution was concentrated, and the resulting residue was purified by reverse-phase column chromatography (acetonitrile / water (0.1% ammonium bicarbonate) = 0-60%) to afford compound 5d (80 mg, 9.0%).

[0186] LC-MS: (ESI, m / z)=370.10[M+H] +

[0187] Step 4: 3a′-benzyl-2′-methyl-3a′, 4′, 5′, 6′-tetrahydrospiro[cyclopropane-1, 7′-pyrazolo[4, 3-c]pyridine]-3′(2′H)-one 5f

[0188] 5d (80 mg, 0.22 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (0.5 mL) was added. The mixture was allowed to react at room temperature for 2 hours. After completion of the reaction, the reaction solution was concentrated and the resulting residue was dissolved in water. The pH was adjusted to 8 with saturated sodium bicarbonate solution. The product was extracted with dichloromethane (3 × 30 mL), backwashed with saturated sodium chloride solution (30 mL × 1), and dried over anhydrous sodium sulfate. The product was filtered, and the filtrate was concentrated under reduced pressure to afford compound 5f (50 mg, 85.7%).

[0189] LC-MS(ESI+): m / z=270.10[M+H] +

[0190] Step 5: 1-(((2R)-1-(3a′-benzyl-2′-methyl-3′-oxo-2′, 3′, 3a′, 4′-tetrahydrospiro[cyclopropane-1, 7′-pyrazolo[4, 3-c]pyridine]-5′(6′H)-yl)-3-(benzyloxy)-1-oxopropan-2-yl)amino)-2-methyl-1-oxopropan-2-yl)carbamic acid tert-butyl ester 5g

[0191] 5f (50 mg, 0.19 mmol, 1 equiv), (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (106 mg, 0.28 mmol, 1.5 equiv), and N,N-diisopropylethylamine (75 mg, 0.57 mmol, 3 equiv) were dissolved in dichloromethane (5 mL), followed by the addition of 1-propylphosphonic anhydride (240 mg, 50% solution in N,N-dimethylformamide, 0.38 mmol, 2 equiv), and stirring at room temperature for 4 hours. The reaction was complete after liquid chromatography-mass spectrometry (LC-MS / MS), and the reaction solution was concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography (acetonitrile / water (0.1% ammonium bicarbonate) = 0-62%) to afford compound 5g (40 mg, 34.2%).

[0192] LC-MS(ESI+): m / z=632.40[M+H] +

[0193] Step 6: 2-amino-N-((R)-1-((R)-3a′-benzyl-2′-methyl-3′-oxo-2′, 3′, 3a′, 4′-tetrahydrospiro[cyclopropane-1, 7′-pyrazolo[4, 3-c]pyridine]-5′(6′H)-yl)-3-(benzyloxy)-1-oxopropan-2-yl)-2-methylpropanamide G5

[0194] 5 g (40 mg, 0.06 mmol) of the compound was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.5 mL) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated, and the resulting residue was purified by HPLC using the following conditions: column specifications: Xselect CSH™ Prep C18 5 μm 19 mm x 150 mm OBD; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60 mL / min; elution gradient: 15% B to 32% B over 10 min; detection wavelength: UV 254 nm / 220 nm; retention time: 8.72 min. Compound G5 (8.83 mg, 26.2%) was obtained.

[0195] Step 7

[0196] G5 (8.83 mg) was chirally resolved at room temperature to obtain optically pure isomers, two optically pure compounds G5a (2.14 mg) and G5b (2.56 mg).

[0197] G5a

[0198] LC-MS(ESI+): m / z=532.10[M+H] +

[0199] 1 H NMR (400MHz, DMSO-d6) δ8.85-8.56 (m, 1H), 7.96 (s, 3H), 7.36 (t, 2H), 7.30 (d, 2H ), 7.19(d, 2H), 7.05-6.85(m, 2H), 5.16-5.09(m, 1H), 4.69(d, 1H), 4.52(d, 2H), 4.31-4.07(m, 1H), 3.92-3.75(m, 1H), 3.69-3.63(m, 1H), 3.45(t, 1H), 3.03(s, 3 H), 2.97(s, 2H), 2.86(d, 1H), 1.47(s, 6H), 1.27-1.03(m, 2H), 0.74-0.56(m, 2H).

[0200] Retention time (RT) = 3.45 min. (Chromatographic column: CHIRALPAK-IF 2*25 cm, 5 μm; mobile phase A: HEX (0.1% DEA), mobile phase B: ETOH:DCM = 1:1; flow rate: 20 mL / min; gradient: isocratic 50; wavelength: 254 / 220 nm; sample solvent: ETOH:DCM; injection volume: 1.0 mL; number of runs: 2)

[0201] G5b

[0202] LC-MS(ESI+): m / z=532.10[M+H] +

[0203] 1 H NMR (400MHz, DMSO-d6) δ8.63 (s, 1H), 7.98 (s, 2H), 7.36-7.30 (m, 5H), 7.22-7.1 3(m, 2H), 7.03-6.91(m, 2H), 5.21-5.08(m, 1H), 4.74-4.61(m, 1H), 4.61-4.50(m , 2H), 3.85-3.77(m, 1H), 3.67-3.60(m, 1H), 3.52-3.43(m, 1H), 3.24-3.14(m, 1 H), 3.03-2.76(m, 5H), 1.53-1.34(m, 6H), 1.14-0.98(m, 2H), 0.73-0.55(m, 2H).

[0204] Retention time (RT) = 4.65 min. (Chromatographic column: CHIRALPAK-IF 2*25 cm, 5 μm; mobile phase A: HEX (0.1% DEA), mobile phase B: ETOH:DCM = 1:1; flow rate: 20 mL / min; gradient: isocratic 50; wavelength: 254 / 220 nm; sample solvent: ETOH:DCM; injection volume: 1.0 mL; number of runs: 2)

[0205] Example 6

[0206] Step 1: tert-Butyl 3-hydroxy-2-methyl-4H,5H,7H-pyrazolo[3,4-c]pyridine-6-carboxylate 6b

[0207] Under nitrogen, a solution of 6a (1 g, 3.887 mmol, 1 equiv), methylhydrazine (0.21 g, 4.664 mmol, 1.2 equiv), and N,N-diisopropylethylamine (1.51 g, 11.661 mmol, 3.00 equiv) in ethanol (20 mL) was stirred at 80°C for 12 hours. The reaction was monitored for completion by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography using the following conditions: column size (40 g), mobile phase: acetonitrile:water, gradient: 0% to 100% over 30 minutes, UV detection at 254 nm. The resulting residue was concentrated under reduced pressure to yield compound 6b (170 mg, 16.84%).

[0208] LC-MS: (ESI, m / z)=254.30[M+H] +

[0209] Step 2 tert-Butyl 2-methyl-3-(trifluoromethanesulfonyloxy)-4H,5H,7H-pyrazolo[3,4-c]pyridine-6-carboxylate 6c

[0210] To a solution of 6b (150 mg, 0.592 mmol, 1 equiv) in tetrahydrofuran (20 mL) under nitrogen, sodium hydride (17 mg, 0.710 mmol, 1.2 equiv) was slowly added under ice-cooling conditions. The mixture was stirred for half an hour, followed by the addition of N-phenylbis(trifluoromethanesulfonyl)imide (240 mg, 0.671 mmol, 1 equiv). The mixture was allowed to warm to room temperature and stirred for 5 hours. The reaction was monitored for completion by liquid chromatography-mass spectrometry. The reaction mixture was quenched with water (20 mL) at room temperature, and the aqueous phase was extracted with dichloromethane (3 × 10 mL), dried over anhydrous sodium sulfate (5 g), and filtered. The resulting residue was concentrated under reduced pressure, dissolved in N,N-dimethylformamide (3 mL), and purified by reverse-phase column chromatography using the following conditions: column size (20 g), mobile phase: acetonitrile:water, gradient: 0% to 100% over 25 minutes, UV 254 nm detection. The obtained residue was concentrated under reduced pressure to give compound 6c (200 mg, 47.44%).

[0211] LC-MS: (ESI, m / z)=386.25[M+H] +

[0212] Step 3 2-Methyl-3-phenyl-4H,5H,7H-pyrazolo[3,4-c]pyridine-6-carboxylic acid tert-butyl ester 6d

[0213] Under nitrogen, a solution of 6c (220 mg, 0.571 mmol, 1 equiv), tetrakis(triphenylphosphine)palladium (66 mg, 0.057 mmol, 0.1 equiv), phenylboronic acid (84 mg, 0.685 mmol, 1.2 equiv), and sodium carbonate (182 mg, 1.713 mmol, 3 equiv) in water (8.8 mL) and 1,4-dioxane (35 mL) was stirred at 65°C for 18 hours. The reaction was monitored by liquid chromatography-mass spectrometry (LC-MS / MS) to determine completion. The resulting residue was concentrated under reduced pressure, dissolved in N,N-dimethylformamide (3 mL), and filtered. The resulting residue was purified by reverse-phase column chromatography using the following conditions: column size (12 g), mobile phase: acetonitrile:water, gradient: 0% to 50% over 20 minutes, UV detection at 254 nm. The resulting residue was concentrated under reduced pressure to yield compound 6d (105 mg, 54.64%).

[0214] LC-MS: (ESI, m / z)=314.30[M+H] +

[0215] Step 4: 2-Methyl-3-phenyl-4H,5H,6H,7H-pyrazolo[3,4-c]pyridine 6e

[0216] To a solution of 6d (95 mg, 0.303 mmol, 1 equiv) in dichloromethane (3 mL) was added trifluoroacetic acid (1.5 mL). The mixture was stirred at room temperature for 2 hours. The reaction was complete by liquid chromatography-mass spectrometry. The reaction mixture was diluted with water (6 mL) and extracted with dichloromethane (3 × 3 mL). The combined organic phases were dried over sodium sulfate (3 g) and filtered. The filtrate was concentrated under reduced pressure to afford compound 6e (30 mg, crude product).

[0217] LC-MS: (ESI, m / z)=214.25[M+H] +

[0218] Step 5: tert-Butyl N-(1-([(2R)-3-(benzyloxy)-1-(2-methyl-3-phenyl-4H,5H,7H-pyrazolo[3,4-c]pyridin-6-yl-1-oxopropan-2-yl]carbamoyl-1-methylethyl)carbamate 6f

[0219] To a solution of 6e (30 mg, crude), N,N-diisopropylethylamine (55 mg, 0.423 mmol, 3 equiv), and (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (80 mg, 0.211 mmol, 1.5 equiv) in dichloromethane (3 mL) was added 1-propylphosphonic anhydride (90 mg, 0.282 mmol, 2 equiv) under nitrogen atmosphere. The reaction was stirred at room temperature for 3 hours. The reaction was complete after LC / MS analysis. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography using the following conditions: column size (20 g), mobile phase: acetonitrile:water, gradient: 10% to 80% over 20 minutes, with the product peak appearing at 56%. UV detection at 254 nm. The product was concentrated under reduced pressure to afford compound 6f (25 mg, 23.96%).

[0220] LC-MS: (ESI, m / z)=576.50[M+H] +

[0221] Step 6 (R)-2-amino-N-(3-(benzyloxy)-1-(2-methyl-3-phenyl-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)-1-oxopropan-2-yl)-2-methylpropanamide G6

[0222] To a solution of 6f (25 mg, 0.038 mmol, 1 equiv) in dichloromethane (2 mL) was added trifluoroacetic acid (0.2 mL) and stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, dissolved in N,N-dimethylformamide (2 mL), and purified by HPLC using the following conditions: column specifications: Kinetex 5m EVO C18 column, 30 x 150 mm, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 20% B to 55% B over 10 minutes; wavelength: 254 nm / 220 nm; retention time: 7.53 min, to afford compound G6 (5.63 mg, 31.81%).

[0223] LC-MS: (ESI, m / z)=476.05[M+H] +

[0224] 1 H NMR (400MHz, DMSO-d6) δ8.35 (s, 1H), 7.61 (m, 2H), 7.40 (t, 2H), 7.32-7.15 (m, 6H), 5.08 (s, 1H), 4.84-4 .59(m, 2H), 4.47(m, 2H), 3.75(m, 5H), 3.66-3.56(m, 2H), 2.68(s, 2H), 2.07(s, 2H), 1.24-1.11(m, 6H).

[0225] Example 7

[0226] First step 3a-Benzyl-2-(difluoromethyl)-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester 7b

[0227] Under nitrogen, diethyl bromodifluoromethylphosphonate (364.76 mg, 1.367 mmol, 1.5 equiv) and potassium hydroxide (510.97 mg, 9.110 mmol, 10 equiv) were added to a solution of 2a (300 mg, 0.911 mmol, 1 equiv) in acetonitrile (5 mL) at room temperature. The reaction was stirred at room temperature overnight. The desired product was found in the liquid phase. The reaction mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic phases were backwashed with saturated brine (1 x 30 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography using the following conditions: column specifications: C18, mobile phase: water and acetonitrile, gradient: 10% to 100% over 20 minutes, UV 254 nm detector. Compound 7b (50 mg, 14.47%) was obtained.

[0228] LC-MS: (ESI, m / z)=380.30[M+H] +

[0229] Step 2 3a-Benzyl-2-(difluoromethyl)-4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-one 7c

[0230] Under nitrogen, a 4M solution of hydrogen chloride in 1,4-dioxane (2 mL) was added to a solution of 7b (40 mg, 0.105 mmol, 1 equiv) in 1,4-dioxane (2 mL) at room temperature. The reaction was stirred at room temperature for 0.5 hours, and the desired product was observed in the liquid phase. The reaction mixture was concentrated under reduced pressure to afford compound 7c (34 mg, crude product), which was used directly in the next step.

[0231] LC-MS: (ESI, m / z)=280.20[M+H] +

[0232] Step 3: tert-Butyl N-(1-{[(2R)-1-[3a-benzyl-2-(difluoromethyl)-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl]-3-(benzyloxy)-1-oxopropan-2-yl]carbamoyl}-1-methylethyl)carbamate 7d

[0233] To a solution of 7c (34 mg, 0.122 mmol, 1 equiv) in dichloromethane (2 mL) under nitrogen at 0°C were added (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (69.47 mg, 0.183 mmol, 1.5 equiv), 1-hydroxybenzotriazole (24.67 mg, 0.183 mmol, 1.5 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (35.01 mg, 0.183 mmol, 1.5 equiv), and triethylamine (18.48 mg, 0.183 mmol, 1.5 equiv). The mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction was complete after liquid chromatography-mass spectrometry. The reaction mixture was extracted with dichloromethane (3×5 mL). The organic phases were combined, backwashed with saturated brine (1×20 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-50%) to obtain compound 7d (20 mg, 25.60%).

[0234] LC-MS: (ESI, m / z)=642.10[M+H] +

[0235] Step 4: 2-amino-N-((2R)-1-(3a-benzyl-2-(difluoromethyl)-3-oxo-2,3,3a,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)-3-(benzyloxy)-1-oxopropan-2-yl)-2-methylpropanamide G7

[0236] Under nitrogen, a 4M solution of hydrogen chloride in 1,4-dioxane (2 mL) was added to a solution of 7d (20 mg, 0.031 mmol, 1 equiv) in 1,4-dioxane (1 mL) at room temperature and stirred for 1 hour. The desired product was found in the liquid phase. The reaction mixture was concentrated in vacuo. The resulting residue was purified by HPLC using the following conditions: XBridge BEH Shield RP18 5μm, 30mm x 150mm column; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 35% B to 60% B over 10 minutes; wavelength: 254 nm / 220 nm; retention time: 8.74 minutes. Compound G7 (2 mg, 9.85%) was obtained.

[0237] LC-MS: (ESI, m / z)=542.10[M+H] +

[0238] 1 H NMR (400MHz, DMSO-d6) δ8.30 (s, 1H), δ7.46-7.22 (m, 6H), 7.22-6.98 (m, 5H), 5.49 (m, 2H), 4.9 8(s, 1H), 4.40(m, 2H), 3.87(m, 2H), 3.60(s, 4H), 2.65(s, 2H), 1.93(s, 2H), 1.24-0.95(m, 6H).

[0239] Example 8

[0240] Step 1: 3a-Benzyl-2-(cyclopropylmethyl)-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester 8b

[0241] Under nitrogen, sodium hydride (72.85 mg, 3.036 mmol, 2 equiv) was added to a solution of 2a (500 mg, 1.518 mmol, 1 equiv) in tetrahydrofuran (5 mL) at room temperature. After stirring for 0.5 h, (iodomethyl)cyclopropane (331.52 mg, 1.822 mmol, 1.2 equiv) was added at room temperature. The reaction was stirred for 2 h, and the reaction was complete after liquid chromatography-mass spectrometry. The reaction mixture was quenched with saturated aqueous ammonium chloride at 0°C. The reaction mixture was extracted with ethyl acetate (3 x 5 mL). The combined organic phases were backwashed with saturated brine (1 x 20 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-20%) to afford compound 8b (100 mg, 17.18%).

[0242] LC-MS: (ESI, m / z)=384.20[M+H] +

[0243] Step 2 3a-Benzyl-2-(cyclopropylmethyl)-4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-one 8c

[0244] Under nitrogen, a 4M solution of hydrogen chloride in 1,4-dioxane (2 mL) was added to a solution of 8b (200 mg, 0.522 mmol, 1 equiv) in 1,4-dioxane (5 mL) at room temperature. The mixture was stirred for 1 hour, and the reaction was complete by liquid chromatography-mass spectrometry. The resulting residue was concentrated in vacuo to afford crude compound 8c (150 mg, 95.50%).

[0245] LC-MS: (ESI, m / z)=284.10[M+H] +

[0246] Step 3: tert-Butyl N-(1-{[(2R)-1-[3a-benzyl-2-(cyclopropylmethyl)-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl]-3-(benzyloxy)-1-oxopropan-2-yl]carbamoyl}-1-methylethyl)carbamate 8d

[0247] To a solution of 8c (150 mg, 0.529 mmol, 1 equiv) in dichloromethane (2 mL) under nitrogen atmosphere were added (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (302.07 mg, 0.794 mmol, 1.5 equiv), 1-propylphosphonic anhydride (842.12 mg, 2.645 mmol, 5 equiv), and N,N-diisopropylethylamine (342.07 mg, 2.645 mmol, 5 equiv) at room temperature. The reaction mixture was stirred overnight and the reaction was complete after liquid chromatography-mass spectrometry. The reaction mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic phases were backwashed with saturated brine (1 x 30 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to yield crude compound 8d (140 mg, 40.95%).

[0248] LC-MS: (ESI, m / z)=646.35[M+H] +

[0249] Step 4: N-[(2R)-1-[3a-benzyl-2-(cyclopropylmethyl)-3-oxo-4H, 6H, 7H-pyrazolo[4, 3-c]pyridin-5-yl]-3-(benzyloxy)-1-oxopropan-2-yl]-2-amino-2-methylpropionamide G8

[0250] Under nitrogen, a 4M solution of hydrogen chloride in 1,4-dioxane (6 mL) was added to a solution of compound 8d (400 mg, 0.619 mmol, 1 equiv) in 1,4-dioxane (6 mL) at room temperature. The mixture was stirred for 1 hour, and the reaction was monitored for completion by HPLC-MS. The resulting residue was concentrated under reduced pressure. The crude product was purified by HPLC using the following conditions: column: YMC Triart C18 ExRs 5μm, 30mm x 150mm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 35% B to 61% B over 8 minutes; wavelength: 254 nm / 221 nm; retention time 1: 7.47 min) to obtain compound G8 (300 mg, 88.76%).

[0251] LC-MS: (ESI, m / z)=546.30[M+H] +

[0252] Step 5

[0253] Compound G8 (200 mg, 0.367 mmol, 1 equiv) was subjected to chiral separation at room temperature to obtain compound G8a (29.65 mg, 14.60%) and compound G8b (130.66 mg, 64.68%).

[0254] G8a

[0255] LC-MS: (ESI, m / z)=546.05[M+H] +

[0256] 1 H NMR (400MHz, DMSO-d6) δ8.54 (s, 1H), 7.42-7.02 (m, 10H), 5.09 (s, 1H), 4.69 (d, J=12.6H z, 1H), 4.60-4.41 (m, 3H), 3.68 (m, 2H), 3.41-2.82 (t, J=6.4Hz, 12H), 2.63 (s, 1H), 1.36 1.09 (m, 6H), 0.37-0.03 (m, 2H).

[0257] Chromatographic column: CHIRAL ART Cellulose-SC 2*25cm, 5μm; mobile phase A: n-hexane (0.1% diethylamine), mobile phase B: methanol: dichloromethane = 1:1; flow rate: 20mL / min; gradient: isocratic 30; wavelength: 254nm / 220nm; retention time: 5.68min; number of runs: 5

[0258] G8b

[0259] LC-MS: (ESI, m / z)=546.05[M+H] +

[0260] 1H NMR (400MHz, DMSO-d6) δ8.34 (s, 1H), 7.46-7.24 (m, 5H), 7.24-7.07 (m, 3H), 7.07-6.77 (m, 2H), 5.1 7 (t, J=6.2Hz, 1H), 4.95-4.18 (m, 4H), 3.83-3.52 (m, 2H), 3.29 (d, J=7.0Hz, 1H), 3.20-3.12 (m, 1H) , 3.11-3.04 (m, 1H), 3.04-2.80 (m, 2H), 2.66 (dt, J=16.5, 12.8Hz, 3H), 2.10 (s, 2H), 1.18 (d, J=4.4 Hz, 6H), 0.62 (pd, J=7.5, 3.4Hz, 1H), 0.30-0.12 (m, 2H), 0.08-0.01 (m, 1H), -0.06 (d, J=4.9Hz, 1H).

[0261] Chromatographic column: CHIRAL ART Cellulose-SC 2*25cm, 5μm; mobile phase A: n-hexane (0.1% diethylamine), mobile phase B: methanol: dichloromethane = 1:1; flow rate: 20mL / min; gradient: isocratic 30; wavelength: 254nm / 220nm; retention time: 6.77min; number of runs: 5

[0262] Example 9

[0263] Step 1: 3a-Benzyl-2-tert-butyl-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester 9b

[0264] Under nitrogen, tert-butylhydrazine hydrochloride (538.03 mg, 4.317 mmol, 1 equiv) and N,N-diisopropylethylamine (744.06 mg, 5.757 mmol, 2.00 equiv) were added to a solution of 9a (1 g, 2.878 mmol, 1 equiv) in ethanol (10 mL) at room temperature. The mixture was stirred at 80°C overnight. Liquid chromatography-mass spectrometry (LC-MS) indicated that the starting material had reacted completely and converted entirely to the intermediate. The mixture was then concentrated under reduced pressure to remove ethanol, and toluene (10 mL) was added. The mixture was stirred at 120°C overnight. LC-MS monitoring indicated that the reaction was complete. The reaction mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic phases were backwashed with saturated brine (1 x 30 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-10%) to yield compound 9b (800 mg, 72.09%).

[0265] LC-MS: (ESI, m / z)=386.20[M+H] +

[0266] Step 2 3a-Benzyl-2-tert-butyl-4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-one 9c

[0267] Under nitrogen, a 4M solution of hydrogen chloride in 1,4-dioxane (5 mL) was added to a solution of 9b (400 mg, 1.038 mmol, 1 equiv) in 1,4-dioxane (5 mL) at room temperature. The mixture was stirred for 1 hour, and the reaction was complete by liquid chromatography-mass spectrometry. The resulting residue was concentrated in vacuo to afford crude compound 9c (300 mg, 101.31%).

[0268] LC-MS: (ESI, m / z)=286.10[M+H] +

[0269] Step 3: tert-Butyl N-(1-{[(2R)-1-{3a-benzyl-2-tert-butyl-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl}-3-(benzyloxy)-1-oxopropan-2-yl]carbamoyl}-1-methylethyl)carbamate 9d

[0270] To a solution of 9c (300 mg, 1.051 mmol, 1 equiv) in dichloromethane (5 mL) under nitrogen atmosphere were added (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (599.87 mg, 1.576 mmol, 1.5 equiv), 1-propylphosphonic anhydride (1672.35 mg, 5.255 mmol, 5 equiv), and N,N-diisopropylethylamine (679.32 mg, 5.255 mmol, 5 equiv) at room temperature. The reaction mixture was stirred overnight and the reaction was complete after liquid chromatography-mass spectrometry. The reaction mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic phases were backwashed with saturated brine (1 x 30 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to yield crude compound 9d (300 mg, 44.05%).

[0271] LC-MS: (ESI, m / z)=648.40[M+H] +

[0272] Step 4: N-[(2R)-1-{3a-benzyl-2-tert-butyl-3-oxo-4H, 6H, 7H-pyrazolo[4, 3-c]pyridin-5-yl}-3-(benzyloxy)-1-oxopropan-2-yl]-2-amino-2-methylpropionamide G9

[0273] Under nitrogen, a 4M solution of hydrogen chloride in 1,4-dioxane (5 mL) was added to a solution of 9d (300 mg, 0.463 mmol, 1 equiv) in 1,4-dioxane (5 mL) at room temperature. The mixture was stirred for 1 hour, and the reaction was monitored for completion by HPLC-MS. The resulting residue was concentrated under reduced pressure. The crude product was purified by HPLC using the following conditions (column: Sunfire C18 5 μm, 30 mm x 150 mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 14% B to 37% B over 10 minutes; wavelength: 254 nm / 221 nm; retention time: 9.05 min). Compound G9 (200 mg, 78.85%) was obtained.

[0274] LC-MS: (ESI, m / z)=548.05[M+H] +

[0275] Step 5

[0276] G9 (200 mg, 0.365 mmol, 1 equiv) was subjected to chiral separation at room temperature to obtain compound G9a (54.96 mg, 27.48%) and compound G9b (135.25 mg, 67.63%).

[0277] G9a

[0278] LC-MS: (ESI, m / z)=548.05[M+H] +

[0279] 1 H NMR (400MHz, DMSO-d6) δ8.64-8.18(m, 1H), 7.43-7.23(m, 5H), 7.23-7.12(m, 3H), 7.11-6.81(m, 2H), 5.17-5.02(m, 1H) , 4.95-4.09 (m, 4H), 3.80-3.44 (m, 2H), 3.17-2.75 (m, 4H), 2.61 (d, J = 15.3Hz, 1H), 2.46 (d, J = 6.9Hz, 1H), 1.20 (m, 15H).

[0280] Chromatographic column: CHIRAL ART Cellulose-SC 2*25cm, 5μm; mobile phase A: n-hexane (0.1% diethylamine), mobile phase B: methanol: dichloromethane = 1:1; flow rate: 20mL / min; gradient: isocratic 30; wavelength: 254nm / 220nm; retention time: 4.94min; sample solvent: methanol; injection volume: 2.0mL; number of runs: 3

[0281] G9b

[0282] LC-MS: (ESI, m / z)=548.05[M+H] +

[0283] 1 H NMR (400MHz, DMSO-d6) δ8.61-8.09 (m, 1H), 7.58-7.24 (m, 5H), 7.24-7.07 (m, J=5.1, 3.7Hz, 3H), 7.07-6.70 (m, 2H), 5.16 (t, J=6.4Hz, 1H), 4.9 7-4.31(m, 4H), 3.70(d, J=6.2Hz, 2H), 3.59(s, 1H), 3.14-2.75(m, 4H), 2.74-2.53(m, 2H), 2.49-2.29(m, 1H), 1.26-1.14(m, 6H), 1.13(s, 9H).

[0284] Chromatographic column: CHIRAL ART Cellulose-SC 2*25cm, 5μm; mobile phase A: n-hexane (0.1% diethylamine), mobile phase B: methanol: dichloromethane = 1:1; flow rate: 20mL / min; gradient: isocratic 30; wavelength: 254nm / 220nm; retention time: 6.37min; sample solvent: methanol; injection volume: 2.0mL; number of runs: 3

[0285] Example 10

[0286] Step 1: 3a-Benzyl-2-cyclopentyl-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester 10b

[0287] Under nitrogen, NaH (43.71 mg, 1.822 mmol, 2 equiv) was added to a solution of 2a (300 mg, 0.911 mmol, 1 equiv) in tetrahydrofuran at 0°C. The reaction was stirred at 0°C for 30 minutes. Iodocyclopentane (357.07 mg, 1.822 mmol, 2 equiv) was then added, and stirring was continued for 20 hours. Liquid chromatography-mass spectrometry indicated the reaction was complete. The reaction mixture was quenched with ice water at room temperature. The reaction mixture was extracted with ethyl acetate (3 x 5 mL). The combined organic phases were backwashed with saturated sodium chloride solution (1 x 5 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to yield compound 10b (240 mg, 66.29%).

[0288] LC-MS: (ESI, m / z)=398.55[M+H]+

[0289] Step 2: tert-Butyl N-(1-([(2R)-1-(3a-benzyl-2-cyclopentyl-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl-3-(benzyloxy)-1-oxopropan-2-yl)carbamoyl-1-methylethyl)carbamate 10d

[0290] To a solution of 10b (240 mg, 0.604 mmol, 1 equiv) in dichloromethane was added a solution of hydrochloric acid in 1,4-dioxane (5 mL, 164.564 mmol, 272.57 equiv) under nitrogen at room temperature, and the mixture was stirred at room temperature for 1 hour to afford compound 10c. To a solution of 10c (200.75 mg, 0.676 mmol, 1.2 equiv) and (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropylamino}propanoic acid (214 mg, 0.563 mmol, 1.00 equiv) in dichloromethane were added T3P (357.96 mg, 1.126 mmol, 2 equiv) and N,N-diisopropylethylamine (218.11 mg, 1.689 mmol, 3 equiv) in portionwise order under nitrogen at room temperature. After the addition was complete, the system was stirred at room temperature for 1 hour. The reaction was complete as determined by liquid chromatography-mass spectrometry. The reaction mixture was quenched with water at room temperature. The reaction mixture was extracted with ethyl acetate (3 × 8 mL). The combined organic phases were backwashed with saturated sodium chloride solution (1 × 8 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to afford compound 10d (220 mg, 59.27%).

[0291] LC-MS: (ESI, m / z)=660.65[M+H] +

[0292] Step 3: 2-amino-N-((2R)-1-(3a-benzyl-2-cyclopentyl-3-oxo-2,3,3a,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)-3-(benzyloxy)-1-oxopropan-2-yl)-2-methylpropanamide G10

[0293] Under nitrogen, zinc bromide (136.48 mg, 606.061 mmol, 5 equiv) was added to a dichloromethane solution of 10d (80 mg, 0.121 mmol, 1 equiv) at room temperature. The reaction was stirred at room temperature for 2 hours. The reaction was complete as monitored by HPLC. The crude product was purified by HPLC to yield compound G10 (46 mg, 67.81%).

[0294] LC-MS: (ESI, m / z)=560.30[M+H] +

[0295] Step 4

[0296] G10 (40 mg, 0.071 mml, 1 equiv) was subjected to chiral resolution to obtain compound G10a (8 mg, 19.94%) and compound G10b (3.9 mg, 9.63%).

[0297] G10a

[0298] LC-MS: (ESI, m / z)=560.30[M+H] +

[0299] 1 H NMR (400MHz, DMSO-d6) δ8.33 (s, 1H), 7.42-7.23 (m, 5H), 7.14 (dq, J=13.0, 5.9Hz, 3H), 7.03-6.76 (m, 2H), 5.17 (t, J=6.4Hz, 1H), 4.88-4.67 (m, 1H), 4.66-4.41 (m, 3H), 4.25 (p, J=7.1Hz, 1H), 3.76- 3.54 (m, 2H), 3.07 (ddt, J=20.2, 11.0, 6.8Hz, 1H), 2.86 (q, J=13.3Hz, 2H), 2.77-2.55 (m, 3H), 2.3 9(s, 2H), 1.77-1.53(m, 3H), 1.49-1.27(m, 4H), 1.19(d, J=3.7Hz, 6H), 0.80(q, J=6.6, 5.9Hz, 1H).

[0300] Chromatographic column: CHIRALART Cellulose-SB 3*25cm, 5μm; mobile phase A: n-hexane (0.1% diethylamine), mobile phase B: methanol: dichloromethane = 1:1; flow rate: 40mL / min; gradient: isocratic 30; wavelength: 254nm / 220nm; retention time: 6.8min; sample solvent: methanol; number of runs: 1

[0301] G10b

[0302] LC-MS: (ESI, m / z)=560.30[M+H] +

[0303] 1H NMR (400MHz, DMSO-d6) δ8.39 (d, J=37.4Hz, 1H), 7.33 (dq, J=13.8, 7.1Hz, 5H), 7.18 (d, J=6.6Hz, 3H), 7.09-6.88 (m, 2H), 5.10 (s, 1H), 4.69 (d, J=12.6Hz, 1H), 4.51 (d, J=3.3Hz, 2H), 4.48-4.39 (m, 1H), 4.36-4.18 (m, 1H), 3.90 -3.68 (m, 1H), 3.63 (dd, J=9.8, 6.2Hz, 1H), 3.26 (s, 1H), 3.05-2.92 (m, 2H), 2.91-2.71 (m, 2H), 2.69-2.60 (m, 1H ), 2.52 (d, J = 2.2Hz, 2H), 1.80-1.54 (m, 3H), 1.41 (dd, J = 10.4, 6.9Hz, 4H), 1.21 (d, J = 22.1Hz, 6H), 0.87 (s, 1H).

[0304] Chromatographic column: CHIRAL ART Cellulose-SB 3*25cm, 5μm; mobile phase A: n-hexane (0.1% diethylamine), mobile phase B: methanol: dichloromethane = 1:1; flow rate: 40mL / min; gradient: isocratic 30; wavelength: 254nm / 220nm; retention time: 7.25min; sample solvent: methanol; number of runs: 1

[0305] Example 11

[0306] Step 1: 3a-Benzyl-2-cyclohexyl-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester 11b

[0307] Under nitrogen, N,N-diisopropylethylamine (781.26 mg, 6.045 mmol, 3 equiv) was added to a solution of 9a (700 mg, 2.015 mmol, 1 equiv) and cyclohexylhydrazine hydrochloride (364.25 mg, 2.418 mmol, 1.2 equiv) in ethanol at room temperature, and stirring was continued for 12 hours. Liquid chromatography-mass spectrometry (LC-MS) revealed the formation of an intermediate with an MS of 444. The solvent was then removed, and toluene (7 mL, 65.790 mmol, 32.65 equiv) was added. The reaction system was stirred at 100°C for 24 hours. The reaction was complete as determined by LC-MS. The reaction mixture was quenched with water at room temperature. The reaction mixture was extracted with ethyl acetate (3 x 7 mL). The combined organic phases were backwashed with saturated sodium chloride solution (1 x 7 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1) to give compound 11b (610 mg, 73.56%).

[0308] LC-MS: (ESI, m / z)=412.25[M+H] +

[0309] Step 2: tert-Butyl N-(1-([(2R)-1-(3a-benzyl-2-cyclohexyl-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl-3-(benzyloxy)-1-oxopropan-2-yl)carbamoyl-1-methylethyl)carbamate 11d

[0310] Under nitrogen, a solution of 11b (410 mg, 0.996 mmol, 1 equiv) in dichloromethane was added with 1,4-dioxane hydrochloride (5.00 mL, 164.519 mmol, 165.18 equiv) at room temperature. The reaction was stirred at room temperature for 1 hour. Liquid chromatography-mass spectrometry (LC-MS / MS) confirmed the reaction was complete. This afforded crude compound 11c, which was used in the next step without further purification. Under nitrogen, T3P (597.15 mg, 1.876 mmol, 2 equiv) and N,N-diisopropylethylamine (363.85 mg, 2.814 mmol, 3 equiv) were added portionwise to a solution of 11c (350 mg, 1.124 mmol, 1.20 equiv) and (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropylamino}propanoic acid (357 mg, 0.938 mmol, 1.00 equiv) in dichloromethane at room temperature. After the addition was complete, the system was stirred at room temperature for 1 hour. Liquid chromatography-mass spectrometry indicated the reaction was complete. The reaction mixture was quenched with water at room temperature. The reaction mixture was extracted with ethyl acetate (3 x 4 mL). The combined organic phases were backwashed with saturated sodium chloride solution (1 x 4 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give compound 11d (360 mg, 61.46%).

[0311] LC-MS: (ESI, m / z)=674.05[M+H] +

[0312] Step 3: 2-amino-N-((2R)-1-(3a-benzyl-2-cyclohexyl-3-oxo-2,3,3a,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)-3-(benzyloxy)-1-oxopropan-2-yl)-2-methylpropanamide G11

[0313] 11d (300 mg, 0.445 mmol, 1 equiv) was dissolved in dichloromethane (30.00 mL, 471.704 mmol, 1060.01 equiv) and ZnBr2 (145.54 mg, 2.225 mmol, 5 equiv) was added. The desired product was found in the liquid phase. The reaction mixture was quenched with water at room temperature. The reaction mixture was extracted with ethyl acetate (3 x 20 mL). The combined organic phases were backwashed with saturated sodium chloride (1 x 30 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography to afford compound G11 (80 mg, 31.1%).

[0314] LC-MS: (ESI, m / z)=574.60[M+H]+

[0315] Step 4

[0316] G11 was subjected to chiral resolution to obtain compound G11a (14.9 mg, 5.81%) and compound G11b (39.0 mg, 15.18%).

[0317] G11a

[0318] LC-MS: (ESI, m / z)=574.60[M+H] +

[0319] 1 H NMR (400MHz, DMSO-d6) δ8.42 (s, 1H), 7.32 (dq, J=13.8, 7.1Hz, 5H), 7.23-7.11 (m, 3H), 7.09- 6.90 (m, 2H), 5.10 (s, 1H), 4.70 (d, J=12.6Hz, 1H), 4.64-4.03 (m, 3H), 3.98-3.47 (m, 3H), 2.9 6 (q, J=18.0, 15.1Hz, 3H), 2.84 (d, J=13.4Hz, 1H), 2.59 (d, J=12.6Hz, 2H), 2.21 (s, 2H), 1.70 (d, J=13.1Hz, 1H), 1.53 (dd, J=25.4, 12.0Hz, 4H), 1.19 (d, J=22.5Hz, 8H), 1.08-0.77 (m, 3H)

[0320] Chromatographic column: CHIRALPAK-IA 2*25cm, 5μm; mobile phase A: n-hexane (0.1% diethylamine), mobile phase B: methanol: dichloromethane = 1:1; flow rate: 20mL / min; gradient: isocratic 30; wavelength: 254nm / 220nm; retention time: 5.1min; sample solvent: methanol; number of runs: 1

[0321] G11b

[0322] LC-MS: (ESI, m / z)=574.60[M+H] +

[0323] 1H NMR (400MHz, DMSO-d6) δ8.32 (s, 1H), 7.43-7.23 (m, 5H), 7.22-7.07 (m, 3H), 7.04-6.74 ( m, 2H), 5.15 (d, J=6.4Hz, 1H), 4.90-4.68 (m, 1H), 4.65-4.20 (m, 3H), 3.83-3.50 (m, 3H), 3.13-2.80(m, 3H), 2.76-2.57(m, 2H), 2.54(s, 1H), 2.05(s, 2H), 1.68(d, J=13.0Hz, 1H) , 1.61-1.39 (m, 4H), 1.31-1.05 (m, 8H), 0.97 (qd, J=12.3, 3.3Hz, 2H), 0.88-0.74 (m, 1H).

[0324] Chromatographic column: CHIRALPAK-IA 2*25cm, 5μm; mobile phase A: n-hexane (0.1% diethylamine), mobile phase B: methanol: dichloromethane = 1:1; flow rate: 20mL / min; gradient: isocratic 30; wavelength: 254nm / 220nm; retention time: 7.8min; sample solvent: methanol; number of runs: 1

[0325] Example 12

[0326] The first step is 1-tert-butyl 3-methyl 4-oxo-3-(pyridin-2-ylmethyl)piperidine-1,3-dicarboxylate 12b

[0327] Under nitrogen, sodium hydride (186.55 mg, 7.774 mmol, 2 equiv) was added to a solution of 12a (1 g, 3.887 mmol, 1 equiv) in N,N-dimethylformamide (10 mL) at room temperature. After stirring for 1 hour, 2-(bromomethyl)pyridine hydrobromide (983.08 mg, 3.887 mmol, 1 equiv) was added dropwise at room temperature. Stirring was continued at room temperature for 3 hours. Liquid chromatography-mass spectrometry indicated the reaction was complete. The reaction mixture was quenched with saturated aqueous ammonium chloride at room temperature. The reaction mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic phases were backwashed with saturated brine (1 x 100 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-30%) to afford compound 12b (800 mg, 59.08%).

[0328] LC-MS: (ESI, m / z)=350.10[M+H] +

[0329] Step 2: tert-Butyl 2-cyclopropyl-3-oxo-3a-(pyridin-2-ylmethyl)-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylate 12c

[0330] Under nitrogen, cyclopropylhydrazine hydrochloride (498.60 mg, 4.592 mmol, 2 equiv) and N,N-diisopropylethylamine (890.34 mg, 6.888 mmol, 3 equiv) were added to a solution of 12b (800 mg, 2.296 mmol, 1 equiv) in ethanol (10 mL) at room temperature. The mixture was stirred at 80°C overnight and the reaction was complete after liquid chromatography-mass spectrometry. The reaction mixture was cooled to room temperature. The resulting residue was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-35%) to afford compound 12c (700 mg, 82.29%).

[0331] LC-MS: (ESI, m / z)=371.15[M+H] +

[0332] Step 3 2-cyclopropyl-3a-(pyridin-2-ylmethyl)-4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-one 12d

[0333] Under nitrogen, a 4M solution of hydrogen chloride in 1,4-dioxane (5 mL) was added to a solution of 12c (330 mg, 0.891 mmol, 1 equiv) in 1,4-dioxane (5 mL) at room temperature. The mixture was stirred for 1 hour, and the reaction was complete by liquid chromatography-mass spectrometry. The resulting residue was concentrated in vacuo to afford crude compound 12d (280 mg, 93.02%).

[0334] LC-MS: (ESI, m / z)=271.10[M+H] +

[0335] Step 4: tert-Butyl N-(1-{[3-(benzyloxy)-1-[2-cyclopropyl-3-oxo-3a-(pyridin-2-ylmethyl)-4H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl]-1-oxopropan-2-yl]carbamoyl}-1-methylethyl)carbamate 12e

[0336] To a solution of 12d (280 mg, 1.036 mmol, 1 equiv) in dichloromethane (5 mL) under nitrogen atmosphere were added (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (591.06 mg, 1.554 mmol, 1.5 equiv), 1-propylphosphonic anhydride (1647.78 mg, 5.180 mmol, 5 equiv), and N,N-diisopropylethylamine (669.34 mg, 5.180 mmol, 5 equiv) at room temperature. The reaction mixture was stirred overnight and the reaction was complete after liquid chromatography-mass spectrometry. The reaction mixture was extracted with ethyl acetate (300 mg, 10 mL). The combined organic phases were backwashed with saturated brine (1 x 30 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to yield crude compound 12e (300 mg, 45.77%).

[0337] LC-MS: (ESI, m / z)=633.35[M+H] +

[0338] Step 5: 2-amino-N-[3-(benzyloxy)-1-[2-cyclopropyl-3-oxo-3a-(pyridin-2-ylmethyl)-4H, 6H, 7H-pyrazolo[4, 3-c]pyridin-5-yl]-1-oxopropan-2-yl]-2-methylpropanamide G12

[0339] To a solution of 12e (300 mg, 0.474 mmol, 1 equiv) in 1,4-dioxane (3 mL) was added a 4 M solution of hydrogen chloride in 1,4-dioxane (3 mL) at room temperature under nitrogen atmosphere. The mixture was stirred for 1 hour, and the reaction was monitored for completion by HPLC-MS. The resulting residue was concentrated in vacuo. The crude product was purified by HPLC-HPLC to afford compound G12 (200 mg, 79.20%) using the following conditions (column: Kinetex 5m EVO C18, 30 mm x 150 mm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 15% B to 40% B over 8 min; wavelength: 254 nm / 220 nm; retention time: 7.45 min).

[0340] LC-MS: (ESI, m / z)=533.30[M+H] +

[0341] Step 6

[0342] G12 (200 mg, 0.375 mmol, 1 equiv) was subjected to chiral separation at room temperature to obtain compound G12a (11.03 mg, 5.42%) and compound G12b (15.88 mg, 7.90%).

[0343] G12a

[0344] LC-MS: (ESI, m / z)=533.30[M+H] +

[0345] 1 H NMR (400MHz, DMSO-d6) δ8.59-8.15 (m, 2H), 7.60 (t, J=7.6Hz, 1H), 7.46-7.11 (m, 6H) , 6.98 (m, 1H), 5.11 (s, 1H), 4.93-4.62 (m, 1H), 4.62-4.15 (m, 3H), 3.66 (t, J=5.8Hz, 2 H), 3.21-2.91 (m, 3H), 2.90-2.59 (m, 3H), 2.43 (d, J=13.9Hz, 1H), 2.14 (m, 2H), 1.18 (d, J=5.5Hz, 6H), 0.68 (dq, J=6.9, 3.6Hz, 2H), 0.55-0.43 (m, 1H), 0.42-0.25 (m, 1H).

[0346] Retention time: 4.49 min. Chromatographic column: CHIRAL ART Cellulose-SB 3*25 cm, 5 μm; Mobile phase A: n-hexane (0.1% diethylamine), Mobile phase B: methanol: dichloromethane = 1:1; Flow rate: 40 mL / min; Gradient: Isocratic 30; Wavelength: 254 nm / 220 nm; Sample solvent: methanol; Number of runs: 7

[0347] G12b

[0348] LC-MS: (ESI, m / z)=533.30[M+H] +

[0349] 1H NMR (400MHz, DMSO-d6) δ8.87-7.87 (m, 2H), 7.81-7.53 (m, 1H), 7.31 (hept, J=7.2Hz, 4H), 7.25-6.6 8(m, 3H), 5.08(t, J=6.1Hz, 1H), 4.87-4.59(m, 1H), 4.58-4.45(m, 2H), 4.45-4.19(m, 1H), 3.67(m, 2 H), 3.02 (p, J=12.7, 11.6Hz, 3H), 2.83 (tt, J=6.9, 3.5Hz, 1H), 2.70-2.57 (m, 2H), 2.42-2.34 (m, 1H) , 2.33-1.84 (m, 2H), 1.20 (s, 6H), 0.78-0.63 (m, 2H), 0.56 (d, J = 11.6Hz, 1H), 0.38 (d, J = 9.9Hz, 1H).

[0350] Retention time: 5.66 min. Chromatographic column: CHIRAL ART Cellulose-SB 3*25 cm, 5 μm; Mobile phase A: n-hexane (0.1% diethylamine), Mobile phase B: methanol: dichloromethane = 1:1; Flow rate: 40 mL / min; Gradient: Isocratic 30; Wavelength: 254 nm / 220 nm; Sample solvent: methanol; Number of runs: 7

[0351] Example 13

[0352] The first step is tert-butyl 3a-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate 13b

[0353] To a solution of 2a (1000 mg, 3.0 mmol, 1 equiv) in tetrahydrofuran (10 mL) was added sodium hydroxide (146 mg, 6.1 mmol, 2 equiv) at 0°C under nitrogen protection. After stirring for 0.5 hours, iodomethane (862 mg, 6.1 mmol, 2 equiv) was added dropwise at 0°C. The resulting residue was stirred at room temperature under nitrogen protection for 1 hour. The reaction mixture was quenched by the addition of water (200 mL) at room temperature. The reaction mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with saturated brine (1 × 100 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to give compound 13b (750 mg, 71.9%).

[0354] LC-MS: (ESI, m / z)=343.90[M+H] +

[0355] Step 2 3a-Benzyl-2-methyl-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one 13c

[0356] Under nitrogen, 4M 1,4-dioxane hydrochloride (5 mL) was added to a solution of 13b (750 mg, 2.3 mmol, 1 equiv) in dichloromethane (5 mL) at room temperature and stirred for 1 hour. The resulting residue was concentrated under reduced pressure to afford compound 13c (500 mg, crude product).

[0357] LC-MS: (ESI, m / z)=244.10[M+H] +

[0358] Step 3: Tert-butyl ((2R)-1-(3a-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)-3-(benzyloxy)-1-oxopropan-2-yl)carbamate 13d

[0359] Under nitrogen, to a solution of 13c (500 mg, 2.1 mmol, 1 equiv) and N,N-diisopropylethylamine (1593 mg, 12.3 mmol, 6 equiv) in dichloromethane (10 mL) were added (2R)-3-(benzyloxy)-2-([(tert-butoxy)carbonyl]aminopropionic acid (425 mg, 1.4 mmol, 0.7 equiv) and 1-propylphosphonic acid cyclic anhydride (2615 mg, 4.1 mmol, 2 equiv, 50%) at room temperature. The resulting residue was stirred for 3 hours. The reaction mixture was quenched by addition of water (100 mL) at room temperature. The reaction mixture was extracted with ethyl acetate (3×100 mL). The combined organic phases were washed with saturated brine (1×100 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by preparative chromatography (petroleum ether / ethyl acetate = 1:1) to give compound 13d (800 mg, 74.7%).

[0360] LC-MS: (ESI, m / z)=521.35[M+H] +

[0361] Step 4: 3a-Benzyl-5-(O-Benzyl-D-seryl)-2-methyl-2,3a,4,5,6,7-hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one 13e

[0362] Under nitrogen, a solution of 13d (800 mg, 1.5 mmol, 1 equiv) and 4M 1,4-dioxane hydrochloride (5 mL) in dichloromethane (5 mL) was stirred at room temperature for 1 hour. The resulting residue was concentrated in vacuo to afford compound 13e (700.0 mg, crude product).

[0363] LC-MS: (ESI, m / z)=421.20[M+H] +

[0364] Step 5: tert-Butyl 4-(((2R)-1-(3a-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)-3-(benzyloxy)-1-oxopropan-2-yl)carbamoyl)piperidine-1-carboxylate 13f

[0365] Under nitrogen, a solution of 13e (195 mg, 0.8 mmol, 0.7 equiv), 1-propylphosphonic acid cyclic anhydride (772 mg, 2.4 mmol, 2 equiv), and N,N-diisopropylethylamine (941 mg, 7.3 mmol, 6 equiv) in dichloromethane (10 mL) was stirred overnight at room temperature. The reaction mixture was quenched by the addition of water (100 mL) at room temperature. The reaction mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic phases were backwashed with saturated brine (1 x 100 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to afford compound 13f (420 mg, 54.8%).

[0366] LC-MS: (ESI, m / z)=532.40[M+H] +

[0367] Step 6:

[0368] Under nitrogen protection, a solution of compound 13f (420 mg, 0.7 mmol, 1 equiv) and trifluoroacetic acid (1 mL) in dichloromethane (5 mL) was stirred and reacted for 1 hour at room temperature. The resulting residue was concentrated under reduced pressure to obtain crude compound G13 (N-((2R)-1-(3-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)-3-(benzyloxy)-1-oxopropan-2-yl)piperidine-4-carboxamide). The crude product was purified by high-performance liquid chromatography to afford compound G13a (75.3 mg, 20.7%) and compound G13b (59.1 mg, 16.6%).

[0369] G13a

[0370] LC-MS: (ESI, m / z)=531.90[M+H] +

[0371] 1 H NMR (400MHz, DMSO-d6, ppm) δ8.42 (d, J=7.8Hz, 1H), 7.32 (dq, J=12.4, 6.5, 5.5Hz, 5H), 7. 18 (t, J=7.1Hz, 3H), 7.09-7.02 (m, 2H), 5.11 (q, J=7.1Hz, 1H), 4.67 (d, J=12.6Hz, 1H), 4. 51(s, 3H), 3.71(t, J=8.4Hz, 1H), 3.63-3.54(m, 1H), 2.98(s, 3H), 2.92(s, 4H), 2.87(s, 1 H), 2.64 (d, J=12.6Hz, lH), 2.45 (d, J=13.7Hz, 5H), 1.63-1.44 (m, 2H), 1.41-1.39 (m, 2H).

[0372] Chromatographic column specifications: Kinetex 5m EVO C18, 20mm*250mm; mobile phase A: petroleum ether (0.1% diethanolamine), mobile phase B: ethanol: dichloromethane = 1:1; flow rate: 20mL / min; elution gradient: isocratic 30; detection wavelength: UV 254nm / 220nm; retention time: 6.6min; number of runs: 3

[0373] G13b

[0374] LC-MS: (ESI, m / z)=531.90[M+H] +

[0375] 1H NMR (400MHz, DMSO-d6, ppm) δ8.13 (d, J=8.1Hz, 1H), 7.30 (dd, J=15.5, 8.6Hz, 5H), 7.17-7.11 (m, 3H), 6.88 -6.81 (m, 2H), 5.21 (d, J = 7.4Hz, 1H), 4.83 (s, 1H), 4.67 (d, J = 12.6Hz, 1H), 4.64-4.53 (m, 2H), 3.88-3.79 ( m, 1H), 3.76 (t, J = 8.4Hz, 1H), 3.65-3.57 (m, 1H), 3.54 (s, 1H), 2.95-2.81 (m, 3H), 2.64 (d, J = 12.6Hz, 3H), 2.44 (s, 1H), 2.42 (d, J = 10.7Hz, 2H), 2.38 (s, 2H), 2.33-2.23 (m, 1H), 1.43 (d, J = 12.2Hz, 2H), 1.39 (s, 2H).

[0376] Chromatographic column specifications: Kinetex 5m EVO C18, 20mm*250mm; mobile phase A: petroleum ether (0.1% diethanolamine), mobile phase B: ethanol: dichloromethane = 1:1; flow rate: 20mL / min; elution gradient: isocratic 30; detection wavelength: UV 254nm / 220nm; retention time: 7.5min; number of runs: 3

[0377] Example 14

[0378] Step 1: 1-tert-Butyl 3-methyl-6-methyl-4-oxopiperidine-1,3-dicarboxylate 14b

[0379] Under nitrogen, sodium hydride (2.81 g, 117.218 mmol, 2.5 equiv) was slowly added to a solution of 14a (10 g, 46.887 mmol, 1 equiv) in tetrahydrofuran (100 mL). After stirring at room temperature for 30 minutes, dimethyl carbonate (10.56 g, 117.218 mmol, 2.5 equiv) was added dropwise and the mixture was stirred at room temperature overnight. Liquid chromatography-mass spectrometry confirmed the reaction was complete. The reaction mixture was quenched with water (200 mL), extracted with ethyl acetate (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-20%) to afford a mixture of compound 14bb and compound 14b (9 g, crude product).

[0380] LC-MS: (ESI, m / z)=270.20[M+H] +

[0381] Step 2: 1-tert-Butyl 3-methyl-2-methyl-4-oxopiperidine-1,3-dicarboxylate 14c

[0382] To a solution of a mixture of 14b and 14bb (7 g, crude) in N,N-dimethylformamide (70 mL) under nitrogen was slowly added sodium hydride (0.62 g, 25.800 mmol, 2 equiv). The mixture was stirred at room temperature for 1 hour, followed by the addition of benzyl bromide (2.32 g, 13.545 mmol, 1.05 equiv). The reaction mixture was stirred for 5 hours, and the reaction was complete by liquid chromatography-mass spectrometry. The reaction mixture was quenched with water (100 mL), and the aqueous phase was extracted with ethyl acetate (3 × 150 mL), dried over anhydrous sodium sulfate (40 g), filtered, and the filtrate concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-15%) to afford the mixed product (5.8 g, crude). The crude product was purified by HPLC using the following conditions: column size: Ultimate μXB-C18 column, 30 x 150 mm, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 100 mL / min; gradient: 45% B to 75% B over 20 minutes; wavelength: 254 / 220 nm; retention time: 18 / 20.5 min). Compound 14c (1.7 g, 36.46%, retention time: 18 min) and compound 14cc (2.5 g, 53.62%, retention time: 20.5 min) were obtained.

[0383] LC-MS: (ESI, m / z)=362.30[M+H] +

[0384] Step 3: 3a-Benzyl-2,4-dimethyl-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester 14d

[0385] Under nitrogen, a solution of 14c (1.7 g, 4.703 mmol, 1 equiv), methylhydrazine (0.26 g, 5.644 mmol, 1.2 equiv), and N,N-diisopropylethylamine (1.82 g, 14.109 mmol, 3 equiv) in ethanol (200 mL) was stirred at 80°C overnight. After liquid chromatography-mass spectrometry (LC-MS / MS) monitoring, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-25%) to afford compound 14d (800 mg, 44.63%).

[0386] LC-MS: (ESI, m / z)=358.5[M+H] +

[0387] Step 4: 3a-Benzyl-2,4-dimethyl-4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-one 14e

[0388] To a 7 mL sample vial were added 14d (300 mg, 0.839 mmol, 1 equiv) and a 1,4-dioxane solution of hydrochloric acid (4 M, 4 mL) at room temperature. The reaction was stirred for 1 hour. The reaction was completed by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain compound 14e (250 mg, crude product).

[0389] LC-MS: (ESI, m / z)=258.3[M+H] +

[0390] Step 5: N-(1-([(2R)-1-(3a-benzyl-2,4-dimethyl-3-oxo-4H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl-3-(benzyloxy)-1-oxopropan-2-yl]carbamoyl-1-methylethyl)carbamic acid tert-butyl ester 14f

[0391] To a solution of 14e (250 mg, crude), N,N-diisopropylethylamine (1.26 g, 9.710 mmol, 10 equiv) and (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (628 mg, 1.651 mmol, 1.7 equiv) in dichloromethane (10 mL) was added 1-propylphosphonic anhydride (618 mg, 1.942 mmol, 2 equiv), and the mixture was stirred at room temperature for 3 h. The reaction was completed by liquid chromatography-mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in N,N-dimethylformamide (1 mL) and filtered. The filter cake was washed with N,N-dimethylformamide (2 mL), and the filtrate was purified by reverse-phase column chromatography using the following conditions: 20 g column, acetonitrile:water mobile phase, gradient from 0% to 100% over 20 minutes, with the product peak appearing at 65% gradient, detection wavelength: UV 254 nm. The resulting residue was concentrated under reduced pressure to afford compound 14f (100 mg, 13.39%).

[0392] LC-MS: (ESI, m / z)=620.6[M+H] +

[0393] Step 6: 2-amino-N-((2R)-1-(3a-benzyl-2,4-dimethyl-3-oxo-2,3,3a,4,6,7-hexahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)-3-(benzyloxy)-1-oxopropan-2-yl)-2-methylpropanamide G14

[0394] To a solution of 14f (100 mg, 0.161 mmol, 1 equiv) in dichloromethane (1.5 mL) was added trifluoroacetic acid (0.15 mL) and stirred at room temperature for 1 hour. Following LC / MS monitoring of the reaction, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high-performance liquid chromatography using the following conditions: column specifications: XBridge BEH Shield RP18 5 μm, 30 x 150 mm, 5 μm; mobile phase A: water (10 mmol / L NH₄HCO₃), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 25% B to 45% B over 10 minutes; wavelength: 220 nm; retention time: 9.05 min. Compound G14 (12.01 mg, 14.24%) was obtained.

[0395] LC-MS: (ESI, m / z)=520.1[M+H] +

[0396] 1 H NMR(400MHz, DMSO-d6, ppm)δ 8.37(s, 1H), 7.36-7.27(m, 5H), 7.21-7.12(m, 3H), 7.01-6.81(m, 2H), 5.18-5.03( m, 2H), 4.82-4.58 (m, 2H), 4.31 (dd, J=13.9, 6.4Hz, 1H), 3.79-3.66 (m, 2H), 3.65-3 .48(m, 1H), 3.13-2.94(m, 1H), 2.90(d, J=3.7Hz, 4H), 2.86-2.68(m, 1H), 2.63(dd, J=12.8, 6.4Hz, 1H), 2.45-2.11 (m, 2H), 1.23-1.13 (m, 6H), 0.81 (dd, J=6.9, 3.5Hz, 3H).

[0397] Example 15

[0398] Step 1-tert-Butyl 3-methyl 5,5-dimethyl-4-oxopiperidine-1,3-dicarboxylate 15b

[0399] To a solution of tert-butyl 3,3-dimethyl-4-oxopiperidine-1-carboxylate (7 g, 30.796 mmol, 1 equiv) in tetrahydrofuran (80 mL) was added sodium hydride (2.4 g, 61.592 mmol, 2 equiv, 60%) at 0°C. After the addition was complete, the system was stirred at room temperature for 1 hour. Dimethyl carbonate (8.3 g, 92.388 mmol, 3 equiv) was added dropwise at room temperature. After the addition was complete, the system was stirred at 60°C overnight. The reaction was complete as determined by liquid chromatography-mass spectrometry. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-35%) to afford compound 15b (4 g, 45.52%).

[0400] 1 H NMR (400MHz, DMSO-d6) δ4.09-3.95 (m, 2H), 3.79-3.64 (m, 3H), 3.31 (d, J=13.9Hz, 3H), 1.42 (d, J=10.3Hz, 9H), 1.09 (s, 6H).

[0401] Step 2: 1-tert-Butyl 3-methyl 3-benzyl-5,5-dimethyl-4-oxopiperidine-1,3-dicarboxylate 15c

[0402] 15b (3 g, 10.514 mmol, 1 equiv) was dissolved in tetrahydrofuran (30 mL). Sodium hydride (0.50 g, 12.617 mmol, 1.2 equiv, 60%) was added under ice-cooling. The nitrogen atmosphere was replaced three times. After 30 minutes of reaction, benzyl bromide (3.60 g, 21.028 mmol, 2 equiv) was slowly added dropwise and allowed to react overnight. The reaction was monitored for completion by liquid chromatography-mass spectrometry. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The reaction mixture was quenched by the addition of saturated aqueous ammonium chloride (20 mL) at room temperature. The reaction mixture was extracted with ethyl acetate (4 x 20 mL). The combined organic phases were backwashed with saturated aqueous sodium chloride (1 x 50 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography using the following conditions: C18 column, water (0.01% sodium bicarbonate) / acetonitrile (0%-70%). Compound 15c (1.5 g, 38.00%) was obtained.

[0403] LC-MS (ESI, m / z):=376.05[M+H] +

[0404] Step 3 3a-Benzyl-7,7-dimethyl-3-oxo-2H,4H,6H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester 15d

[0405] To a solution of 15c (1 g, 2.663 mmol, 1 equiv) in ethanol (15 mL) was added hydrazine hydrate (0.75 g, 14.993 mmol, 5.63 equiv) at room temperature. The atmosphere was replaced with nitrogen, and after completion of the addition, the system was stirred in a microwave oven at 100°C for 2 hours. The reaction was monitored for completion by liquid chromatography-mass spectrometry. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography using a C18 column in water (0.01% sodium bicarbonate) / acetonitrile (0%-70%) to afford compound 15d (450 mg, 47.27%).

[0406] LC-MS (ESI, m / z):=358.15[M+H] +

[0407] Step 4: 3a-Benzyl-2,7,7-trimethyl-3-oxo-4H,6H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester 15e

[0408] Under nitrogen, sodium hydride (89 mg, 2.238 mmol, 2 equiv, 60%) was added to a solution of 15e (400 mg, 1.119 mmol, 1 equiv) in N,N-dimethylformamide (15 mL) at 0°C. The mixture was stirred at room temperature for 1 h, and then iodomethane (190 mg, 1.343 mmol, 1.2 equiv) was added dropwise at 0°C. The mixture was allowed to react for 2 h at room temperature. Liquid chromatography-mass spectrometry (LC-MS / MS) confirmed the reaction was complete. The reaction mixture was diluted with ethyl acetate (20 mL) and poured into an ice-cold saturated ammonium chloride solution (20 mL). The aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined organic phases were backwashed with saturated sodium chloride solution (30 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%-35%) to afford compound 15e (240 mg, 57.73%).

[0409] LC-MS (ESI, m / z):=372.15[M+H] +

[0410] Step 5: 3a-Benzyl-2,7,7-trimethyl-4H,5H,6H-pyrazolo[4,3-c]pyridin-3-one 15f

[0411] Compound 15e (240 mg, 0.646 mmol, 1 equiv) was dissolved in a solution of hydrogen chloride in 1,4-dioxane (4 M, 2 mL) at room temperature and allowed to react for 2 h. The reaction was monitored for completion by LCMS. The reaction solution was concentrated under reduced pressure, and the resulting residue was basified to pH 8 with saturated sodium bicarbonate solution. The aqueous solution was extracted with dichloromethane (3 x 20 mL). The combined organic phases were backwashed with saturated sodium chloride solution (20 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to afford compound 15f (150 mg, 85.56%).

[0412] LC-MS (ESI, m / z):=272.10[M+H] +

[0413] Step 6: 15g of tert-butyl N-(1-([(2R)-1-(3a-benzyl-2,7,7-trimethyl-3-oxo-4H,6H-pyrazolo[4,3-c]pyridin-5-yl-3-(benzyloxy)-1-oxopropyl-2-yl]carbamoyl-1-methylethyl)carbamate

[0414] To a solution of 15f (150 mg, 0.553 mmol, 1 equiv) and (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (315 mg, 0.830 mmol, 1.5 equiv) in dichloromethane (4 mL) were added dropwise 1-propylphosphonic anhydride (703 mg, 1.106 mmol, 2 equiv, 50%) and N,N-diisopropylethylamine (214 mg, 1.659 mmol, 3 equiv) at room temperature. The reaction was allowed to react at room temperature for 2 h. The reaction was complete after liquid chromatography-mass spectrometry. The reaction mixture was cooled to room temperature, poured into water, and extracted with dichloromethane (3 x 20 mL). The combined organic phases were backwashed with saturated sodium chloride solution (1 x 20 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography under the following conditions: C18 column, water (sodium bicarbonate 0.01%) / acetonitrile = (0%-55%) to obtain compound 15g (200 mg, 67.80%).

[0415] LC-MS (ESI, m / z):=634.30[M+H] +

[0416] Step 7: N-[(2R)-1-{3a-benzyl-2,7,7-trimethyl-3-oxo-4H,6H-pyrazolo[4,3-c]pyridin-5-yl}-3-(benzyloxy)-1-oxopropan-2-yl]-2-amino-2-methylpropionamide G15

[0417] To a solution of 15 g (300 mg, 0.473 mmol, 1 equiv) in dichloromethane (10 mL) at room temperature was added trifluoroacetic acid (1 mL, 13.463 mmol, 28.44 equiv). After the addition was complete, the system was stirred at room temperature for 1 hour. The reaction was monitored for completion by HPLC. The mixture was filtered, the filter cake washed with dichloromethane (1 x 2 mL), and the filtrates combined. The crude product was purified by HPLC using the following conditions: column specifications: Kinetex EVO C18 column, 30 x 150 mm, 5 μm; mobile phase A: water (10 mmol / L NH₄HCO₃), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 30% B to 50% B over 10 minutes; wavelength: 220 nm; retention time: 8.32 min. Compound G15 (156.7 mg, 61.41%) was obtained.

[0418] LC-MS (ESI, m / z):=534.15[M+H] +

[0419] 1 H NMR (400MHz, DMSO-d6) δ8.35 (s, 1H), 7.46-7.09 (m, 5H), 7.21-7.02 (m, 3H), 6.98 (d, J=6.9Hz, 1H), 6.81 (s, 1H), 5.19-5.02 (m, 1H), 4.78-4.37 ( m, 3H), 4.33-4.00 (m, 1H), 4.01-3.34 (m, 2H), 3.21-3.00 (m, 5H), 2.99- 2.73(m, 1H), 2.73-2.54(m, 1H), 2.38-1.47(m, 2H), 1.41-0.95(m, 12H).

[0420] Example 16

[0421] Step 1: tert-Butyl 1-benzyl-4H,5H,7H-pyrrolo[2,3-c]pyridine-6-carboxylate 16b

[0422] Under nitrogen, 16a (300 mg, 1.350 mmol, 1 equiv) was dissolved in dimethylformamide (3 mL, 38.765 mmol, 28.72 equiv). Sodium hydride (129.55 mg, 2.700 mmol, 2 equiv, 50%) was added portionwise at 0°C. The mixture was stirred at room temperature for 1 hour, and then benzyl bromide (277.00 mg, 1.620 mmol, 1.2 equiv) was added dropwise to the reaction mixture. The reaction system was stirred at room temperature for 3 hours. The reaction mixture was quenched by the addition of water (10 mL) at 0°C. The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated brine (3 × 50 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50:1) to provide compound 16b (330 mg, 78.27%).

[0423] LC-MS: (ESI, m / z)=313.05[M+H] +

[0424] Step 2 1-Benzyl-4H,5H,6H,7H-pyrrolo[2,3-c]pyridine 16c

[0425] Under nitrogen protection, 16b (110 mg, 0.352 mmol, 1 equiv) was dissolved in trifluoroacetic acid (0.5 mL) and dichloromethane (1 mL) at room temperature, and the reaction system was stirred at room temperature for 2 hours. The resulting residue was concentrated under reduced pressure to obtain compound 16c (60 mg, 80.27%).

[0426] LC-MS: (ESI, m / z)=211.10[M+H] -

[0427] Step 3: tert-Butyl N-(1-{[(2R)-1-{1-benzyl-4H,5H,7H-pyrrolo[2,3-c]pyridin-6-yl}-3-(benzyloxy)-1-oxopropan-2-yl]carbamoyl}-1-methylethyl)carbamate 16d

[0428] Under nitrogen, to a solution of 16c (60 mg, 0.283 mmol, 1 equiv), (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (118.27 mg, 0.311 mmol, 1.1 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (81.27 mg, 0.424 mmol, 1.5 equiv), and triethylamine (85.80 mg, 0.849 mmol, 3 equiv) in dichloromethane (1 mL) at 25°C was added 1-hydroxybenzotriazole (57.28 mg, 0.424 mmol, 1.5 equiv). The reaction system was stirred at room temperature for 6 hours. The reaction mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic phases were dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 70:1) to give compound 16d (70 mg, 43.10%).

[0429] LC-MS: (ESI, m / z)=575.30[M+H] +

[0430] Step 4: 2-amino-N-[(2R)-1-{1-benzyl-4H, 5H, 7H-pyrrolo[2, 3-c]pyridin-6-yl}-3-(benzyloxy)-1-oxopropan-2-yl]-2-methylpropanamide G16

[0431] Under nitrogen, 16d (50 mg, 0.087 mmol, 1 equiv) was dissolved in trifluoroacetic acid (0.5 mL) and dichloromethane (1 mL) at room temperature. The reaction system was stirred at room temperature for 2 hours. The resulting residue was concentrated under reduced pressure. The crude product was purified by HPLC using the following conditions: column: YMC Triart C18 ExRs 5 μm, 30 mm x 150 mm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 45% B to 70% B over 8 minutes; wavelength: 254 nm / 220 nm; retention time: 6.48 minutes. This afforded G16 (2.3 mg, 5.57%).

[0432] LC-MS: (ESI, m / z)=475.20[M+H] +

[0433] 1H NMR (400MHz, DMSO-d6, ppm) δ8.27 (s, 1H), δ7.39-7.21 (m, 8H), 7.20-7.05 (m, 2H), 6.81-6.71 (m, 1H), 5.90-5.82 (s, 1H), 5.04 (s, 2H), 5.01-4.88 (m, 1H), 4.58-4.27 (s, 4H), 3.83-3.42 (m, 5H), 2.02 (s, 2H), 1.33-1.06 (m, 6H).

[0434] Example 17

[0435] Step 1: tert-Butyl 3-bromo-2-methyl-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylate 17b

[0436] Under nitrogen, 17a (1 g, 3.309 mmol, 1 equiv) was dissolved in dimethylformamide (10 mL, 129.215 mmol, 39.05 equiv). Sodium hydride (317.67 mg, 6.618 mmol, 2 equiv, 50%) was added portionwise at 0°C. The mixture was stirred at room temperature for 1 hour, and then iodomethane (563.67 mg, 3.971 mmol, 1.2 equiv) was added dropwise to the reaction mixture. The reaction system was stirred at room temperature for 4 hours. The reaction mixture was quenched by the addition of water (10 mL) at 0°C. The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated brine (3 × 50 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50:1) to afford compound 17b (550 mg, 52.56%).

[0437] LC-MS: (ESI, m / z)=316.05[M+H] +

[0438] Step 2 tert-Butyl 2-methyl-3-phenyl-4H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylate 17c

[0439] Under nitrogen, to a solution of 17b (100 mg, 0.316 mmol, 1 equiv), phenylboronic acid (57.84 mg, 0.474 mmol, 1.5 equiv), and sodium carbonate (67.04 mg, 0.632 mmol, 2 equiv) in 1,4-dioxane (1 mL, 11.804 mmol, 37.32 equiv) and water (0.2 mL, 11.102 mmol, 35.10 equiv) at 25°C was added [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (25.76 mg, 0.032 mmol, 0.1 equiv). The reaction system was stirred at 65°C for 18 hours. The reaction mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic phases were dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 70:1) to give compound 17c (50 mg, 50.45%).

[0440] LC-MS: (ESI, m / z)=314.15[M+H] +

[0441] Step 3 2-Methyl-3-phenyl-4H,5H,6H,7H-pyrazolo[4,3-c]pyridine 17d

[0442] Under nitrogen, 17c (50 mg, 0.160 mmol, 1 equiv) was dissolved in trifluoroacetic acid (0.5 mL) and dichloromethane (1 mL) at room temperature. The reaction system was stirred at room temperature for 2 hours. The resulting residue was concentrated under reduced pressure to afford compound 17d (30 mg, 88.16%).

[0443] LC-MS: (ESI, m / z)=214.10[M+H] +

[0444] Step 4: tert-Butyl N-(1-{[(2R)-3-(benzyloxy)-1-{2-methyl-3-phenyl-4H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl}-1-oxopropan-2-yl]carbamoyl}-1-methylethyl)carbamate 17e

[0445] Under nitrogen, to a solution of 17d (30 mg, 0.141 mmol, 1 equiv), (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (58.86 mg, 0.155 mmol, 1.1 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (40.45 mg, 0.211 mmol, 1.5 equiv), and triethylamine (42.70 mg, 0.423 mmol, 3 equiv) in dichloromethane (1 mL) at 25°C was added 1-hydroxybenzotriazole (28.51 mg, 0.211 mmol, 1.5 equiv). The reaction system was stirred at room temperature for 6 hours. The reaction mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic phases were dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 70:1) to give compound 17e (60 mg, 74.09%).

[0446] LC-MS: (ESI, m / z)=576.35[M+H] +

[0447] Step 5: 2-amino-N-[(2R)-3-(benzyloxy)-1-{2-methyl-3-phenyl-4H, 6H, 7H-pyrazolo[4,3-c]pyridin-5-yl}-1-oxopropan-2-yl]-2-methylpropanamide G17

[0448] Under nitrogen, 17e (50 mg, 0.074 mmol, 1 equiv) was dissolved in trifluoroacetic acid (0.5 mL) and dichloromethane (1 mL) at room temperature and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by HPLC using the following conditions: XBridge BEH C18 OBD Preparative Column 130, 5 μm, 30 mm x 150 mm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 25% B to 47% B over 8 minutes; wavelength: 254 nm / 220 nm; retention time: 7.43 minutes. Compound G17 (20 mg, 56.38%) was obtained.

[0449] LC-MS: (ESI, m / z)=476.25[M+H] +

[0450] 1H NMR (400MHz, DMSO-d6, ppm) δ8.29 (s, 2H), 7.61 (d, J=7.6Hz, 2H), 7.49-7.36 (m , 2H), 7.35-7.08(m, 5H), 5.10-4.94(m, 1H), 4.80-4.64(m, 2H), 4.48(s, 1H), 4. 42-4.28(m, 1H), 4.00-3.78(m, 2H), 3.71(d, J=17.7Hz, 3H), 3.64-3.49(m, 2H) , 2.79-2.60 (m, 2H), 2.00 (s, 2H), 1.17 (d, J=3.6Hz, 3H), 1.12 (d, J=8.8Hz, 3H).

[0451] Example 18

[0452] First step 3a-Benzyl-7,7-dimethyl-2H,4H,5H,6H-pyrazolo[4,3-c]pyridin-3-one 18b

[0453] At room temperature, 15d (300 mg, 0.839 mmol, 1 equiv) was dissolved in a 4 M solution of hydrogen chloride in 1,4-dioxane (3 mL). The mixture was stirred at room temperature for 1 hour. The reaction was complete after liquid chromatography-mass spectrometry. The resulting residue was concentrated under reduced pressure to afford compound 18b (150 mg, 69.45%).

[0454] LC-MS (ESI, m / z):=258.10[M+H] +

[0455] Step 2: tert-Butyl N-(1-([(2R)-1-(3a-benzyl-7,7-dimethyl-3-oxo-2H,4H,6H-pyrazolo[4,3-c]pyridin-5-yl-3-(benzyloxy)-1-oxopropan-2-yl]carbamoyl-1-methylethyl)carbamate 18c

[0456] To a solution of 18b (150 mg, 0.583 mmol, 1 equiv) and (2R)-3-(benzyloxy)-2-{2-[(tert-butoxycarbonyl)amino]-2-methylpropionamido}propanoic acid (333 mg, 0.874 mmol, 1.5 equiv) in dichloromethane (2 mL) were added dropwise 1-propylphosphonic anhydride (742 mg, 1.166 mmol, 2 equiv, 50%) and N,N-diisopropylethylamine (226 mg, 1.749 mmol, 3 equiv) at room temperature. After the addition was complete, the system was stirred at room temperature for 2 hours. The reaction was complete as monitored by liquid chromatography-mass spectrometry. The resulting residue was concentrated under reduced pressure. The reaction mixture was dissolved in N,N-dimethylformamide (2 mL). The resulting residue was purified by reverse-phase column chromatography using the following conditions: C18 column, water (0.01% sodium bicarbonate) / acetonitrile (0%-50%). Compound 18c (100 mg, 33.01%) was obtained.

[0457] LC-MS (ESI, m / z):=620.25[M+H] +

[0458] Step 3 N-[(2R)-1-{3a-benzyl-7,7-dimethyl-3-oxo-2H,4H,6H-pyrazolo[4,3-c]pyridin-5-yl}-3-(benzyloxy)-1-oxopropan-2-yl]-2-amino-2-methylpropanamide G18

[0459] To a solution of 18c (100 mg, 0.161 mmol, 1 equiv) in dichloromethane (5 mL) was added trifluoroacetic acid (0.5 mL, 6.732 mmol, 41.72 equiv) at room temperature. After the addition was complete, the system was stirred at room temperature for 1 hour. The reaction was monitored for completion by HPLC. The reaction mixture was dissolved in N,N-dimethylformamide (2 mL). Filtered, the filter cake was washed with N,N-dimethylformamide (1 × 0.5 mL), and the filtrates were combined. The crude product was purified by HPLC using the following conditions: column specifications: Kinetex EVO C18 column, 30×150, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 21% B to 56% B over 10 minutes; wavelength: 220 nm; retention time: 8.55 minutes. Compound G18 (23.3 mg, 26.96%) was obtained.

[0460] LC-MS (ESI, m / z):=520.10[M+H] +

[0461] 1H NMR (400MHz, DMSO-d6) δ9.79 (s, 1H), 8.31 (s, 1H), 7.38-7.23 (m, 4H), 7.23-7.14 (m, 4H), 7.14 (s, 2H), 5.61-5.07 (m, 3H ), 4.99 (s, 1H), 4.48 (s, 1H), 4.42 (s, 1H), 3.68 (s, 2H), 3.66-3.57 (m, 2H), 3.47 (t, J=13.6Hz, 1H), 1.17-1.00 (m, 12H).

[0462] Biological evaluation

[0463] Test Example 1: Determination of the activity of the compounds of the present invention on human GHSR

[0464] This method is used to determine the agonistic effect of the compounds of the present invention on the activity of human GHSR protein expressed in human GHSR / CHO stable transfected cells.

[0465] 1. Test materials and instruments

[0466] 1. Culture medium

[0467] F12 (Gibco, Cat#11765-047);

[0468] FBS (Corning, Cat#35-076-CV);

[0469] Geneticin (Invitrogen, Cat#10131);

[0470] Penicillin / Streptomycin (Invitrogen, Cat#15140).

[0471] 2. Reagents

[0472] Fluo-4 Direct (Invitrogen, Cat#F10471);

[0473] HBSS (Gibco, Cat#14025076);

[0474] HEPES (Gibco, Cat#15630080);

[0475] Bonine Serum Albumin (Sgima, Cat#B2064-100G).

[0476] 3. Instrument consumables

[0477] 384 well Poly-D-Lysine protein coating plate (Greiner, Cat#781946);

[0478] FLIPR (Molecular Devices);

[0479] Vi-cell XR Cell Viability Analyzer (Beckman Coulter);

[0480] Incubator (Thermo).

[0481] 2. Experimental steps

[0482] Compound gradient preparation: Ghrelin and the compound of the present invention were diluted 5-fold to prepare 10 concentration gradients, and then transferred to the compound plate, with 900 nL per well.

[0483] Preparation buffer: HBSS (1X): HEPES (1M) = 49:1, add 0.5% BSA.

[0484] Stable human GHSR / CHO cells were seeded into 384-well plates. After overnight, the cell plates were removed and the culture medium was discarded. 20 μL of buffer was slowly added to each well, followed by 20 μL of 2X Fluo-4 Direct TM No-wash Loading Buffer. Place the cell plate in a 37°C 5% CO2 incubator and incubate for 50 minutes. Take out the cell plate and place it at room temperature for 10 minutes. Add 30 μL of buffer to each well of the compound plate; prepare another buffer plate and add 30 μL of buffer to each well. For the test of compound agonism: use the FLIPR instrument and run the software. Transfer 10 μL of buffer to the cell plate and read the fluorescence signal value. Transfer 10 μL of compound to the cell plate and read the fluorescence signal value. Use the FLIPR program to calculate the maximum-minimum value from the 91st signal point to the 230th signal point. EC of the compound 50 The value can be calculated by software using the fluorescence values ​​corresponding to different concentrations.

[0485] 3. Experimental Results

[0486] The agonist activity of the compounds of the present invention on human GHSR was determined by the above experiments, and the measured EC 50 See Table 1 for values.

[0487] Table 1 EC of the compounds of the present invention for agonistic activity against human GHSR 50 value

[0488] 4. Experimental Conclusions

[0489] The above data show that the compounds of the present invention have good agonist activity on human GHSR, and the activities of some preferred compounds are comparable to or better than the positive drug Capromorelin.

[0490] Test Example 2: Determination of the activity of the compounds of the present invention on canine GHSR

[0491] This method is used to determine the agonistic effect of the compounds of the present invention on the activity of canine GHSR protein expressed in canine GHSR / CHO stable transgenic cells.

[0492] 1. Test materials and instruments

[0493] 1. Culture medium

[0494] F12 (Gibco, Cat#11765-047);

[0495] FBS(Coming,Cat#35-076-CV);

[0496] G418, Gibco, Cat#(10131-027);

[0497] Penicillin / Streptomycin (Invitrogen, Cat#15140).

[0498] 2. Reagents

[0499] Fluo-4 Direct (Invitrogen, Cat#F10471);

[0500] 3. Instrument consumables

[0501] 384 well Poly-D-Lysine protein coating plate (Greiner, Cat#781946);

[0502] FLIPR (Molecular Devices);

[0503] Vi-cell XR Cell Viability Analyzer (Beckman Coulter);

[0504] Incubator (Thermo).

[0505] 2. Experimental Methods

[0506] Compound gradient preparation: Ghrelin and the compound of the present invention were diluted 5-fold to prepare 10 concentration gradients, and then transferred to the compound plate, with 900 nL per well.

[0507] Preparation buffer: HBSS (1X): HEPES (1M) = 49:1, add 0.5% BSA.

[0508] Canine GHSR / CHO stable cells were inoculated into 384-well plates. After overnight, the cell plates were removed and the culture medium was discarded. 20 μL of buffer was slowly added to each well, followed by 20 μL of 2X Fluo-4 Direct TM No-wash Loading Buffer. Place the cell plate in a 37°C 5% CO2 incubator and incubate for 50 minutes. Remove the cell plate and place it at room temperature for 10 minutes. Add 30 μL of buffer to each well of the compound plate; prepare another buffer plate and add 30 μL of buffer to each well. For the test of compound agonism: use the FLIPR instrument and run the software. Transfer 10 μL of buffer to the cell plate and read the fluorescence signal value. Transfer 10 μL of compound to the cell plate and read the fluorescence signal value. Use the FLIPR program to calculate the maximum-minimum value between the signal points. EC of the compound 50 The value can be calculated by software using the fluorescence values ​​corresponding to different concentrations.

[0509] 3. Experimental Results

[0510] The agonist activity of the compounds of the present invention on canine GHSR was determined by the above experiment, and the measured EC 50 See Table 2 for values.

[0511] Table 2 EC of the compounds of the present invention for agonistic activity against canine GHSR 50 value

[0512] 4. Experimental Conclusions

[0513] The above data show that the agonist activity of the preferred compound G1a of the present invention on canine GHSR is significantly stronger than that of capromorelin.

[0514] Test Example 3: Metabolic Stability Study in Human Liver Microsomes

[0515] 1. Experimental Methods

[0516] The incubation system consisted of 100 mM phosphate buffer (pH 7.4), 3 mM MgCl2, 1 mM NADPH, and 0.5 mg mL -1The reaction was performed in a final volume of 100 μL. NADPH was preincubated with human liver microsomes in a 37°C water bath for 5 minutes. The reaction was initiated by adding G1a or capromorelin working solution, with a final test substance concentration of 1 μM. The reaction was terminated by adding 500 μL of glacial acetonitrile to the incubation system at 0, 5, 15, 30, and 60 minutes. The cells were vortexed for 5 minutes and centrifuged at 3700 rpm for 10 minutes. Then, 50 μL of the supernatant was aspirated and diluted with 100 μL of deionized water. The cells were mixed and sampled for analysis. Verapamil served as a positive control, and G1a and capromorelin incubations without NADPH served as negative controls.

[0517] 2. Experimental Results

[0518] The metabolic stability of the compounds of the present invention in human liver microsomes was determined by the above experiments. 1 / 2 See Table 3 for values.

[0519] Table 3 Metabolic stability of the test compounds in human liver microsomes

[0520] 3. Experimental Conclusion

[0521] The preferred compound G1a of the present invention is cleared more slowly in human liver microsomes than capromorelin and has better drugability.

[0522] Test Example 4: In vitro study of PXR activation by test compounds in DPX2 cell culture

[0523] 1. Experimental Methods

[0524] DPX2 cells were cultured in T-75 flasks in a cell culture incubator at 37°C, 5% CO2, and 95% relative humidity. DPX2 cell culture medium was prepared using DPX2 medium supplemented with 10% fetal bovine serum. The cultured cells were rinsed with 10 ml of PBS in a T-75 flask. 3-5 mL of trypsin were added and incubated at 37°C for approximately 5 minutes or until the cells detached and floated. The trypsin was inactivated by adding medium containing excess serum. The cell suspension was transferred to a conical tube and centrifuged at 150 g for 5 minutes. The cells were resuspended in the inoculation medium at a density of 4.0 x 10 5 Transfer 100 μL to each well of a 96-well cell culture plate. Place the plate in an incubator and incubate at 37°C for 24 hours. After adding the test compound and incubating for 2 days, the culture is ready for quantitative analysis of PXR activation.

[0525] 2. Experimental Results

[0526] The in vitro study on the PXR activation of the compounds of the present invention in DPX2 cell culture was conducted by the above experiments. The results are shown in Table 4.

[0527] Table 4 PXR agonist activity of the test compounds

[0528] 3. Experimental Conclusion

[0529] The preferred compound G1a of the present invention has weaker PXR activation in DPX2 cell culture than Capromorelin and has better drugability.

[0530] Test Example 5: Pharmacokinetic Study of the Test Compound in Beagle Dogs

[0531] 1. Experimental Methods

[0532] Eight beagle dogs (half male and half female) were randomly divided into two groups (two females and two males in each group). Group A received a single oral gavage of 7 mg / kg G1a, while Group B received a single oral gavage of 7 mg / kg capromorelin. Jugular vein blood was collected before administration and at 0.167, 0.333, 0.666, 1, 2, 4, 6, 8, and 24 hours after administration. G1a and capromorelin concentrations in beagle plasma were determined using a validated LC-MS / MS method.

[0533] 2. Experimental Results

[0534] The pharmacokinetics of the compounds of the present invention in beagle dogs were determined by the above experiments. The results are shown in Table 5.

[0535] Table 5 Pharmacokinetic parameters of G1a and Capromorelin administered orally to beagle dogs at 7 mg / kg

[0536] 3. Experimental Conclusion

[0537] The preferred compound G1a of the present invention was administered to beagle dogs by single oral gavage. max and AUC 0-t Both are stronger than Capromorelin and have better pharmacokinetic properties.

[0538] Test Example 6: Tissue distribution study of the test compound in rat pituitary

[0539] 1. Experimental Methods

[0540] Twenty-four SD rats (half male and half female) were randomly divided into four groups and gavage-administered 20 mg / kg of G1a and capromorelin. Plasma samples and pituitary tissue were collected from each group at the following time points:

[0541] A validated LC-MS / MS method was used to detect G1a and capromorelin in plasma and tissues.

[0542] 2. Experimental Results

[0543] The tissue distribution of the compounds of the present invention in rat pituitary was determined by the above experiments. The results are shown in Table 6.

[0544] Table 6 G1a and Capromorelin concentrations in pituitary tissue of rats after oral administration

[0545] 3. Experimental Conclusion

[0546] The preferred compound G1a of the present invention has a stronger distribution in the pituitary gland than capromorelin in a single oral administration as the drug distribution time in the body is prolonged, and may have a better growth hormone release advantage.

[0547] Test Example 7: In vivo pharmacodynamics test of the preferred compounds of the present invention in rats

[0548] 1. Experimental Methods

[0549] In humans and rodents, growth hormone is stimulated by growth hormone-releasing hormone (GHRH) and secreted into the circulation by the pituitary gland. It acts on liver tissue to produce effector factors such as insulin-like growth factor-1 (IGF-1), exerting metabolic regulatory effects such as promoting growth and development. The rat model was used to test the growth hormone (GH)-stimulating effects of compounds of the present invention and to evaluate their effects on circulating GH levels.

[0550] Experimental animals:

[0551] Species: SD rats, Grade: SPF, Age: 6-8 weeks, Gender: 10 rats per group, half male and half female, Quality Certificate Number: No. 110324231103101327, Supplier: Sibeifu (Beijing) Biotechnology Co., Ltd., License Number: SCXK (Beijing) 2019-0010. Animals were acclimated for one week upon arrival and had free access to labeled chow and sterilized water. Cage bedding was changed twice weekly. Animals were fasted for 12 hours on the day before the study, but drinking water was not permitted.

[0552] Capromorelin and the compound of the present invention were accurately weighed into a glass bottle, and an appropriate amount of DMSO was added and vortexed until most of the particles were dissolved. Then, an appropriate amount of HS15, vortex mix, heat in a 60℃ water bath for 5 min, then add an appropriate amount of normal saline, vortex mix, and administer to animals. Prepare the drug immediately before use.

[0553] The experimental animals were administered the drug by oral gavage. Blood was collected from the jugular vein before administration (0 min), and 10, 20, 40, and 1 h after administration, with a blood volume of 0.2 mL each time. All collected whole blood samples were placed in 0.5 mL centrifuge tubes. After standing for half an hour, the tubes were centrifuged at 3000 rpm for 10 minutes. The supernatant was collected and stored at -80°C for GH content determination. GH levels in the plasma of each experimental animal were determined using the GH ELISA Kit (Cat. No.: EZRMGH-45K, Batch No.: 3891309) and Sigma-Aldrich enzyme-linked immunosorbent assay.

[0554] 2. Experimental Results

[0555] Table 7 shows the effects of the preferred compounds of the present invention on the GH levels in the blood circulation of SD rats.

[0556] Table 7 Effects of preferred compounds on GH concentration in rats

[0557] 3. Experimental Conclusion

[0558] As shown in Figure 1, 10 minutes after administration, compound G1a significantly increased circulating GH levels in rats, with the magnitude of the increase being much greater than that observed with capromorelin. Compound G1a exhibited greater efficacy than capromorelin at lower doses, demonstrating its superior ability to stimulate GH secretion compared to capromorelin.

[0559] Test Example 8: Subacute toxicity test of the preferred compounds of the present invention in rats

[0560] 1. Experimental Methods

[0561] Twenty-four SD rats (half male and half female) were randomly divided into four groups, with three males and three females in each group. The rats were administered orally by gavage at the following doses: Group 1 received the vehicle (once daily for 14 days), while Groups 2, 3, and 4 received compound G1a (10 mg / kg, 30 mg / kg, and 100 mg / kg, respectively, once daily for 14 days).

[0562] 2. Experimental Results

[0563] All animals were autopsied at the end of the dosing period. All animals survived until the scheduled autopsy, and no gross abnormalities or histological changes related to Compound G1a were observed.

[0564] 3. Experimental Conclusion

[0565] In the subacute toxicity test of the preferred compound G1a of the present invention in rats, no obvious abnormalities were observed at a dose of 100 mg / kg administered by oral gavage, indicating that compound G1a has good safety at a dose of at least 1000 times (compared to 0.1 mg / kg in Test Example 7).

[0566] The above is an exemplary description of the implementation methods of the technical solution of the present invention. It should be understood that the scope of protection of the present invention is not limited to the above implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the scope of protection of the claims of this application.

Claims

1. A compound of formula I, its racemate, stereoisomer, tautomer, nitrogen oxide, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof: in, X is selected from N or CR x ; R x is selected from oxo (=O) or phenyl; Y is selected from NR y or O; R y Selected from H, C 1-6 Alkyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, halogenated C 1-6 Alkyl, C 3-6 Cycloalkyl-C 1-6 alkyl; Z is selected from N or C-Ph; W is selected from N or CH; R1 and R2 are the same or different and are independently selected from H or C 1-6 Alkyl; or, R1, R2 together with the carbon atom to which they are attached form a 3-8 membered N-containing heterocyclic group; R3 is selected from H or amino; R4 is absent or selected from methylene substituted by R5; R5 is selected from unsubstituted or optionally substituted by 1, 2 or more R 51 Substituted phenyl or pyridyl; each R 51 are the same or different and are independently selected from halogen or CN; Each R a The same or different, independently selected from halogen, CN, C 1-6 alkyl; Each R b The same or different, independently selected from halogen, CN, C 1-6 Alkyl; or two R attached to the same carbon atom b Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; or two non-adjacent R groups attached to different carbon atoms b Connected by their end groups, together forming C 1-3 of alkylene; represents a single bond or a double bond; and when X is C(=O), is a single bond; m is selected from 0, 1, 2 or 3; n is selected from 0, 1, 2 or 3.

2. The compound according to claim 1, characterized in that X is selected from C(═O); Preferably, Y is selected from SRy; Ry is selected from C 3-6 Cycloalkyl or 3-6 membered O heterocyclic group; Preferably, Y is selected from NR y ; R y Selected from H, methyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 3-oxacyclobutane methyl, cyclopropylmethyl, tert-butyl, difluoromethyl; Preferably, Y is selected from NRy; Ry is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or 3-oxetanyl; Preferably, Z is selected from N; Preferably, W is selected from CH; Preferably, R1 and R2 are selected from methyl groups, and R3 is selected from amino groups; Preferably, R4 is absent or selected from benzyl, Preferably, R4 is benzyl.

3. The compound according to claim 1 or 2, characterized in that The compound represented by formula I is selected from the following structures: Among them, R y The method has the meaning as defined in any one of claims 1 or 2.

4. The compound according to any one of claims 1 to 3, characterized in that The compound represented by formula I is selected from the following structures:

5. The compound according to any one of claims 1 to 3, characterized in that The compound represented by formula I is selected from the following structures: Preferably, the compound represented by formula I is selected from the following structures:

6. A pharmaceutical composition comprising at least one of the compound according to any one of claims 1 to 5, its racemate, stereoisomer, tautomer, nitrogen oxide, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

7. Use of at least one of the compound according to any one of claims 1 to 5, its racemate, stereoisomer, tautomer, nitrogen oxide, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof, or the pharmaceutical composition according to claim 6 in the preparation of a drug, Preferably, the drug is a GHSR 1a agonist; Preferably, the medicament is used for diagnosing, preventing and / or treating diseases or conditions related to growth hormone deficiency or growth hormone dependence.

8. A method for diagnosing, preventing and / or treating a disease or condition requiring stimulation of the production or secretion of growth hormone, the method comprising administering to a patient in need of such treatment an effective amount of at least one compound according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 alone or, optionally, in combination with at least one other type of therapeutic agent.

9. The use according to claim 7 or the method according to claim 8, characterized in that The disease or condition is related to growth hormone deficiency or growth hormone dependence; preferably, the compound can promote the growth hormone level in the plasma of humans and animals after administration; preferably, it is used for the diagnosis and treatment of physiological or medical symptoms characterized by growth hormone secretion deficiency, such as the diagnosis of growth hormone deficiency patients, slow growth and short stature of children with growth hormone deficiency, energy balance and food intake regulation; treatment of fat formation, obesity and weight loss; treatment of cachexia; Improvement of gastrointestinal motility, treatment of gastroparesis and diabetic gastroparesis, postoperative ileus; increase in muscle mass and skin thickness, reduction in fat material and slight increase in bone density in the elderly patient population; treatment of burns, AIDS and cancer conditions, and healing of wounds and bones.

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