Steroidal compounds, uses and preparation methods thereof

Abstract

The present invention relates to steroid compounds, uses and preparation methods thereof; it is expected that such compounds can effectively treat psychoneurological diseases and have good activity efficacy, pharmacokinetic (PK) properties, oral bioavailability, stability, safety, clearance rate and / or metabolic properties, etc.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to steroid compounds, uses and preparation methods thereof. Technical Background

[0002] Neuropsychiatric diseases, including anxiety, depression, schizophrenia, etc., rank first in the total disease burden in my country, accounting for about 20% of the total disease burden (Wang Juncheng et al., China Health Management, 2009(5):348-350). With the continuous development of social modernization, the accelerated pace of work and increased pressure in life have led to a significant increase in the number of patients with various neuropsychiatric diseases, and the progression of symptoms has accelerated significantly. The development, research and production of drugs for neuropsychiatric diseases have become more urgent. At the same time, clinical data show that many patients suffer from multiple neuropsychiatric diseases at the same time. The high comorbidity rate of neuropsychiatric diseases makes clinical treatment face greater challenges.

[0003] In animals, GABA (gamma-aminobutyric acid) γ-aminobutyric acid only exists in nerve tissue. Immunological studies have shown that the area with the highest concentration is the substantia nigra in the brain. GABA is an important inhibitory neurotransmitter that has been studied in depth. It participates in a variety of metabolic activities and has high physiological activity. The GABA system is the main inhibitory signaling pathway in the brain and central nervous system, and is of great significance in regulating the function of the central nervous system. GABA (A) receptor (GABAAR) is an ionotropic receptor and ligand-gated ion channel. Its endogenous ligand γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. After the GABA (A) receptor is activated, it selectively allows Cl - Through its pores. - It flows out of neurons when the internal voltage is less than the resting potential and flows into neurons when the internal voltage is greater than the resting potential (i.e., -75 mV). This successfully reduces the chances of action potentials occurring, inhibiting neurotransmission. Anxiety and depression have a high comorbidity rate and are believed to overlap and co-occur. Anxiety and depression are also key emotional symptoms in schizophrenia. Research findings confirm that the GABAergic system and GABA(A) receptors play a crucial role in the etiology and pathology of all three disorders, suggesting that a pathophysiological process related to the GABAergic system may be a common determinant of these three diseases. The GABAergic system and GABA(A) receptors have been shown to be involved in the pathological processes of anxiety, depression, and schizophrenia at the molecular, preclinical, and clinical levels, and GABA(A) receptors have long been considered an important drug target for the treatment of these diseases.

[0004] Clinical trials have confirmed that GABA(A) receptors can not only bind to GABA itself to form a receptor complex (GRC) to change brain excitability, but also bind to other small molecule compounds with specific structures, such as barbiturates (trade name: succinate) drugs, benzodiazepines, These drugs, such as diazepam (trade name diazepam), bind to specific allosteric sites on the GABA(A) receptor to produce their therapeutic effects. Furthermore, studies have shown that the GABA(A) receptor also has a unique site for steroid compounds (Lan, NC et al., Neurochem. Res. 16:347-356 (1991)).

[0005] Neuroactive steroids can occur endogenously. The most potent endogenous neuroactive steroids are 3α-hydroxy-5-reduced pregnane-20-one and 3α-21-dihydroxy-5-reduced pregnane-20-one, which are metabolites of the hormonal steroids progesterone and deoxycorticosterone, respectively. The ability of these steroid metabolites to alter brain excitability was recognized in 1986 (Majewska, MD et al, Science 232: 1004-1007 (1986); Harrison, NL et al, J Pharmacol. Exp. Ther. 241: 346-353 (1987)). The ovarian hormone progesterone and its metabolites have been shown to have a profound effect on brain excitability (Backstrom, T. et al, Acta Obstet. Gynecol. Scand. Suppl. 130:19-24 (1985); Pfaff, D. Wan and McEwen, BS, et al, Science 219:808-814 (1983); Gyermek et al, J Med Chem. 11:117 (1968); Lambert, J. et al, Trends Pharmacol. Sci. 8:224-227 (1987)). The levels of progesterone and its metabolites vary with the phase of the menstrual cycle. It has been well documented that the levels of progesterone and its metabolites decrease before the onset of menstruation. It has also been well documented that some physical symptoms that recur monthly before the onset of menstruation have occurred. These symptoms, which have become associated with premenstrual syndrome (PMS), include stress (tension), anxiety, and migraines (Dalton, K., Premenstrual Syndrome and Progesterone Therapy, 2nd edition, Chicago Yearbook, Chicago (1984)). Subjects with PMS have monthly recurring symptoms that are present before their period and absent after their period.

[0006] Other studies have also found that a decrease in progesterone is associated with an increase in the frequency of seizures in female epileptic patients, namely catamenial epilepsy (Laidlaw, J., Lancet, 1235-1237 (1956)). A more direct correlation has been observed for a decrease in progesterone metabolites (Rosciszewska et al., J. Neurol. Neurosurg. Psych. 49:47-51 (1986)). In addition, for patients with primary generalized petit mal epilepsy, the incidence of disease has been correlated with the incidence of premenstrual symptoms (Backstrom, T. et al., J. Psychosom. Obstet. Gynaecol. 2:8-20 (1983)). The steroid deoxycorticosterone has been found to be effective in treating patients with epilepsy related to the menstrual cycle (Aird, R. B. and Gordan, G., J. Amer. Med. Soc. 145:715-719 (1951)).

[0007] In addition to the above symptoms, postpartum depression (PND) is also associated with low progesterone levels. Immediately after childbirth, progesterone levels drop sharply, leading to the onset of PND. The symptoms of PND range from mild depression to psychosis requiring hospitalization. PND is also associated with severe anxiety and irritability. PND-related depression cannot be treated with classic antidepressants, and the probability of PMS occurring in women experiencing PND increases (Dalton, K., Premenstrual Syndrome and Progesterone Therapy, 2nd edition, Chicago Yearbook, Chicago (1984)). These observations more or less suggest that progesterone and deoxycorticosterone, particularly their metabolites, play a key role in regulating brain excitability, and their related clinical indications are shown as catamenial epilepsy, PMS, and PND.

[0008] Numerous studies have focused on GABA(A) receptors in an effort to identify effective drugs for treating related diseases. CN103958540A and CN10533928A, among others, disclose a series of neurosteroid compounds for the treatment of neuropsychiatric disorders. GABA(A) is a subtype of the γ-aminobutyric acid receptor (GABAR). There are three GABA receptor subtypes: GABA(A), GABA(B), and GABA(C). The GABA(A) receptor is a chloride ion channel.

[0009] GABA(A) is composed of five subunits (polypeptide chains) assembled into a pentagonal hetero-oligomer structure. Two pairs of these subunits are identical, so a specific GABA(A) receptor contains three types of subunits: the most common α, β, and γ. Each subunit has several different subtypes, such as α, which includes α1, α2, α3, α4, α5, and α6; β, which includes β1, β2, and β3; and γ, which includes γ1, γ2, γ3, and γ4. In addition, subunits such as δ, ε, ρ1-3, θ, and π can form pentamers with α and β.

[0010] According to statistics, there are currently 23 different subunit combinations of GABA(A) receptors. The specific combination of ligands is related to the distribution area of ​​brain neurons. For example, GABA(A) receptors containing γ subunits are mainly distributed in synapses, while GABA(A) receptors containing δ subunits are mainly distributed outside synapses (extrasynaptic). (Activation of GABA(A)Receptors:Views from Outside the Synaptic Cleft, Neuron.vol 56, 2007, 763-770); According to existing research (Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites: Nature.Vol 444.23 November.2006.486-489), endogenous steroid compounds such as tetrahydrodeoxycorticosterone (THDOC) can directly activate the receptor subtype α1β2γ2 (hereinafter referred to as GABA) in synapses at high concentrations (micromolar level) A1 ), while at lower concentrations (submicromolar), it can enhance the amplitude of GABA's effect on α1β2γ2.

[0011]

[0012] Steroid compounds act on extrasynaptic receptor subtypes (e.g., α4β3δ, hereinafter referred to as GABA A4 ) There are no reports of direct activation. Summary of the Invention

[0013] One of the objectives of the present invention is to provide a steroidal compound with enhanced efficacy for the more effective treatment of neuropsychiatric disorders. The compounds of the present invention are expected to provide excellent potency, pharmacokinetic (PK) properties, oral bioavailability, formulatability, stability, safety, clearance and / or metabolic properties, and reduced side effects such as anesthesia and sedation.

[0014] Based on the above purpose, the present invention provides a compound of formula I and a pharmaceutically acceptable salt thereof:

[0015]

[0016] wherein R1 is hydrogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, or substituted or unsubstituted C3-6 carbocyclyl;

[0017] R2 is hydrogen, halogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted

[0018] substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-6 carbocyclyl;

[0019] R3 is hydrogen, unsubstituted C1-6 alkyl or -CH2OR a , where R a is hydrogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted

[0020] or an unsubstituted C3-6 carbocyclyl;

[0021] L is -C(Rb)(Rb)-, each Rb is independently hydrogen or C1-C6 alkyl, n is an integer from 0 to 3,

[0022] R4 is halogen or substituted or unsubstituted heteroaryl or heterocyclic group.

[0023] Further preferred compounds, wherein R1 is hydrogen, substituted or unsubstituted C1-6 alkyl; R2 is hydrogen, substituted or unsubstituted C1-6 alkyl; R3 is hydrogen, unsubstituted C1-6 alkyl; Rb is hydrogen, n is an integer of 1-2; R4 is heteroaryl, and is replaced by cyano, nitro, hydroxyl, halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, -C(O)Rd, -C(O)N(Re)(Rf), -C(O)O(Rd), -N(Re)(Rf), -OC(O)N(Re)(Rf), -OC(O)O(Rd), -OC(O)Rd, -S(O) 0-2 Rd, -S(O) 0-2 ORd or -S(O) 0-2 N(Re)(Rf) is optionally substituted; each Rd is hydrogen or C1-C6 alkyl; each Re and Rf is independently hydrogen, C1-C6 alkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl.

[0024] Further preferred compounds are those in which R1 is hydrogen or methyl; R2 is hydrogen; R3 is hydrogen; L is CH2; and R4 is a monocyclic or bicyclic heteroaryl or a monocyclic or bicyclic heterocyclic group, wherein the heteroatom is oxygen, nitrogen or sulfur, and the bicyclic ring is selected from a spirocyclic ring or a fused ring.

[0025] In a further preferred compound, R4 is a 5-membered or 6-membered heteroaryl group containing 2-4 nitrogen atoms, and is arbitrarily substituted by cyano, nitro, hydroxyl, halo, C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 haloalkyl.

[0026] Or as shown in the compound of formula II:

[0027]

[0028]

[0029] wherein R1 is hydrogen, C1-3 alkyl substituted or unsubstituted by halogen; R2 is hydrogen, halogen, C1-6 alkyl substituted or unsubstituted by halogen, or C1-6 alkoxy; X is CH2, N, O, or S; and R5 is selected from the following groups:

[0030]

[0031] Wherein R6 is H, halogen, CN, CF3, NO2, C1-6 alkyl or C1-6 alkoxy which is substituted or unsubstituted by halogen.

[0032] Alternatively, the compound of the present invention is preferably selected from:

[0033]

[0034]

[0035]

[0036] The present invention also provides a pharmaceutical composition comprising any one of the above compounds or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable excipient.

[0037] The compounds described herein can act as GABA modulators, e.g., affecting GABA(A) receptors in a positive or negative manner. As modulators of central nervous system (CNS) excitability, such compounds are expected to have CNS activity when mediated through their ability to modulate GABA(A) receptors.

[0038] The present invention further provides the use of the above-mentioned compound or pharmaceutical composition in the preparation of a medicament for preventing or treating a nervous system disease; wherein the nervous system disease is preferably selected from the group consisting of sleep disorders, mood disorders, schizophrenia spectrum disorders, spastic disorders, memory disorders and / or cognitive disorders, movement disorders, personality disorders, autism spectrum disorders, pain, traumatic brain injury, vascular disease, substance abuse disorders, and / or truncation syndrome or tinnitus; and the mood disorder is depression; wherein the depression is disruptive mood dysregulation disorder, major depressive disorder, persistent depressive disorder, premenstrual dysphoric disorder, a disorder caused by a substance or medication, a disorder caused by another medical condition, other specified depressive disorders, and unspecified depressive disorders, preferably mild depression, moderate depression, severe depression, or postpartum depression. Severe depression or postpartum depression is further preferred. Furthermore, the above-mentioned compound or pharmaceutical composition can be administered orally, subcutaneously, intravenously, or intramuscularly.

[0039] definition

[0040] Chemical definition

[0041] The compounds described herein may include one or more asymmetric centers and may therefore exist in a variety of isomeric forms, for example, enantiomers and / or diastereomeric forms. For example, the compounds described herein may be individual enantiomers, diastereomers, or geometric isomers, or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be separated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis.

[0042] The following terms are intended to have the meanings provided below and are useful in understanding the description and intended scope of the present invention. When describing the present invention, compounds, pharmaceutical compositions containing the compounds, and test methods for the compounds and compositions may be included. The definitions of terms involved in the present invention may refer to the following description. Any of the moieties defined below may be substituted with a variety of substituents, and the corresponding definitions are within their scope listed below, including such substituted moieties. Unless otherwise indicated, the term "substituted" is defined as follows.

[0043] "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 20 carbon atoms ("C1-20 alkyl"). In some embodiments, the alkyl group has 1 to 12 carbon atoms ("C1-12 alkyl"). In some embodiments, the alkyl group has 1 to 10 carbon atoms ("C1-10 alkyl"). In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C1-9 alkyl"). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C1-8 alkyl"). In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C1-7 alkyl"). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C1-6 alkyl", also referred to herein as "lower alkyl"). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C1-5 alkyl"). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C1-4 alkyl"). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C1-3 alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C1-2 alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("C1 alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-6 alkyl"). Examples of C1-6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). Other examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Unless otherwise specified, each of the alkyl groups is independently optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"); for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In some embodiments, the alkyl group is an unsubstituted C1-10 alkyl group (e.g., -CH3). In some embodiments, the alkyl group is a substituted C1-10 alkyl group.

[0044] "Alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds), and optionally one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds) ("C2-20 alkenyl"). In some embodiments, the alkenyl group does not contain any triple bonds. In some embodiments, the alkenyl group has 2 to 10 carbon atoms ("C2-10 alkenyl"). In some embodiments, the alkenyl group has 2 to 9 carbon atoms ("C2-9 alkenyl"). In some embodiments, the alkenyl group has 2 to 8 carbon atoms ("C2-8 alkenyl"). In some embodiments, the alkenyl group has 2 to 7 carbon atoms ("C2-7 alkenyl"). In some embodiments, the alkenyl group has 2 to 6 carbon atoms ("C2-6 alkenyl"). In some embodiments, the alkenyl group has 2 to 5 carbon atoms ("C2-5 alkenyl"). In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("C2-4 alkenyl"). In some embodiments, an alkenyl group has 2 to 3 carbon atoms ("C2-3 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl"). One or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). Examples of C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include the C2-4 alkenyl groups described above, as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Other examples of alkenyl groups include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each alkenyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents; for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In some embodiments, an alkenyl group is an unsubstituted C2-10 alkenyl group. In some embodiments, an alkenyl group is a substituted C2-10 alkenyl group.

[0045] "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds) ("C2-20 alkynyl"). In some embodiments, the alkynyl group does not contain any double bonds. In some embodiments, the alkynyl group has 2 to 10 carbon atoms ("C2-10 alkynyl"). In some embodiments, the alkynyl group has 2 to 9 carbon atoms ("C2-9 alkynyl"). In some embodiments, the alkynyl group has 2 to 8 carbon atoms ("C2-8 alkynyl"). In some embodiments, the alkynyl group has 2 to 7 carbon atoms ("C2-7 alkynyl"). In some embodiments, the alkynyl group has 2 to 6 carbon atoms ("C2-6 alkynyl"). In some embodiments, the alkynyl group has 2 to 5 carbon atoms ("C2-5 alkynyl"). In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C2-4 alkynyl"). In some embodiments, an alkynyl group has 2 to 3 carbon atoms ("C2-3 alkynyl"). In some embodiments, an alkynyl group has 2 carbon atoms ("C2 alkynyl"). One or more carbon triple bonds can be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of C2-4 alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groups include the above-mentioned C2-4 alkynyl groups, as well as pentynyl (C5), hexynyl (C6), and the like. Other examples of alkynyl groups include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each alkynyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents (a "substituted alkynyl"); for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In some embodiments, the alkynyl group is an unsubstituted C2-10 alkynyl group. In some embodiments, the alkynyl group is a substituted C2-10 alkynyl group.

[0046] "Aryl" refers to a group having a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) with 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ("C6-14 aryl"). In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has fourteen ring carbon atoms ("C14 aryl"; e.g., anthracenyl). "Aryl" also includes ring systems in which the aryl ring is fused to one or more carbocyclic or heterocyclic groups, wherein the radical or point of attachment is on the aryl ring, in which case the number of carbon atoms continues to refer to the number of carbon atoms in the aryl ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, coronene, fluoranthene, fluorene, hexacene, indane, indene, naphthalene, octacene, octalene, octalene, olphene, penta-2,4-diene, pentacene, pentalene, pentalene, perylene, phenalene, phenanthren, chrysene, septalene, pyrene, pyranthracene, rubrocene, triphenylene, and terapthyl. Specifically, aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Unless otherwise specified, each aryl group is independently optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted ("substituted aryl") with one or more substituents. In some embodiments, aryl groups are unsubstituted C6-14 aryl groups. In some embodiments, aryl groups are substituted C6-14 aryl groups. In some embodiments, the aryl group is substituted with one or more groups selected from halogen, C1-C8 alkyl, C1-C8 haloalkyl, cyano, hydroxy, C1-C8 alkoxy, and amino.

[0047] "Heteroaryl" refers to a group having a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms provided in an aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom as valence permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which the above-mentioned heteroaryl rings are fused to one or more carbocyclyls or heterocyclyls, wherein the point of attachment is on the heteroaryl ring, and in such cases, the number of ring members continues to represent the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which the above-mentioned heteroaryl rings are fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in such cases, the number of ring members represents the number of ring members in the fused (aryl / heteroaryl) ring system. For bicyclic heteroaryls in which one ring does not contain heteroatoms (e.g., indolyl, quinolyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., on the ring with heteroatoms (e.g., 2-indolyl) or on the ring without heteroatoms (e.g., 5-indolyl). In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms (present in the aromatic ring system), wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In some embodiments, heteroaryl is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms (present in the aromatic ring system), wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some embodiments, heteroaryl is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms (present in the aromatic ring system), wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl"). In some embodiments, 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each heteroaryl group is independently optionally substituted, i.e., unsubstituted ("unsubstituted heteroaryl") or substituted ("substituted heteroaryl") with one or more substituents. In some embodiments, heteroaryl is an unsubstituted 5-14 membered heteroaryl group. In some embodiments, heteroaryl is a substituted 5-14 membered heteroaryl group. 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl.Five-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Six-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Six-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Six-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Seven-membered heteroaryl groups containing one heteroatom include, but are not limited to, azacycloheptatrienyl, oxepantatrienyl, and thiepantatrienyl. 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indanizinyl, and purinyl. 6,6-Bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0048] "Carbocyclyl" or "carbocycle" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms in a non-aromatic ring system ("C3-10 carbocyclyl"). In some embodiments, the carbocyclyl has 3 to 8 ring carbon atoms ("C3-8 carbocyclyl"). In some embodiments, the carbocyclyl has 3 to 6 ring carbon atoms ("C3-6 carbocyclyl"). In some embodiments, the carbocyclyl has 3 to 6 ring carbon atoms ("C3-6 carbocyclyl"). In some embodiments, the carbocyclyl has 5 to 10 ring carbon atoms ("C5-10 carbocyclyl"). Exemplary C3-6 carbocyclyls include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-8 carbocyclyl groups include, but are not limited to, the aforementioned C3-6 carbocyclyl groups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), etc. Exemplary C3-10 carbocyclyl groups include, but are not limited to, the aforementioned C3-8 carbocyclyl groups, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthyl (C10), spiro[4.5]decyl (C10), etc. As illustrated in the foregoing examples, in some embodiments, carbocyclyl is a monocycle ("monocyclic carbocyclyl") or a carbocyclyl comprising a fused ring system, a bridged ring system or a spirocyclic ring system, such as a bicyclic system ("bicyclic carbocyclyl"), and may be saturated or may be partially unsaturated carbocyclyl. "Carbocyclyl" also includes a ring system in which the above-mentioned carbocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the carbocyclyl ring, and in such a case, the number of carbons continues to represent the number of carbons in the carbocyclyl system. Unless otherwise stated, each of the carbocyclyl groups is independently optionally substituted, that is, unsubstituted ("unsubstituted carbocyclyl") or substituted ("substituted carbocyclyl") by one or more substituents. In some embodiments, the carbocyclyl group is an unsubstituted C3-10 carbocyclyl. In some embodiments, the carbocyclyl group is a substituted C3-10 carbocyclyl. In some embodiments, a “carbocyclyl” is a monocyclic, saturated carbocyclyl having 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”).Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C6). Examples of C3-6 cycloalkyl groups include the above-mentioned C5-6 cycloalkyl groups, as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the above-mentioned C3-6 cycloalkyl groups, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of cycloalkyl is independently unsubstituted ("unsubstituted cycloalkyl") or substituted ("substituted cycloalkyl") with one or more substituents. In some embodiments, cycloalkyl groups are unsubstituted C3-10 cycloalkyl groups. In some embodiments, cycloalkyl groups are substituted C3-10 cycloalkyl groups.

[0049] "Heterocyclyl" or "heterocycle" refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3-10 membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. A heterocyclyl group may be monocyclic ("monocyclic heterocyclyl") or a fused ring system, a bridged ring system, or a spirocyclic ring system, such as a bicyclic ring system ("bicyclic heterocyclyl"), and may be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems may include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which the above-mentioned heterocyclyl ring is fused to one or more carbocyclyl groups, wherein the point of attachment is on the carbocyclyl or heterocyclyl ring, or ring systems in which the above-mentioned heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each of the heterocyclyl groups is independently optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In some embodiments, the heterocyclyl group is an unsubstituted 3-10 membered heterocyclyl group. In some embodiments, the heterocyclyl group is a substituted 3-10 membered heterocyclyl group. In some embodiments, the heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon ("5-10 membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, the heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl group has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl group has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl group has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0050] "Hetero" when used to describe a compound or a group present on a compound means that one or more carbon atoms in the compound or group have been replaced by nitrogen, oxygen, or sulfur heteroatoms. Hetero can be applied to any of the above-mentioned hydrocarbon groups: for example, alkyl groups, such as heteroalkyl; cycloalkyl groups, such as heterocyclyl; aryl groups, such as heteroaryl; cycloalkenyl groups, such as cycloheteroalkenyl, etc.; which have 1 to 5 heteroatoms, especially 1 to 3 heteroatoms.

[0051] "Alkoxy" refers to the group -OR, where R is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. Specific alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy. Specific alkoxy groups are lower alkoxy groups, i.e., having 1 to 6 carbon atoms. Further specific alkoxy groups have 1-4 carbon atoms. Exemplary "substituted alkoxy groups" include, but are not limited to: -O-(CH 2 )t(C6-C10 aryl), -O-(CH2)t(5-10 membered heteroaryl), -O-(CH2)t(C3-C10 cycloalkyl) and -O-(CH2)t(4-10 membered heterocyclyl), wherein t is an integer from 0 to 4, and any aryl, heteroaryl, cycloalkyl or heterocyclyl present may itself be substituted with unsubstituted C1-C4 alkyl, halogen, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy.

[0052] "Carboxy" refers to the group -C(O)OH.

[0053] "Cyano" refers to the radical -CN.

[0054] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). In some embodiments, the halo group is fluorine or chlorine.

[0055] "Hydroxy" refers to the radical -OH.

[0056] "Nitro" refers to the radical -NO2.

[0057] Other definitions

[0058] The term "pharmaceutically acceptable salt" refers to salts that are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed with an amino group and inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or salts formed with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts formed using methods used in the art, such as ion exchange methods. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and tetraalkylammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts and amine cations formed using counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates, if appropriate. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The change of plasma and brain content of the reference substance over time

[0060] Figure 2 The changes of KH022 plasma and brain levels over time DETAILED DESCRIPTION

[0061] In order to better explain the technical solution of the present invention, the present invention provides synthesis or biological examples of some compounds, which are not intended to further limit the scope of protection of the invention.

[0062] Materials and methods

[0063] The compounds provided herein can be prepared from readily available starting materials using the following general methods and processes. It should be understood that under the typical or preferred process conditions given (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.), other process conditions may also be used unless otherwise stated. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by conventional optimization by those skilled in the art.

[0064] Additionally, it will be apparent to those skilled in the art that conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesirable reactions. The selection of suitable protecting groups for specific functional groups and suitable conditions for protection and deprotection are well known in the art.

[0065] The compounds provided herein can be isolated and purified by known standard methods. Such methods include, but are not limited to, recrystallization, column chromatography, HPLC, or supercritical fluid chromatography (SFC). The following reaction schemes are provided for the preparation details of the representative pyrazoles listed herein. The compounds provided herein can be prepared by those skilled in the art of organic synthesis from known or commercially available raw materials and reagents.

[0066] The compounds of the present invention can be prepared according to methods described in the art and using appropriate reagents, starting materials and purification methods known to those skilled in the art.

[0067] Example 1 Synthesis of Compound KH001

[0068]

[0069] Compound KHC-2: To a 4 L methanol solution of compound KHC-1 (100.0 g, 0.3 mol) was added potassium cyanide (600.0 g, 9.2 mol) and acetic acid (640 mL), respectively. The reaction was stirred at room temperature overnight. The reaction solution was then poured into water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The resulting crude product was dissolved in acetic acid (2.5 L). Platinum dioxide (24.0 g, 0.1 mol) was added to the reaction solution and hydrogenated with hydrogen. TLC indicated the reaction was complete. The reaction solution was filtered, the filtrate concentrated to approximately 200 mL, and diluted with 2.4 L of ice water. An aqueous solution of sodium nitrite (46.0 g, 0.7 mol) was slowly added dropwise to the mixture. After the addition was complete, the mixture was stirred at room temperature for 15 hours. The mixture was then extracted with ethyl acetate. The combined organic layers were washed with brine, dried, and concentrated. The crude product was purified by silica gel column chromatography to yield compound KHC-2 (57.3 g, 55% yield).

[0070] Compound KHC-3: Under a nitrogen atmosphere, ethynylmagnesium bromide (395 mL, 0.5 M) was added dropwise to a tetrahydrofuran solution (1 L) of compound KHC-2 (57.3 g, 164.5 mmol) at -78°C, then gradually warmed to room temperature and stirred at room temperature overnight. Saturated aqueous ammonium chloride was slowly added dropwise to the mixture, followed by extraction with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography to obtain compound KHC-3 (33.7 g, 55% yield).

[0071] Compound KHC-4: Compound KHC-3 (33.7 g, 90.5 mmol) was dissolved in a mixed solvent of acetic acid (200 mL) and water (20 mL). Dowex-50 (10.0 g) was then added and the mixture was heated under reflux. After monitoring the reaction for completion, the mixture was cooled to room temperature, the resin was filtered, washed with ethyl acetate, and the filtrate was neutralized with a 40% aqueous sodium hydroxide solution. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography to obtain compound KHC-4 (23.6 g, 70% yield).

[0072] Compound KHC-5: Compound KHC-4 (23.6 g, 63.3 mmol) and palladium black (1.2 g) (in tetrahydrofuran (200 mL)) were hydrogenated with hydrogen gas. After stirring at room temperature overnight, the mixture was filtered through a celite pad, washed with ethyl acetate, and the filtrate was concentrated in vacuo to provide a crude compound. Recrystallization from acetone gave compound KHC-5 (19.0 g, 80% yield).

[0073] Compound KH001: To a solution of compound KHC-5 (19.0 g, 50.8 mmol) in tetrahydrofuran (200 mL) was slowly added dropwise an aqueous sodium hydroxide solution (50 mL, 1 M). After stirring at room temperature overnight, the pH was adjusted with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography to obtain compound KHC (13.5 g, 80% yield). 1 HNMR(400MHz, CDCl3), δ4.06(s,1H),2.32-2.28(dd,1H),2.15(s,3H),1.84-1.77(m,2H),1.74-1.61(m,5H),1.61-1 .54(m,3H),1.48-1.46(m,1H),1.33-1.14(m,10H),1.02-0.95(m,1H),0.93(s,3H),0.85-0.79(m,3H),0.75(s,3H).

[0074] Example 2 Synthesis of Compound KH002

[0075]

[0076] Compound KHC-6: Compound KH001 (1.5 g, 4.5 mmol), synthesized in Example 1, was dissolved in methanol (40 mL) until clear. The mixture was stirred and cooled to an internal temperature of 5°C in an ice bath. Bromine (1.5 g, 9.2 mmol) was then slowly added dropwise. The ice bath was removed and the reaction was allowed to react at room temperature for 4 hours. The orange color of the reaction solution turned pale yellow, and TLC monitoring indicated the reaction was complete. 30 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 35 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. Purification by flash chromatography on a silica gel column (eluting with dichloromethane) afforded a white solid (1.0 g, 56.7%).

[0077] Compound KH002: Compound KHC-6 (80 mg, 0.2 mmol) was dissolved in tetrahydrofuran (5 mL) until clear. K2CO3 (83 mg, 0.6 mmol) and 4-cyanopyrazole (56 mg, 0.6 mmol) were added and allowed to react at room temperature overnight. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. Purification by flash chromatography on a silica gel column (eluted with dichloromethane / methanol = 100:1) followed by recrystallization (ethyl acetate / petroleum ether = 1:10) afforded a white solid (36 mg, 42.5%). 1 HNMR(400MHz, CDCl3)δ7.82(s,1H),7.80(s,1H),5.06-4.93(m,2H),4.05-4.04(m,1H),2.32(dd,1H),1.88-1.83(m,2H),1.79-1.6 6(m,4H),1.64-1.58(m,2H),1.54-1.44(m,4H),1.39-1.13(m,9H),1.04-0.97(m,1H),0.94(s,3H),0.85-0.75(m,3H),0.74(s,3H).

[0078] Example 3 Synthesis of Compound KH003

[0079]

[0080] Compound KH003: Compound KHC-6 (50 mg, 0.1 mmol) was dissolved in tetrahydrofuran (5 mL) until the solution was clear. K2CO3 (50 mg, 0.3 mmol) and 1H-triazole (25 mg, 0.3 mmol) were added. The reaction was allowed to react at room temperature overnight. TLC monitoring indicated the reaction was complete. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. Purification was performed by flash chromatography on a silica gel column (eluted with dichloromethane / methanol = 150:1) followed by recrystallization (ethyl acetate / petroleum ether = 1:10) to obtain a white solid (17 mg, 35.5%). 1 HNMR(400MHz, CDCl3)δ7.76(s,1H),7.60(s,1H),5.31-5.17(m,2H),4.06-4.05(m,1H),2.37(d,1H),1 .87-1.61(m,8H),1.52-1.10(m,13H),1.04-0.98(m,1H),0.95(s,3H),0.92-0.79(m,3H),0.74(s,3H).

[0081] Example 4 Synthesis of Compounds KH004 and KH005

[0082]

[0083] Compounds KH004 and KH005: Compound KHC-6 (103 mg, 0.2 mmol) was dissolved in acetonitrile (15 mL) until clear, and Cs2CO3 (244 mg, 0.7 mmol) and 1H-tetrazole (53 mg, 0.7 mmol) were added. The reaction was allowed to react overnight at room temperature. TLC monitoring of the reaction showed that the reaction was complete. 10 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (2*25 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a pale yellow solid crude product. Purification by flash chromatography on a silica gel column (eluted with dichloromethane / methanol = 150:1 to 90:1) gave two compounds, which were then recrystallized separately (ethyl acetate / petroleum ether = 1:10) to give white solid 1 (24 mg, 24.0%) and white solid 2 (36 mg, 36.0%). White solid 1 (KH004) 1HNMR (400 MHz, CDCl3) δ 8.57 (s, 1H), 5.50 (s, 2H), 4.06-4.05 (m, 1H), 2.35 (dd, 1H), 1.92-1.81 (m, 3H), 1.74-1.68 (m, 3H), 1.64-1.57 (m, 4H), 1.46-1.11 (m, 10H), 1.05-0.98 (m, 2H), 0.96 (s, 3H), 0.88-0.78 (m, 3H), 0.75 (s, 3H). White solid 2 (KH005) 1 HNMR(400MHz, CDCl3)δ8.71(s,1H),5.38-5.23(m,2H),4.06-4.05(m,1H),2.41(dd,1H),2.28(dd,1H),1.91-1.7 2(m,4H),1.68-1.51(m,7H),1.47-1.11(m,10H),1.06-0.99(m,1H),0.95(s,3H),0.91-0.78(m,3H),0.75(s,3H).

[0084] Example 5 Synthesis of Compound KH006

[0085]

[0086] Compound KH006: Compound KHC-6 (50 mg, 0.1 mmol) was dissolved in acetonitrile (8 mL) until clear, and Cs2CO3 (117 mg, 0.3 mmol) and 1H-pyrazole (24 mg, 0.3 mmol) were added. The reaction was allowed to react at room temperature overnight. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. The product was purified by flash chromatography on a silica gel column (eluted with dichloromethane / methanol = 100:1) and then recrystallized (ethyl acetate / petroleum ether = 1:10) to obtain a white solid (24 mg, 50.2%). 1 HNMR(400MHz, CDCl3)δ7.56(d,1H),7.39(d,1H),6.34(s,1H),5.03-4.92(m,2H),4.05-4.04(m,1H),2.28(dd,1H),1.86-1.73(m ,4H),1.71-1.61(m,3H),1.60-1.49(m,4H),1.48-1.09(m,10H),1.03-0.97(m,1H),0.95(s,3H),0.81-0.76(m,3H),0.74(s,3H).

[0087] Example 6 Synthesis of Compound KH007

[0088]

[0089] Compound KH007: Compound KHC-6 (150 mg, 0.3 mmol) was dissolved in tetrahydrofuran (10 mL) until clear, and KCO (152 mg, 1.1 mmol) and 1H-imidazole (75 mg, 1.1 mmol) were added. The reaction was allowed to react at room temperature overnight. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. Purification was performed by flash chromatography on a silica gel column (eluted with dichloromethane / methanol = 30:1) and then recrystallized from ethyl acetate to obtain a white solid (35 mg, 24.1%). 1 HNMR(400MHz, CDCl3)δ7.47(s,1H),7.11(s,1H),6.85(s,1H),4.74(d,2H),4.06-4.05(m,1H),2.30(dd,1H),1.88-1.68(m,4H ),1.64-1.56(m,3H),1.52-1.45(m,4H),1.40-1.11(m,10H),1.04-0.97(m,1H),0.95(s,3H),0.83-0.77(m,3H),0.75(s,3H).

[0090] Example 7 Synthesis of Compound KH008

[0091]

[0092] Compound KH008: Compound KHC-6 (100 mg, 0.2 mmol) was dissolved in tetrahydrofuran (10 mL) until clear, and K2CO3 (97 mg, 0.7 mmol) and 3-cyanopyrazole (68 mg, 0.7 mmol) were added. The mixture was heated at 70°C for 2 hours. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was then extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. The product was purified by preparative TLC chromatography (developing solvent: petroleum ether / acetone = 3:1) and then recrystallized from acetonitrile to obtain a white solid (20 mg, 19.4%). 1HNMR(400MHz, CDCl3)δ7.44(d,1H),6.72(d,1H),6.34(s,1H),5.00(q,2H),4.06-4.05(m,1H),2.32(dd,1H),1.88-1.68(m,4H ),1.63-1.60(m,3H),1.50-1.45(m,4H),1.41-1.11(m,10H),1.04-0.97(m,1H),0.95(s,3H),0.87-0.79(m,3H),0.75(s,3H).

[0093] Example 8 Synthesis of Compound KH009

[0094]

[0095] Compound KH009: Compound KHC-6 (50 mg, 0.1 mmol) was dissolved in acetonitrile (5 mL) until clear, and Cs2CO3 (120 mg, 0.3 mmol) and 1H-indole (68 mg, 0.7 mmol) were added. The reaction was allowed to react at room temperature overnight. TLC monitoring indicated the reaction was complete. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. Purification by preparative TLC chromatography (developing solvent: dichloromethane / ethyl acetate = 10:1) afforded a pale yellow solid (34 mg, 62.4%). 1 HNMR(400MHz, CDCl3)δ7.65-7.63(m,1H),7.21-7.18(m,1H),7.13-7.10(m, 2H),7.01-7.00(m,1H),6.57(d,1H),4.83(d,2H),4.06-4.05(m,1H),2.30( dd,1H),1.82-1.72(m,4H),1.64-1.61(m,3H),1.51-1.45(m,4H),1.41-1.1 1(m,10H),1.01-0.96(m,1H),0.96(s,3H),0.92-0.77(m,3H),0.74(s,3H).

[0096] Example 9 Synthesis of Compound KH010

[0097]

[0098] Compound KH010: Compound KHC-6 (100 mg, 0.2 mmol) was dissolved in acetonitrile (5 mL) until clear, and K2CO3 (101 mg, 0.7 mmol) and 4-(trifluoromethyl)-1H-pyrazole (35 mg, 0.2 mmol) were added. The mixture was heated to 50°C for 2 hours. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. The product was purified by flash chromatography on a silica gel column (developing solvent: petroleum ether / acetone = 7:1), followed by recrystallization from acetonitrile to obtain a white solid (61 mg, 53.1%). 1 HNMR(400MHz, CDCl3)δ7.71(d,2H),4.98(q,2H),4.06-4.05(m,1H),2.32(dd,1H),1.87-1.68(m,4H),1.64-1.57 (m,3H),1.52-1.45(m,4H),1.41-1.11(m,10H),1.04-0.97(m,1H),0.95(s,3H),0.87-0.82(m,3H),0.74(s,3H).

[0099] Example 10 Synthesis of Compound KH011

[0100]

[0101] Compound KH011: Compound KHC-6 (50 mg, 0.1 mmol) was dissolved in acetonitrile (5 mL) until clear, and Cs2CO3 (120 mg, 0.3 mmol) and 1H-indazole (43 mg, 0.3 mmol) were added. Stir at room temperature overnight. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. Purification by preparative TLC chromatography (developing solvent: petroleum ether / acetone = 3:1) and then recrystallization (acetonitrile) afforded a white solid (20 mg, 36.6%). 1HNMR(400MHz, CDCl3)δ8.11(s,1H),7.78(d,1H),7.45-7.41(m,1H),7.23-7.19(m,2H),5.31-5.20(m,2H),4.06-4.05(m,1H),2.35(dd,1H),1 .87–1.72(m,4H),1.67-1.63(m,3H),1.54-1.45(m,4H),1.42-1.14(m, 10H),1.01-0.98(m,1H),0.96(s,3H),0.93-0.78(m,3H),0.75(s,3H).

[0102] Example 11 Synthesis of Compound KH012

[0103]

[0104] Compound KH012: Compound KHC-6 (50 mg, 0.1 mmol) was dissolved in acetonitrile (5 mL) until clear, and Cs2CO3 (120 mg, 0.3 mmol) and 1H-purine (44 mg, 0.3 mmol) were added. Stir at room temperature overnight. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. The product was purified by preparative TLC chromatography (developing solvent: dichloromethane / methanol = 20:1) and then recrystallized from acetonitrile to obtain a white solid (5 mg, 9.1%). 1 HNMR(400MHz, CDCl3)δ9.22(s,1H),9.06(s,1H),8.25(s,1H),5.18(q,2H),4.07-4.06(m,1H),2.49(dd,1H),1.93-1.72(m,4H ),1.68-1.65(m,3H),1.58-1.45(m,4H),1.42-1.13(m,10H),1.06-1.00(m,1H),0.96(s,3H),0.90-0.80(m,3H),0.74(s,3H).

[0105] Example 12 Synthesis of Compound KH013

[0106]

[0107] Compound KH013: Compound KHC-6 (80 mg, 0.2 mmol) was dissolved in acetonitrile (8 mL) until clear, and Cs2CO3 (196 mg, 0.6 mmol) and 4-chloropyrazole (62 mg, 0.6 mmol) were added. The reaction was allowed to react at room temperature overnight. TLC monitoring indicated completion of the reaction. 15 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 30 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude pale yellow solid. Purification by flash chromatography on a silica gel column (eluted with dichloromethane / methanol = 100:1) followed by recrystallization (ethyl acetate / petroleum ether = 1:10) afforded a white solid (28 mg, 32.3%). 1 HNMRδ7.46(s,1H),7.38(s,1H),4.96-4.83(m,2H),4.06-4.05(m,1H),2.19(dd,1H),1.86-1.71(m,4H),1.64-1.5 5(m,3H),1.51-1.45(m,4H),1.45-1.10(m,10H),1.03-0.96(m,1H),0.95(s,3H),0.86-0.77(m,3H),0.74(s,3H).

[0108] Example 13 Synthesis of Compound KH014

[0109]

[0110] Compound KH014: Compound KHC-6 (60 mg, 0.15 mmol) was dissolved in 5 mL of N,N-dimethylformamide and clarified. 1 M NaOH (1 mL, 1.00 mmol) was added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 50:1) to obtain a white solid (12 mg, 22.92%).

[0111] 1 HNMR(400MHz,DMSO-d6)δ4.94-4.89(m,1H),4.18(t,1H),4.06-4.02(m,2H),3.80(t,1H),2.27(dd,1H),1.77–0.65(m,27H),0.68(s,3H).

[0112] Example 14 Synthesis of Compound KH015

[0113]

[0114] Compound KH015: Compound KHC-6 (60 mg, 0.15 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (98 mg, 0.30 mmol) and 6-chloropurine (46 mg, 0.30 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 40:1) to obtain a white solid (39 mg, 53.61%).

[0115] 1 HNMR(400MHz,DMSO-d6)δ8.60(s,1H),8.37(s,1H),5.41(q,2H),4.20(d,1H),3.79(t,1H),2.56(dd,1H),1.80–0.75(m,27H),0.69(s,3H).

[0116] Example 15 Synthesis of Compound KH016

[0117]

[0118] Compound KH016: Compound KHC-6 (60 mg, 0.15 mmol) was dissolved in 5 mL of acetonitrile to clarify the solution. Cs2CO3 (98 mg, 0.30 mmol) and 2-mercaptothiophene (17 mg, 0.30 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated the reaction was complete. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 40:1) to obtain a white solid (19 mg, 28.34%).

[0119] 1 HNMR (400MHz, CDCl3) δ7.35(d,1H),7.14(d,1H),6.95(dd,1H),4.04(t,1H),3.62(q,2H),2.45(dd,1H),1.65–0.81(m,27H),0.73(s,3H).

[0120] Example 16 Synthesis of Compound KH017

[0121]

[0122] Compound KH017: Compound KHC-6 (60 mg, 0.15 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (98 mg, 0.30 mmol) and 6-methoxypurine (45 mg, 0.30 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 40:1) to obtain a white solid (46 mg, 63.76%).

[0123] 1 HNMR(400MHz,DMSO-d6)δ8.57(s,1H),8.32(s,1H),5.37(q,2H),4.20(d,1H ),3.91(s,3H),3.79(t,1H),2.53(dd,1H),1.79–0.75(m,27H),0.69(s,3H).

[0124] Example 17 Synthesis of Compounds KH018 and KH019

[0125]

[0126] Compounds KH018 and KH019: Compound KHC-6 (60 mg, 0.15 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (98 mg, 0.30 mmol) and 5-methyl-1H-tetrazole (25 mg, 0.30 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude products. Purification by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 40:1) afforded KH6070053-1 (13 mg, 20.88%) and KH6070053-2 (27 mg, 43.37%) as white solids, respectively.

[0127] 1 HNMR(400MHz, CDCl3)δ4.39(s,2H),4.05(t,1H),2.56(s,3H),2.34(dd,1H),1.92–

[0128] 0.76(m,27H),0.74(s,3H).

[0129] 1HNMR(400MHz, CDCl3)δ5.15(dd,2H),4.06(t,1H),2.43(s,3H),2.39(dd,1H),1.95–

[0130] 0.78(m,27H),0.75(s,3H).

[0131] Example 18 Synthesis of Compound KH020

[0132]

[0133] Compound KH020: Compound KHC-6 (60 mg, 0.15 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (98 mg, 0.30 mmol) and 2-oxa-6-aza-spiro[3,3]heptane (30 mg, 0.30 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 40:1) to obtain a white solid (21 mg, 32.56%).

[0134] 1 HNMR (400MHz, CDCl3) δ4.75(s,4H),4.04(t,1H),3.57(dd,4H),3.40(dd,2H),2.13(dd,1H),1.90–0.75(m,27H),0.73(s,3H).

[0135] Example 19 Synthesis of Compound KH021

[0136]

[0137] Compound KHC-7: Dissolve compound KH001 (0.70 g, 2.11 mmol) in 100 mL of dichloromethane and add DMP (1.78 g, 4.20 mmol) in portions. Under N₂ protection, allow to react overnight at room temperature. TLC monitoring indicates completion. Add 40 mL of water to the reaction solution, extract with ethyl acetate (2 x 80 mL). The combined organic phases are washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure to obtain the crude product. Purify by flash chromatography on a silica gel column (petroleum ether / acetone = 40:1 → 30:1) to obtain KHC-7 (0.62 g, 89.47%) as a white solid.

[0138] Compound KH021: FeCl3 (0.61 g, 3.75 mmol) and LiCl (0.34 g, 8.07 mmol) were placed in a 100 mL three-necked flask under nitrogen protection. 40 mL of anhydrous tetrahydrofuran was added and stirred at room temperature for 5 minutes. The mixture was then cooled to -45°C and slowly added dropwise with CH3MgBr (15.20 mL, 15.20 mmol). The mixture was stirred at -40°C for 15 minutes. A solution of compound KHC-7 (0.62 g, 1.88 mmol) in anhydrous tetrahydrofuran was then added dropwise. After the addition was complete, the mixture was heated to -20°C and stirred for 2 hours. TLC monitoring indicated the reaction was complete. The reaction mixture was quenched by the addition of approximately 40 mL of saturated NH4Cl and extracted with ethyl acetate (2 x 80 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. The product was purified by flash chromatography on a silica gel column (eluted with petroleum ether / acetone = 40:1→35:1) to give KH021 (0.52 g, 78.50%) as a white solid.

[0139] 1 HNMR(400MHz, CDCl3)2.29(dd,1H),2.13(s,3H),1.88–1.25(m,21H),1.20(s,3H),0.95(s,3H),0.88–0.78(m,3H),0.70(s,3H).

[0140] Example 20 Synthesis of Compounds KH022 and KH023

[0141]

[0142]

[0143] Compounds KHC-8 and KHC-9: Compound KH021 (1.25 g, 3.60 mmol) was dissolved in 40 mL of methanol to clarify the solution, followed by the dropwise addition of bromine (0.86 g, 5.40 mmol). The reaction mixture was allowed to react at room temperature for 4 hours, with the orange color turning pale yellow. TLC monitoring indicated the reaction was complete. 30 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 65 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to yield a crude pale yellow solid. Purification by flash chromatography on a silica gel column (eluting with dichloromethane) afforded KHC-8 (0.93 g, 60.78%) and KHC-9 (0.36 g, 19.89%) as white solids.

[0144] Compounds KH022 and KH023: Compound KHC-8 (100 mg, 0.24 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (234 mg, 0.72 mmol) and 1H-tetrazole (50 mg, 0.72 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude products. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 30:1) to obtain KH022 (20 mg, 20.08%) and KH023 (47 mg, 47.19%) as white solids, respectively.

[0145] 1 H NMR (400MHz, CDCl3) δ8.56(s,1H),5.54–5.45(m,2H),2.35(dd,1H),1.89–1.25(m,21H),1.20(s,3H),0.95(s,3H),0.88–0.80(m,3H),0.72(s,3H).

[0146] 1 H NMR (400MHz, CDCl3) δ8.71(s,1H),5.30(dd,2H),2.35(dd,1H),1.83–1.23(m,21H),1.20(s,3H),0.95(s,3H),0.87–0.78(m,3H),0.72(s,3H).

[0147] Example 21 Synthesis of Compound KH024

[0148]

[0149] Compound KH024: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of acetonitrile to clarify the solution. Cs2CO3 (91 mg, 0.28 mmol) and 3-cyanopyrazole (26 mg, 0.28 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 40:1) to obtain a white solid (16 mg, 26.09%).

[0150] 1HNMR(400MHz, CDCl3)7.45(d,1H),6.73(d,1H),5.06(q,2H),2.30(dd,1H),1.86–

[0151] 1.23(m,21H),1.20(s,3H),0.95(s,3H),0.87–0.77(m,3H),0.72(s,3H).

[0152] Example 22 Synthesis of Compound KH025

[0153]

[0154] Compound KH025: Compound KHC-8 (100 mg, 0.24 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. Cs2CO3 (235 mg, 0.72 mmol) and 3-hydroxybenzisoxazole (97 mg, 0.72 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. The product was purified by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 5:1) to obtain a white solid (70 mg, 60.76%).

[0155] 1 HNMR(400MHz, CDCl3)δ7.72(d,1H),7.58–7.49(m,1H),7.42(d,1H),7.29(d,1H),5.11–4.95(m,2 H),2.35(dd,1H),1.87–1.22(m,21H),1.20(s,3H),0.99(s,3H),0.87–0.80(m,3H),0.72(s,3H).

[0156] Example 23 Synthesis of Compound KH026

[0157]

[0158] Compound KH026: Compound KHC-8 (53 mg, 0.12 mmol) was dissolved in 5 mL of acetonitrile to clarify the solution. KCO (100 mg, 0.72 mmol) and 2-mercaptobenzoxazole (24 mg, 0.16 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated the reaction was complete. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. The product was purified by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 6:1) to obtain a white solid (30 mg, 50.40%).

[0159] 1 HNMR(400MHz, CDCl3)δ7.55(d,1H),7.42(d,1H),7.34-7.24(m,2H),4.33(m,2H),2.54( dd,1H),1.83–1.23(m,21H),1.18(s,3H),0.94(s,3H),0.85–0.78(m,3H),0.70(s,3H).

[0160] Example 24 Synthesis of Compound KH027

[0161]

[0162] Compound KH027: Compound KHC-9 (110 mg, 0.22 mmol) was dissolved in 10 mL of acetonitrile to clarify the solution. Cs2CO3 (215 mg, 0.66 mmol) and 4-cyanopyrazole (61 mg, 0.66 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 70:1) to obtain a white solid (76 mg, 65.30%).

[0163] 1 HNMR(400MHz, CDCl3)δ8.23(s,1H),8.13(s,1H),7.92(s,1H),7.83(s,1H),7.03(s,1H),2. 11(dd,1H),1.83–1.23(m,21H),1.20(s,3H),0.91(s,3H),0.82–0.75(m,3H),0.70(s,3H).

[0164] Example 25 Synthesis of Compound KH028

[0165]

[0166] Compound KH028: Compound KHC-8 (70 mg, 0.16 mmol) was dissolved in 8 mL of dimethyl sulfoxide (DMSO) to clarify the solution. Cs2CO3 (160 mg, 0.49 mmol) and 2-hydroxybenzoxazole (66 mg, 0.49 mmol) were added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 60:1) to obtain a white solid (55 mg, 71.61%).

[0167] 1 HNMR (400MHz, CDCl3) δ7.24–7.18(m,1H),7.17–7.06(m,2H),6.75–6.64(m,1H),4.58(dd,2H) ,2.38(dd,1H),1.92–1.22(m,21H),1.20(s,3H),0.97(s,3H),0.89-0.78(m,3H),0.72(s,3H).

[0168] Example 26 Synthesis of Compound KH029

[0169]

[0170] Compound KH029: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. Cs2CO3 (138 mg, 0.42 mmol) and 8-methyl-4-hydroxyquinazoline (68 mg, 0.42 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 60:1) to obtain a white solid (50 mg, 70.72%).

[0171] 1HNMR(400MHz, CDCl3)δ8.13(d,1H),7.90(s,1H),7.61(d,1H),7.39(t,1H),4.81(s,2H),2.63(s, 3H),2.43(dd,1H),1.88–1.22(m,21H),1.20(s,3H),0.97(s,3H),0.90–0.81(m,3H),0.72(s,3H).

[0172] Example 27 Synthesis of Compound KH030

[0173]

[0174] Compound KH030: Compound KHC-8 (50 mg, 0.12 mmol) was dissolved in 5 mL of dimethyl sulfoxide to clarify the solution. Cs2CO3 (78 mg, 0.24 mmol) and 3-hydroxy-4-methyl-5-methoxy-4H-1,2,4-triazole (31 mg, 0.24 mmol) were added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 4:1) to obtain a white solid (20 mg, 35.16%).

[0175] 1 HNMR(400MHz, CDCl3)δ4.47(q,2H),3.93(s,3H),3.12(s,3H),2.28(dd,1H),1.86–

[0176] 1.22(m,21H),1.19(s,3H),0.94(s,3H),0.85–0.78(m,3H),0.70(s,3H).

[0177] Example 28 Synthesis of Compound KH031

[0178]

[0179] Compound KH031: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of acetonitrile to clarify. KCO (58 mg, 0.42 mmol) and 1-methyl-5-mercapto-1H-tetrazole (68 mg, 0.42 mmol) were added. The reaction was allowed to react at room temperature overnight. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 4:1) to obtain a white solid (50 mg, 77.47%).

[0180] 1 HNMR(400MHz, CDCl3)4.40(dd,2H),3.97(s,3H),2.47(dd,1H),1.88–1.22(m,21H),1.19(s,3H),0.93(s,3H),0.87–0.79(m,3H),0.71(s,3H).

[0181] Example 29 Synthesis of Compound KH032

[0182]

[0183] Compound KH032: Compound KHC-8 (50 mg, 0.12 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. KCO (50 mg, 0.36 mmol) and 2-hydroxy-1H-imidazole (30 mg, 0.36 mmol) were added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated the reaction was complete. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 2:1) to obtain a white solid (11 mg, 21.37%).

[0184] 1 HNMR(400MHz, CDCl3)δ9.26(s,1H),6.32(s,1H),6.17(s,1H),4.53–4.32(m,2H),2.31( dd,1H),1.86–1.25(m,21H),1.21(s,3H),0.95(s,3H),0.88–0.78(m,3H),0.71(s,3H).

[0185] Example 30 Synthesis of Compound KH033

[0186]

[0187] Compound KH033: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. Cs2CO3 (138 mg, 0.42 mmol) and 2-hydroxyquinoline (61 mg, 0.42 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 4:1) to obtain a white solid (15 mg, 21.87%).

[0188] 1 HNMR(400MHz, CDCl3)δ8.05(d,1H),7.75(dd,2H),7.61(t,1H),7.39(t,1H),7.05(d,1H),5.15(s, 2H),2.43(dd,1H),1.98–1.23(m,21H),1.21(s,3H),0.97(s,3H),0.91–0.78(m,3H),0.74(s,3H).

[0189] Example 31 Synthesis of Compound KH034

[0190]

[0191] Compound KH034: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. KCO (58 mg, 0.42 mmol) and 3-hydroxy-1H-1,2,4-triazole (36 mg, 0.42 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (dichloromethane / methanol = 40:1) to obtain a white solid (40 mg, 66.45%).

[0192] 1 HNMR(400MHz, CDCl3)δ9.30(s,1H),7.42(s,1H),4.47(q,2H),2.33(dd,1H),1 .87–1.25(m,21H),1.20(s,3H),0.95(s,3H),0.87–0.79(m,3H),0.71(s,3H).

[0193] Example 32 Synthesis of Compound KH035

[0194]

[0195] Compound KH035: Compound KHC-8 (80 mg, 0.19 mmol) was dissolved in 6 mL of dimethyl sulfoxide (DMSO) to clarify the solution. Cs2CO3 (184 mg, 0.56 mmol) and 8-fluoro-4-hydroxyquinazoline (92 mg, 0.56 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 2:1) to obtain a white solid (65 mg, 67.21%).

[0196] 1 HNMR(400MHz, CDCl3)δ8.06(d,1H),7.88(s,1H),7.55–7.40(m,2H),4.79(m,2H),2.43( dd,1H),1.88–1.22(m,21H),1.21(s,3H),0.96(s,3H),0.90–0.81(m,3H),0.72(s,3H).

[0197] Example 33 Synthesis of Compound KH036

[0198]

[0199] Compound KH036: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. KCO (58 mg, 0.42 mmol) and 6-fluoropyrido[3,4-d]pyrimidin-4-one (69 mg, 0.42 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (dichloromethane / methanol = 40:1) to obtain a white solid (40 mg, 56.02%).

[0200] 1 HNMR(400MHz, CDCl3)δ8.82(s,1H),7.80(s,1H),7.66(d,1H),4.80(q,2H),2.43(dd ,1H),1.89–1.23(m,21H),1.20(s,3H),0.95(s,3H),0.90–0.80(m,3H),0.71(s,3H).

[0201] Example 34 Synthesis of Compound KH037

[0202]

[0203] Compound KH037: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of acetonitrile to clarify. KCO (58 mg, 0.42 mmol) and 4-methyl-4H-3-mercapto-1,2,4-triazole (49 mg, 0.42 mmol) were added. The reaction was allowed to react at room temperature overnight. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 3:2) to obtain a white solid (53 mg, 82.30%).

[0204] 1 HNMR(400MHz, CDCl3)δ8.16(s,1H),4.34(dd,2H),3.65(s,3H),2.45(dd,1H), 1.88–1.22(m,21H),1.19(s,3H),0.91(s,3H),0.87–0.79(m,3H),0.70(s,3H).

[0205] Example 35 Synthesis of Compound KH038

[0206]

[0207] Compound KH038: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of dimethyl sulfoxide to clarify the solution. Cs2CO3 (138 mg, 0.42 mmol) and 3-hydroxyisoxazole (36 mg, 0.42 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated the reaction was complete. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification by flash chromatography on a silica gel column (petroleum ether / acetone = 15:1) afforded a white solid (39 mg, 65.00%).

[0208] 1 HNMR(300MHz, CDCl3)δ8.12(d,1H),6.06(d,1H),4.87(s,2H),2.29(dd,1H),1.90–

[0209] 1.22(m,21H),1.20(s,3H),0.97(s,3H),0.90–0.74(m,3H),0.71(s,3H).

[0210] Example 36 Synthesis of Compound KH039

[0211]

[0212] Compound KH039: Compound KHC-8 (75 mg, 0.18 mmol) was dissolved in 5 mL of acetonitrile to clarify the solution. Cs2CO3 (171 mg, 0.53 mmol) and 5-phenyltetrazolyl (77 mg, 0.53 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 3:1) to obtain a white solid (79 mg, 89.39%).

[0213] 1 HNMR(400MHz, CDCl3)δ8.19–8.07(m,2H),7.55–7.42(m,3H),5.54–5.40(m,2H),2.37( dd,1H),1.92–1.23(m,21H),1.21(s,3H),0.97(s,3H),0.90–0.78(m,3H),0.73(s,3H).

[0214] Example 37 Synthesis of Compound KH040

[0215]

[0216] Compound KH040: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of acetonitrile to clarify the solution. Cs2CO3 (91 mg, 0.28 mmol) and 4-cyanopyrazole (26 mg, 0.28 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 40:1) to obtain a white solid (16 mg, 26.09%).

[0217] 1HNMR(400MHz, CDCl3)δ7.82(s,1H),7.81(s,1H),5.00(q,2H),2.32(dd,1H),1.88–

[0218] 1.22(m,21H),1.20(s,3H),0.94(s,3H),0.87–0.79(m,3H),0.71(s,3H).

[0219] Example 38 Synthesis of Compound KH041

[0220]

[0221] Compound KH041: Compound KHC-8 (70 mg, 0.16 mmol) was dissolved in 5 mL of acetonitrile to clarify the solution. Cs2CO3 (160 mg, 0.49 mmol) and 2-mercaptothiophene (57 mg, 0.49 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification by flash chromatography on a silica gel column (eluted with dichloromethane / petroleum ether = 1:1) afforded an off-white solid (27 mg, 35.53%).

[0222] 1 HNMR(400MHz, CDCl3)δ7.35(dd,1H),7.14(dd,1H),6.95(dd,1H),3.62(dd,2H) ,1.90–1.21(m,21H),1.20(s,3H),0.90(s,3H),0.84-0.74(m,3H),0.70(s,3H).

[0223] Example 39 Synthesis of Compound KH042

[0224]

[0225] Compound KH042: Compound KHC-8 (150 mg, 0.35 mmol) was dissolved in 30 mL of acetone, 15 mL of water, and K2CO3 (49 mg, 0.35 mmol) to clarify. Refluxed at 70°C overnight, TLC monitoring indicated completion. Added 10 mL of water to the reaction solution, extracted with ethyl acetate (2 x 30 mL), and the combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and evaporated to remove the solvent under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 50:1) to afford a white solid (21 mg, 16.54%).

[0226] 1 HNMR(400MHz,DMSO-d6)δ4.92(t,1H),4.06-4.04(m,2H),3.85(t,1H),2.27(dd,1 H),1.82–1.10(m,21H),1.05(s,3H),0.87(s,3H),0.85-0.70(m,3H),0.65(s,3H).

[0227] Example 39 Synthesis of Compounds KH043 and KH044

[0228]

[0229] Compounds KH043 and KH044: Compound KHC-8 (60 mg, 0.14 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (91 mg, 0.28 mmol) and 1H-triazole (15 mg, 0.21 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude products. Purification by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 40:1) afforded KH6070100-1 (12 mg, 20.70%) and KH6070100-2 (27 mg, 46.58%) as white solids, respectively.

[0230] 1 HNMR (400MHz, CDCl3) δ7.68(s,2H),5.34–5.20(m,2H),2.25(dd,1H),1.88–1.23(m,21H),1.20(s,3H),0.95(s,3H),0.89–0.79(m,3H),0.71(s,3H).

[0231] 1 HNMR(400MHz, CDCl3)δ7.76(s,1H),7.61(s,1H),5.25(dd,2H),2.37(dd,1H), 1.86–1.22(m,21H),1.20(s,3H),0.94(s,3H),0.86–0.76(m,3H),0.71(s,3H).

[0232] Example 40 Synthesis of Compound KH045

[0233] Compound KHC-11: Compound KHC-10 (150.0 g, 183.8 mmol) was dissolved in 500 mL of tetrahydrofuran. 10% Pd / C (6.0 g) was added and the reaction mixture was hydrogenated three times with H2 displacement. Stir at room temperature for 24 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was then slurried with a 1:1 solution of petroleum ether:acetone to obtain compound KHC-11 (150.0 g, 99.26%).

[0234] Compound KHC-12: Compound KHC-11 (150.0 g, 547.4 mmol) was dissolved in 1000 mL of methanol, and I2 (14.0 g, 54.8 mmol) was added. The mixture was heated at 60°C and stirred for 12 hours. The reaction solution was concentrated and purified by chromatography (PE:EA=10:1) to obtain compound KHC-12 (130.0 g, 74.20%) as a white solid.

[0235] Compound KHC-13: Compound KHC-12 (130.0 g, 406.2 mmol) was dissolved in 1000 mL of N,N-dimethylformamide. t-BuOK (91.0 g, 812.4 mmol) and trimethyl iodide sulfide (165.8 g, 812.7 mmol) were added. The reaction mixture was stirred at room temperature under nitrogen overnight. Brine was added to the reaction mixture, and the mixture was extracted with ethyl acetate (1.0 L x 3). The organic phases were combined, dried, and the solvent was removed to obtain compound KHC-13 (120.0 g, 88.50%) as a white solid.

[0236] Compound KHC-14: Compound KHC-13 (120.0 g, 359.4 mmol) was dissolved in 800 mL of ethanol and 160 mL of water. Sodium azide (70.2 mg, 1077.9 mmol) and ammonium chloride (67.2 g, 1257.9 mmol) were added, and the mixture was heated at 90°C with stirring overnight. Brine was added to the reaction mixture, and the mixture was extracted with ethyl acetate (1.0 L x 3). The combined organic phases were dried, the solvent removed, and purified by column chromatography (PE:EA = 10:1) to obtain compound KHC-14 (130.0 g, 95.59%) as a white solid.

[0237] Compound KHC-15: Compound KHC-14 (125.6 g, 332.4 mmol) was dissolved in 1000 mL of acetonitrile. Sodium iodide (249.3 g, 1662.0 mmol) was added and stirred at room temperature for 0.5 hours. TMSCl (144.9 g, 1329.6 mmol) was added dropwise, and the reaction mixture was allowed to react at room temperature for 4 hours. Brine was added to the reaction mixture, and the mixture was extracted with ethyl acetate (1000 mL x 3). The organic phases were combined, dried, and the solvent removed. The mixture was purified by column chromatography (PE:EA = 10:1) to obtain compound KHC-15 (72.0 g, 75.20%) as a white solid. Compound KHC-16: Compound KHC-15 (72.0 g, 250.0 mmol) was dissolved in 1000 mL of methanol, and I2 (19.0 g, 75.0 mmol) was added. The mixture was heated at 60°C and stirred for 12 hours. The reaction solution was concentrated and purified by chromatography (PE:EA=10:1) to obtain a white solid compound KHC-16 (53.0 g, 63.50%).

[0238] Compound KHC-17: Trimethylsilylacetylene (77.8 g, 793.0 mmol) was added to 1000 mL of tetrahydrofuran. n-Butyllithium solution (1.6 M, 329.0 mmol, 205.6 ml) was added at -78°C and stirred for 2 hours. Compound KHC-16 (53.0 g, 158.6 mmol) was dissolved in 400 mL of tetrahydrofuran and added to the reaction mixture. The mixture was stirred at -78°C for 2 hours. The reaction mixture was gradually warmed to 0°C and poured into saturated ammonium chloride solution. The mixture was extracted with ethyl acetate (800 mL x 3). The reaction mixture was concentrated and purified by column chromatography (PE:EA = 10:1) to obtain compound KHC-17 (56.0 g, 81.55%) as a white solid.

[0239] Compound KHC-18: Compound KHC-17 (56.0 g, 129.3 mmol) was dissolved in 800 mL of tetrahydrofuran. 1.0 M TBAF solution (129.3 mL, 129.3 mmol) was added dropwise at 0°C and stirred for 1 hour. The reaction mixture was added with brine and extracted with ethyl acetate (1000 mL x 3). The combined organic phases were dried, the solvent removed, and purified by column chromatography (PE:EA = 5:1) to obtain compound KHC-18 (43.0 g, 92.30%) as a white solid.

[0240] Compound KHC-19: Compound KHC-18 (38.0 g, 105.6 mmol) was dissolved in 800 mL of tetrahydrofuran and adjusted to pH 3 with 1N HCl solution. The mixture was stirred at room temperature for 12 hours. Brine (1.0 L) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (1000 mL x 3). The combined organic phases were dried, the solvent removed, and purified by column chromatography (PE:EA = 5:1) to yield compound KHC-19 (32.0 g, 96.50%) as a white solid.

[0241] Compound KHC-20: Methanesulfonic acid (148.0 g, 1541.6 mmol) was dissolved in 1000 mL of dichloromethane. Compound KHC-19 (32.0 g, 101.9 mmol) was added dropwise to the reaction mixture, and the mixture was heated at 60°C with stirring for 2 hours. The reaction mixture was cooled to room temperature, and brine (1.0 L) was added. The organic phases were combined, dried, and the solvent removed. The mixture was purified by column chromatography (PE:EA = 10:1) to obtain compound KHC-20 (20.0 g, 62.50%) as a white solid. Compound KHC-21: Compound KHC-20 (20.0 g, 63.7 mmol) was dissolved in 500 mL of tetrahydrofuran, and 10% Pd / C (3.0 g) was added. The reaction mixture was hydrogenated by H2 substitution three times, and stirred at room temperature for 2 days. The reaction solution was filtered and the solvent was evaporated from the filtrate under reduced pressure to obtain a crude product, which was then purified by column chromatography (PE:EA=15:1) to obtain compound KHC-21 (17.0 g, 84.40%) as a white solid.

[0242] Compound KHC045: FeCl3 (23.0 g, 142.0 mmol) and LiCl (13.5 g, 321.4 mmol) were placed in a 2000 mL three-necked flask under N2 protection. Anhydrous tetrahydrofuran (800 mL) was added and stirred at room temperature for 20 minutes. The mixture was then cooled to -40°C and CH3MgBr (3 M, 190 mL, 570.0 mmol) was slowly added dropwise. The mixture was stirred at approximately -40°C for 30 minutes. A solution of compound KHC-21 (17.0 g, 53.8 mmol) in anhydrous tetrahydrofuran (200 mL) was then added dropwise. After the addition was complete, the mixture was heated to approximately -20°C and stirred for 4 hours. TLC monitoring indicated the reaction was complete. The reaction mixture was quenched by the addition of approximately 40 mL of saturated NH4Cl and extracted with ethyl acetate (1.0 L x 3). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification by column chromatography (PE:EA=15:1) gave compound KH045 (10.2 g, 57.10%) as a white solid.

[0243] 1HNMR (400MHz, CDCl3) δ2.34–2.25(m,1H),2.14(s,3H),2.00(t,J=11.4Hz,1H),1.87–1.78(m,1H) ,1.75–1.54(m,9H),1.51–1.43(m,2H),1.39–1.30(m,4H),1.27–1.13(m,7H),1.09–0.84(m,8H).

[0244]

[0245] Example 41 Synthesis of Compound KH046

[0246]

[0247] Compounds KHC-22 and KHC-23: Compound KH045 (1.50 g, 4.50 mmol) was dissolved in 40 mL of methanol to clarify the solution, followed by the dropwise addition of bromine (1.08 g, 6.75 mmol). The reaction mixture was allowed to react at room temperature for 4 hours, during which time the orange color of the reaction solution turned pale yellow. TLC monitoring indicated the reaction was complete. 30 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 65 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to yield a crude pale yellow solid. Purification by flash chromatography on a silica gel column (eluting with dichloromethane) afforded KHC-22 (1.19 g, 64.34%) and KHC-23 (0.33 g, 17.84%) as white solids.

[0248] Compound KHC046: Compound KHC-22 (50 mg, 0.12 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. KCO (21 mg, 0.15 mmol) and 8-fluoro-4-hydroxyquinazoline (25 mg, 0.15 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 4:1) to obtain a white solid (33 mg, 55.56%).

[0249] 1 HNMR(400MHz, CDCl3)δ8.06(d,1H),7.88(s,1H),7.55–7.40(m,2H),4.80(q,2H),2.44 (dd,1H),2.04-2.01(m,1H),1.91-1.88(m,1H),1.84–1.16(m,21H),1.15–0.80(m,8H).

[0250] Example 42 Synthesis of Compound KH047

[0251]

[0252] Compound KH047: Compound KHC-22 (50 mg, 0.12 mmol) was dissolved in 5 mL of acetonitrile to clarify the solution. Cs2CO3 (49 mg, 0.15 mmol) and 5-mercapto-1-methyl-1H-tetrazole (17 mg, 0.15 mmol) were added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 4:1) to obtain a white solid (51 mg, 50.12%).

[0253] 1 HNMR(400MHz, CDCl3)4.40(dd,2H),3.97(s,3H),2.48(dd,1H),2.05-1.99(m,1H),1.93-1.82(m,1H),1.83–1.14(m,21H),1.13–0.70(m,8H).

[0254] Example 43 Synthesis of Compound KH048

[0255]

[0256] Compound KH048: Compound KHC-22 (60 mg, 0.14 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. KCO (58 mg, 0.42 mmol) and 6-fluoropyrido[3,4-d]pyrimidin-4-one (69 mg, 0.42 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (dichloromethane / methanol = 40:1) to obtain a white solid (37 mg, 53.28%).

[0257] 1HNMR(400MHz, CDCl3)δ8.82(s,1H),7.79(s,1H),7.66(d,1H),4.80(dd,2H),2.44(d d,1H),2.05-1.99(m,1H),1.93-1.82(m,1H),1.83–1.14(m,21H),1.13–0.70(m,8H).

[0258] Example 44 Synthesis of Compound KH049

[0259]

[0260] Compound KH049: Compound KHC-22 (60 mg, 0.15 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (62 mg, 0.19 mmol) and 2-mercaptobenzoxazole (29 mg, 0.19 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 4:1) to obtain a white solid (42 mg, 42.92%).

[0261] 1 HNMR(400MHz, CDCl3)δ7.55(d,1H),7.42(d,1H),7.34-7.24(m,2H),4.33(dd,2H),2.57 (dd,1H),2.05-1.99(m,1H),1.93-1.82(m,1H),1.83–1.14(m,21H),1.13–0.70(m,8H).

[0262] Example 45 Synthesis of Compound KH050

[0263]

[0264] Compound KH050: Compound KHC-22 (60 mg, 0.15 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (62 mg, 0.19 mmol) and 3-mercapto-4-methyl-4H-1,2,4-triazole (22 mg, 0.19 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 70:1) to obtain a white solid (17 mg, 25.41%).

[0265] 1 HNMR(400MHz, CDCl3)δ8.22(s,1H),4.37(dd,2H),3.68(s,3H),2.50(dd,1H), 2.10–1.97(m,1H),2.06-2.02(m,1H),1.79–1.14(m,21H),1.12–0.79(m,8H).

[0266] Example 46 Synthesis of Compound KH051

[0267]

[0268] Compound KH051: Compound KHC-22 (60 mg, 0.14 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. KCO (58 mg, 0.42 mmol) and 3-hydroxy-1H-1,2,4-triazole (36 mg, 0.42 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (dichloromethane / methanol = 40:1) to obtain a white solid (27 mg, 46.36%).

[0269] 1 HNMR(400MHz, CDCl3)δ9.57(s,1H),7.42(s,1H),4.46(q,2H),2.34(dd,1H),2.07–

[0270] 1.96(m,1H),1.88-1.82(m,1H),1.79(s,21H),1.11–0.81(m,8H).

[0271] Example 47 Synthesis of Compounds KH052 and KH053

[0272]

[0273] Compounds KH052 and KH053: Compound KHC-22 (100 mg, 0.24 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (234 mg, 0.72 mmol) and 1H-tetrazole (50 mg, 0.72 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude products. Purification by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 30:1) afforded KH6070069-1 (19 mg, 19.74%) and KH6070069-2 (44 mg, 45.72%) as white solids, respectively.

[0274] 1 HNMR (400MHz, CDCl3) δ8.57(s,1H),5.49(s,2H),2.33(dd,1H),2.05-2.01(m,1H),1.94–1.84(m,1H),1.83–1.15(m,21H),1.14–0.82(m,8H).

[0275] 1 HNMR (400MHz, CDCl3) δ8.72(s,1H),5.29(dd,2H),2.41(dd,1H),2.06-2.01(m,1H),1.95–1.85(m,1H),1.85–1.15(m,21H),1.14–0.82(m,8H).

[0276] Example 48 Synthesis of Compound KH054

[0277]

[0278] Compound KH054: Compound KHC-22 (60 mg, 0.15 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (62 mg, 0.19 mmol) and 2-hydroxyoxazolo[4,5-b]pyridine (26 mg, 0.19 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 70:1) to obtain a white solid (17 mg, 42.92%).

[0279] 1HNMR(400MHz, CDCl3)δ7.25(d,1H),7.01(d,1H),6.69(t,1H),5.11(q,2H),2.40(dd ,1H),2.08–1.98(m,1H),1.93–1.83(m,1H),1.82–1.14(m,21H),1.15–0.81(m,8H).

[0280] Example 49 Synthesis of Compound KH055

[0281]

[0282] Compound KH055: Compound KHC-22 (100 mg, 0.24 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (156 mg, 0.48 mmol) and 4-cyanopyrazole (45 mg, 0.48 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated the reaction was complete. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. The product was purified by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 30:1) to obtain a white solid (51 mg, 50.12%).

[0283] 1 HNMR(400MHz, CDCl3)δ7.83(s,1H),7.81(s,1H),4.99(q,2H),2.32(dd,1H), 2.05-2.01(m,1H),1.92–1.83(m,1H),1.82–1.15(m,21H),1.14–0.81(m,8H).

[0284] Example 50 Synthesis of Compound KH056

[0285]

[0286] Compound KH056: Compound KHC-23 (40 mg, 0.10 mmol) was dissolved in 5 mL of acetonitrile to clarify. Cs2CO3 (98 mg, 0.30 mmol) and 4-cyanopyrazole (28 mg, 0.30 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (developed with dichloromethane / methanol = 30:1) to obtain a white solid (12 mg, 23.30%).

[0287] 1 HNMR(400MHz, CDCl3)δ8.22(s,1H),8.14(s,1H),7.92(s,1H),7.84(s,1H),7.02(s,1H),2 .10(dd,1H),2.05-2.00(m,1H),1.87–1.78(m,1H),1.78–1.14(m,21H),1.12–0.79(m,8H).

[0288] Example 51 Synthesis of Compound KH057

[0289]

[0290] Compound KH057: Compound KHC-22 (60 mg, 0.15 mmol) was dissolved in 5 mL of dimethyl sulfoxide to clarify the solution. Cs2CO3 (143 mg, 0.44 mmol) and 2-hydroxybenzoxazole (59 mg, 0.44 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated the reaction was complete. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 3:1) to obtain a white solid (30 mg, 42.92%).

[0291] 1 HNMR(400MHz, CDCl3)δ7.24–7.19(m,1H),7.17–7.07(m,2H),6.75–6.65(m,1H),4.58(s,2H) ,2.40(dd,1H),2.08–1.98(m,1H),1.93–1.83(m,1H),1.82–1.14(m,21H),1.15–0.81(m,8H).

[0292] Example 52 Synthesis of Compound KH058

[0293]

[0294] Compound KH058: Compound KHC-22 (80 mg, 0.19 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. Cs2CO3 (190 mg, 0.58 mmol) and 8-methyl-4-quinazolinone (93 mg, 0.58 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 3:1) to obtain a white solid (28 mg, 30.01%).

[0295] 1 HNMR(400MHz, CDCl3)δ8.13(d,1H),7.88(s,1H),7.61(d,1H),7.39(t,1H),4.80(dd,2H),3.49(s ,3H),2.45(dd,1H),2.03-2.01(m,1H),1.92–1.84(m,1H),1.83–1.16(m,21H),1.14–0.80(m,8H).

[0296] Example 53 Synthesis of Compound KH059

[0297]

[0298] Compound KH059: Compound KHC-22 (80 mg, 0.19 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. Cs2CO3 (190 mg, 0.58 mmol) and 8-hydroxy-1,7-naphthyridine (85 mg, 0.58 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 3:1) to obtain a white solid (28 mg, 30.89%).

[0299] 1 HNMR(400MHz, CDCl3)δ8.86(d,1H),7.88(d,1H),7.54(dd,1H),6.96(d,1H),6.45(d,1H),4.86(s ,2H),2.45(dd,1H),2.09–1.95(m,1H),1.87-1.83(m,1H),1.80–1.15(m,21H),1.12–0.76(m,8H).

[0300] Example 54 Synthesis of Compound KH060

[0301]

[0302] Compound KH060: Compound KHC-22 (100 mg, 0.24 mmol) was dissolved in 5 mL of dimethyl sulfoxide to clarify the solution. Cs2CO3 (156 mg, 0.48 mmol) and 3-hydroxy-4-methyl-5-methoxy-4H-1,2,4-triazole (62 mg, 0.48 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. The product was purified by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 4:1) to obtain a white solid (56 mg, 50.72%).

[0303] 1 HNMR(400MHz, CDCl3)δ4.54–4.40(m,2H),3.93(s,3H),3.12(s,3H),2.29(dd,1H ),2.03-1.99(m,1H),1.85-1.82(m,1H),1.77–1.13(m,21H),1.10–0.81(m,8H).

[0304] Example 55 Synthesis of Compound KH061

[0305]

[0306] Compound KH061: Compound KHC-22 (80 mg, 0.19 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to clarify the solution. Cs2CO3 (190 mg, 0.58 mmol) and 2-hydroxyquinoline (84 mg, 0.58 mmol) were then added. The reaction was allowed to react overnight at room temperature. TLC monitoring indicated completion of the reaction. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed by thin-layer chromatography on silica gel thick preparative plates (petroleum ether / acetone = 4:1) to obtain a white solid (45 mg, 49.76%).

[0307] 1HNMR(400MHz, CDCl3)δ8.05(d,1H),7.75(dd,2H),7.61(t,1H),7.39(t,1H),7.05(d,1H),5.15(s ,2H),2.43(dd,1H),2.04-2.00(m,1H),1.86-1.83(m,1H),1.78–1.12(m,21H),1.10–0.80(m,8H).

[0308] Example 56 In vitro cell activity assay

[0309] The structures of the comparative compounds used in the present invention are as follows:

[0310]

[0311] The specific synthesis process thereof is prepared with reference to CN105339381B.

[0312] The present invention uses recombinant GABA A The in vitro activity of the compounds was evaluated by patch clamp electrophysiology of the α1β2γ2 and α4β3δ subtypes of the receptor.

[0313] 56.1 Electrophysiological testing solution

[0314] Intracellular and extracellular fluid components:

[0315] Extracellular solution: 140 mM NaCl, 5 mM CsCl, 2 mM CaCl2·2H2O, 1 mM MgCl2·6H2O, 5 mM HEPES, 10 mM D-Glucose, pH = 7.4 (NaOH).

[0316] Intracellular solution: 130 mM CsCl, 0.1 mM CaCl2·2H2O, 2 mM MgCl2·6H2O, 1.1 mM EGTA, 5 mM Na 2- ATP, 10mM HEPES, pH=7.2 (CsOH).

[0317] After the intracellular solution is prepared, it is divided into 1 mL per tube and stored in a -20℃ refrigerator. Freshly melted electrode intracellular solution is used for each experiment. All intracellular solutions should be used up within three months.

[0318] 56.2 Cell lines stably expressing ion channels:

[0319]

[0320] 56.3 Cell Culture

[0321] 56.3.1 GABA A (α1β2γ2) cell culture

[0322] Stable expression of GABA A HEK293 cells expressing the (α1β2γ2) receptor were cultured in DMEM medium containing 10% fetal bovine serum, 800 μg / mL G418, 200 μg / mL Hygromycin B, and 100 μg / mL Zeocin at 37°C and a carbon dioxide concentration of 5%.

[0323] Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of 0.25%-Trypsin-EDTA solution and incubate at 37°C for 0.5 min. When the cells detach from the bottom of the dish, add 5 mL of complete culture medium preheated at 37°C. Gently pipette the cell suspension to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes to collect the cells. For expansion or maintenance culture, inoculate the cells in a 6 cm cell culture dish. Each cell culture dish should be inoculated with 2.5×10 5 cells (final volume: 5 mL).

[0324] To maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

[0325] Before the experiment, cells were detached with 0.25%-Trypsin-EDTA and 8×10 3 The cells were plated on coverslips and cultured in 24-well plates (final volume: 500 μL). After 18 h, patch clamp assays were performed.

[0326] 56.3.2 GABA A (α4β3δ) cell culture

[0327] HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and a carbon dioxide concentration of 5%.

[0328] Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of 0.25%-Trypsin-EDTA solution and incubate at 37°C for 0.5 min. When the cells detach from the bottom of the dish, add 5 mL of complete culture medium preheated at 37°C. Gently pipette the cell suspension to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes to collect the cells. For expansion or maintenance culture, inoculate the cells in a 6 cm cell culture dish with a cell count of 2.5×10 5 (Final volume: 5 mL) Cells for transfection and patch clamp assay were seeded in 24-well plates with coverslips placed in advance, with 8×10 cells per well. 3 cells.

[0329] Transfection: GABA was transfected on the second day A4 (The α4, β3, and δ plasmids were transfected in a 1:1:1 ratio using X-tremeGENE HP DAN Transfection Reagent at a plasmid:transfection reagent ratio of 1 μg:2 μL. For each well of a 24-well plate, use 0.5 μg of plasmid and 1 μL of transfection reagent. The following are the specific steps for transfecting 12 wells: Add 600 μL of Opti-MEM to a sterile centrifuge tube, add 6 μg of plasmid, and mix thoroughly. Then, add 12 μL of transfection reagent, mix thoroughly, and incubate at room temperature for 15 minutes. Then, add the transfection complex dropwise to the cells, 50 μL per well, and gently shake to mix thoroughly.

[0330] The medium was changed on the third day, and patch clamp assay was performed on the fourth day.

[0331] 56.4 Patch Clamp Assay

[0332] Recording electrodes were made from capillary glass tubes (BF150-86-10, Sutter Instruments) using a microelectrode puller (P97, Sutter Instruments). Under an inverted microscope (IX71, Olympus), a microelectrode manipulator (MP285, Sutter Instruments) was used to place the recording electrode in contact with the cell. Negative pressure was applied to create a GΩ seal. After the GΩ seal was established, rapid capacitance compensation was performed. Further negative pressure was applied to rupture the cell membrane, establishing whole-cell recording mode. Slow capacitance compensation was then performed, and membrane capacitance and series resistance were recorded. No leakage compensation was performed.

[0333] A coverslip containing cells was placed in the recording bath of an inverted microscope. The test sample working solution and compound-free external solution were gravity-flown through the recording bath, sequentially from low to high concentrations, to act on the cells. A vacuum pump was used to exchange the fluids during recording. Multiple replicates were performed for each concentration. All electrophysiological experiments were performed at room temperature.

[0334] Whole-cell patch clamp recording of GABA A1 The voltage stimulation protocol for receptor currents was as follows: after whole-cell seal was formed, the cell membrane voltage was clamped at -70 mV. GABA was recorded in gap-free mode. A1 Current. Once cells stabilized, 3 μM GABA was administered for stimulation. After incubation at the test concentration for 30 seconds, mixtures of 3 μM GABA and various compound concentrations were sequentially administered, followed by 100 μM GABA. Each administration was separated by 2 minutes. Experimental data were acquired using an EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0335] Whole-cell patch clamp recording of GABA A4 The voltage stimulation protocol for receptor currents was as follows: after whole-cell seal was formed, the cell membrane voltage was clamped at -70 mV. GABA was recorded in gap-free mode. A4 When the cells stabilized, 10 nM GABA was administered for stimulation. After incubation at the tested concentration for 30 s, a mixture of 10 nM GABA and different concentrations of the compound was administered sequentially, and finally 10 μM GABA was administered. The interval between each administration was 2 min. The experimental data were collected by an EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0336] 56.5 Data Quality Standards

[0337] The following criteria are used to determine whether the data is acceptable:

[0338] (1) Electrode resistance <5 MΩ;

[0339] (2) Sealing resistance > 1GΩ;

[0340] (3) The initial access resistance is less than 15 MΩ;

[0341] (4) Access resistance ends <15MΩ;

[0342] (5) There is no obvious spontaneous decay of the current;

[0343] (6) There is no obvious leakage current at a membrane potential of -70 mV;

[0344] 56.6 Data Analysis

[0345] In detecting GABA A1 The currents of 3 μM GABA and mixed solutions with different drug concentrations were normalized with the current of 100 μM GABA. The activation rate corresponding to each drug concentration was then calculated.

[0346] In detecting GABA A4 The currents of 10nM GABA and mixed solutions with different drug concentrations were normalized with the current of 10μM GABA. Then, calculate the activation rate for each drug concentration. Calculate the mean and standard error for each concentration. Calculate the half-activation concentration for each compound using the following equation:

[0347] Y=Bottom+Bottom / (1+10^((LogEC 50 -X)×HillSlope))

[0348] The above equation was used to perform nonlinear fitting of the dose-dependent effect, where EC 50 is the half-activation concentration, and Hillslope represents the Hill coefficient. Curve fitting and EC 50 The calculations were completed using Graphpad 5.0 software.

[0349] 56.7 In vitro activity results

[0350] Table 1: Effects of different compounds on GABA A1 Electrophysiological evaluation of channel agonism

[0351]

[0352]

[0353] A:>80%, B:60%-80% (including 60%), C:40%-60% (including 40%), D:20%-40% (including 20%), E:0-20% Table 1 shows the efficacy of the test samples at 0.1μM and 1μM concentrations, that is, the test samples acted together with 3μmol GABA on GABA at these two concentrations. A1 The ratio of the peak current generated when the receptor is activated to the peak current generated when 100 μmol of GABA is present alone is multiplied by 100%.

[0354] Table 2: Effects of different compounds on GABA A1 Electrophysiological evaluation of channel agonism

[0355] Compound <![CDATA[EC 50 (nmol)]]> <![CDATA[E max ]]> KH002 463.9 74.6％ KH003 478.1 72.8％ KH004 403.7 106.1％ KH022 821.1 93.1％ KH025 619.9 93.7％ Comparative compounds 1261 84.2％

[0356] E max It is defined as the ratio of the peak current produced in the presence of the test sample and 3 μmol GABA to the peak current produced in the presence of 100 μmol GABA alone, multiplied by 100%.

[0357] Table 3: Effects of different compounds on GABA A4 Electrophysiological evaluation of channel agonism

[0358]

[0359] N / A indicates that the current response does not change with concentration.

[0360] E max It is defined as the ratio of the peak current produced by the test sample alone and in the presence of 10 nmol GABA to the peak current produced by 10 μmol GABA alone, multiplied by 100%.

[0361] Example 57 Pharmacokinetics in Brain and Plasma after Administration

[0362] 1. Experimental plan

[0363] 1.1 Investigational Drugs

[0364] The KH022 compound of the present application and the reference compound (the same as the reference compound described in Example 56).

[0365] 1.2 Experimental animals

[0366] The experimental animals were CD-1 mice.

[0367] 1.3 Administration

[0368] The formulation for oral administration was prepared using 30% SBECD in water. On the first day of the experiment, animals in Group 1 were intraperitoneally injected with a solution of KH022 and the control compound at a volume of 5 mL / kg. Animals were weighed before dosing, and the dosing volume was calculated based on body weight. Whole blood samples (approximately 0.03 mL per group) were collected at the designated times by saphenous vein puncture (or other appropriate blood collection sites). After blood collection, the supernatant plasma was aspirated and immediately placed on dry ice at 3200 g for 10 minutes at 4°C for LC-MS / MS analysis. At each time point, animals were euthanized by CO2, and brain tissue was obtained. The samples were rinsed with saline, dried, and homogenized with a 2:1 solution of 15 mM PBS (pH 7.4):MeOH (4:1) equal to four times the brain weight (g). After homogenization, the samples were transferred to labeled centrifuge tubes and immediately placed on dry ice at -20°C or below for LC-MS / MS analysis.

[0369] 2. Data Analysis

[0370] The concentration of the drug preparation was determined by HPLC-UV method, and the calibration curve contained at least 6 concentration levels, and R 2 ≥0.999. The concentration of the test compound in plasma was determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). The retention time of the compound and the internal standard, the acquisition of the chromatogram and the integration of the chromatogram were processed by the software Analyst (Applied Biosystems), and the statistics of the data were processed by the software Watson LIMS (Thermo Fisher Scientific) or Analyst (Applied Biosystems). The unit of analyte concentration in the sample is ng / mL, and 3 significant figures are retained. All values ​​expressed as percentages (such as % deviation and % coefficient of variation, etc.) are retained to one decimal place. WinNonlin TMPlasma concentrations were treated using a non-compartmental model using the pharmacokinetic software Version 6.3 (Pharsight, Mountain View, CA), and pharmacokinetic parameters were calculated using the linear-log trapezoidal method.

[0371] 3. Test results

[0372] As attached Figure 1 and attached Figure 2 As shown, at the same dosage, compared with the control (the substance could no longer be detected in the brain at 8 hours), the clearance rate of compound KH022 in the mouse brain was slower, and the content in the mouse brain at 2 hours was 2.5 times that of the control.

[0373] 57.3 Anti-anxiety test in mice

[0374] 57.3.1 Light-Dark Shuttle Test: The light-dark shuttle test for mice is designed based on the fact that mice prefer to move in a dark box, but their exploratory nature also prompts them to attempt to explore the light box. The bright light stimulation in the light box inhibits the animals' exploratory activity, leading to anxiety, dark avoidance, and other neurotic states. Anxiolytic effects can be assessed by increasing the time animals spend in the light box, which can be used to assess their activity and thus reflect the anxiolytic efficacy of the drug. Animals were randomly divided into five groups based on body weight: a negative control group and two groups (1 and 3 mg / kg) of the test compound (KH004 or KH022). Each group consisted of 10 male animals. Animals were pre-administered with the corresponding dose of the test compound for five consecutive days. Behavioral testing was performed 30 minutes after the last dose. The time spent in the light box was used as the primary indicator for evaluating the anxiolytic effects of KH004 and KH022.

[0375] The test results are shown in Table 4:

[0376] Table 4: Effects of KH004 and KH022 on the light box retention time of experimental animals

[0377]

[0378] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SD), and 10 animals were used for statistical analysis in each experimental group. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates the statistical analysis results of animals in different experimental groups and negative control group.

[0379] 57.3.2 Elevated Plus Maze Test: The elevated plus maze test exploits the conflicting nature of animals' exploratory nature and their fear of suspended open arms. Once placed in the maze, animals will actively explore the open arms, but at the same time, they fear the open environment within the arms, resulting in anxiety-like symptoms. Anxiolytics can alleviate this anxiety-like conflict in animals within this experimental system. Animals were randomly assigned based on body weight to four groups: a negative control group and two groups (1 mg / kg and 3 mg / kg) of the test compound (KH004 or KH022). Each group consisted of 10 male animals. Animals were pre-administered with the corresponding dose of the test compound for five consecutive days. Behavioral testing was performed 30 minutes after the last dose. The anxiolytic effects of KH004 and KH022 were evaluated using the latency percentage as the primary indicator. The results are shown in Table 5.

[0380] Table 5: Effects of different doses of KH004 and KH022 on the open arm retention time of mice

[0381]

[0382] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SD), and 10 animals were used for statistical analysis in each experimental group. ** P<0.01, *** P<0.001, **** P<0.0001 indicates the statistical analysis results of the animals in different experimental groups and the negative control group. Experimental Conclusion: KH004 and KH022 significantly increased the percentage of animals' open arm retention time, demonstrating anxiolytic efficacy.

[0383] 57.3.3 Animal Side Effect Experiments During Administration

[0384] Because drugs that act on GABA(A) can produce behavioral manifestations similar to anesthesia (side effects), the side effects of the animals were observed during the elevated plus maze test. Ten mice were included in each group, and three behavioral indicators were observed: gait, mobility, and prone behavior. "+" represents severity, with three "+"s representing the most severe, and a return to normal activity time of >60 minutes. The positive drug was the control compound described in Example 56. The experimental results are shown in Table 6 below.

[0385] As can be seen from Table 6, compared with the positive drugs, the side effects of the KH004 and KH022 compounds of the present application are significantly smaller.

[0386]

Claims

1. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating depression, characterized in that The compound is selected from:

2. The use according to claim 1, characterized in that The depression is disruptive mood dysregulation disorder, major depressive disorder, persistent depressive disorder, premenstrual dysphoric disorder, drug-induced disorder, or disorder caused by physical illness.

3. The use according to claim 1, characterized in that The depression is mild depression, moderate depression, severe depression or postpartum depression.

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