Patent Application
20240327383
The present application belongs to the field of medicinal chemistry, in particular to a piperidine derivative, and a pharmaceutical composition, a preparation method and use thereof.
Pain is the most common physical sensation in people's daily life, affecting even more people than diabetes, cardiovascular diseases and cancers combined. The pathogenesis of pain is diverse, and it accompanies almost all diseases. There is a huge clinical need for anti-inflammatory analgesia. At present, opioid analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) dominate the market of pain treatment and are widely used in areas such as surgical analgesia, advanced cancer analgesia and long-term chronic pain. Opioid analgesics are the main therapeutic drugs for moderate to severe pain in clinical practice, as they take effect quickly, have strong analgesic effect, etc. However, the side effects such as addiction, respiratory depression, constipation, tolerance, and itching limit the use, which also bring forth social problems such as drug dependence and death from abuse. It is a very urgent social need to study and develop a safe and effective analgesic drug without side effects.
For many years, scientists have been trying to find new analgesics for clinical selection, which have the analgesic effect of opioids without their side effects. Medicinal chemists have used a variety of strategies to develop safe and effective drugs to treat pain and opioid addiction. Many new target mechanisms have been discovered and applied to the research and development of novel analgesics. Numerous studies have shown that the analgesic effect of μ-receptor agonists is mediated through activation of Gi signals, whereas side effects such as addiction, respiratory depression, and constipation are regulated by downstream β-arrestin signaling pathway. Therefore, G protein-biased μ-opioid receptor agonists have been an important direction of research in this field in recent years. Biased agonists of μ-receptors, which selectively activate Gi pathway without affecting β-arrestin signaling pathway, provide possibility of developing desirable opioid drugs.
WO2017/161017A1 discloses a class of G protein-biased MOP receptor agonists of benzimidazolone piperidine compounds, which exhibit good analgesic activity in rodent analgesic models, with less associated side effects than morphine. However, the compounds themselves may have deficiencies in terms of druggability, and no further entry into clinical studies has been reported.
Selective KOP receptor agonists and DOP receptor agonists have their own side effects, although they have no side effects mediated by MOP receptor agonists. KOP receptor agonists may mediate the production of side effects such as restlessness, sedation, and polyuria, but they have also been found to be able to reduce the rewarding effect of addictive drugs, and therefore have the potential to be developed as analgesics which overcome opioid dependence. After being activated by agonists, DOP receptors will produce convulsive effect, but also show good analgesic effect on chronic pain. Compared with MOP receptor agonists, after being agonized, DOP receptors are not physiologically dependent and are less abuse-dependent, and they also have anti-anxiety and anti-depression effects. However, up to now, ligand compounds of selective KOP receptors or DOP receptors have not been successfully developed in clinical practice because of their limited analgesic efficacy.
The analgesic effect mediated by NOP receptors is much more complex than that of other opioid receptor family members. The nociceptin/orphanin FQ-NOP receptor system not only inhibits opioid receptor-mediated analgesic effect, but also mediates analgesic effect by reducing hyperalgesia. Studies have shown that NOP receptor agonists have analgesic effect and the effect of regulating addiction caused by MOP agonists, and it is found in non-human primate tests that selective NOP agonists do not cause respiratory depression and potential addiction. RO 64-6198, the first reported small molecule NOP receptor selective agonist, was developed by Roche and exhibited good analgesic effect in non-human primate models and rodent model experiments, without classical opioid-mediated side effects. Schering discloses in WO01/07050A1 a class of piperidinyl-derived compounds that are used as NOP receptor (ORL-1 receptor) agonists for the treatment of cough. Due to the close interaction between nociceptin/orphanin FQ receptors and classical opioid receptors, in recent years, people's attention has gradually shifted to the study of ligand compounds that act simultaneously on double targets.
BU08028 is an analog of buprenorphine. This molecule has relatively high affinity for all four typical kinds of opioid receptors. Results of in vitro experiments have shown that BU08028 is a partial agonist of MOP/NOP receptor, and compared with buprenorphine, BU08028 has higher biological activity for NOP receptor; and in in vivo experiments in non-human primates, compared with buprenorphine and remifentanil, BU08028 shows very high analgesic and anti-anxiety effects, with lower addiction, and more importantly, it does not show remarkable physiological dependence. Nalorphine is a kind of opioids with bifunctional effects of MOP receptor antagonist and KOP receptor agonist, which can inhibit the effect of morphine in vivo and show analgesic activity. Bifunctional molecules with high activity for KOP receptors and low or moderate activity for MOP receptors are less addictive than MOP receptor agonists alone, but they have the side effects of making the patients irritable and being hallucinogenic. Cebranopadol is a MOP/NOP receptor bifunctional agonist. Preclinical data show that Cebranopadol also has partial agonist activity for KOP receptors. Cebranopadol has recently been reported to show reduced tolerance, physiological dependence, and respiratory depression compared with morphine. Patent WO2017/096323A1 discloses AT-121, which is a kind of piperidinyl spirocyclic compound with high affinity for both MOP and NOP receptors, and is a partial agonist of the two receptors. In non-human primate experiments, this molecule shows good analgesic and reinforcing effect of inhibitory efficacy, and has no side effects such as respiratory depression, opioid-induced hypersensitivity and drug dependence.
As can be seen from the existing reports, different opioid receptors have their own pharmacological functional characteristics, and the molecules with bifunctional or multifunctional effects on opioid receptors show unique pharmacological effects. While producing good analgesic efficacy, they can effectively reduce the side effects of traditional opioids such as morphine, such as respiratory depression, constipation, tolerance, addiction, etc., have anti-depression and anti-anxiety effects, and are potentially effective in the treatment of alcohol addiction. However, at present, they are all in the early stage of research, with few successes. On the other hand, there is an urgent social need for analgesic drugs without side effects. Therefore, there is a necessity to conduct more extensive research in the field of new analgesic drugs with multifunctional or bifunctional opioid receptor effects.
The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the protection scope of the claims.
The present application provides a piperidine derivative, a pharmaceutical composition thereof, a preparation method and a use thereof. This kind of compounds are structurally novel, and have high affinity and agonistic bioactivity for MOP receptor, NOP receptor (ORL-1 receptor) and (or) KOP receptor, high selectivity with bifunctional effect for MOP receptor and KOP receptor, or high selectivity with bifunctional effect for MOP receptor and NOP receptor (ORL-1 receptor); and some of the compounds have a high bias for G protein, and thus have a unique mechanism of pharmacological action. They can be developed into a new kind of opioid receptor drugs for the treatment or prevention of acute and chronic pain, anxiety, depression, alcohol addiction, and substance abuse/dependence.
In an embodiment of the present application, the present application provides a compound shown in Formula (I), or a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof;
wherein,
n is 0 or 1;
m is 0 or 1;
p is 0, 1, or 2;
R1 and R2 are each independently selected from: hydrogen, halogen, C1-3 alkyl, and C1-3 alkoxy; provided that R1 and R2 are not hydrogen at the same time;
R3 is selected from unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, substituted heteroaryl, unsubstituted C3-8 cycloalkyl, substituted C3-8 cycloalkyl, unsubstituted C4-6 heterocycloalkyl, and substituted C4-6 heterocycloalkyl;
R4 is selected from hydrogen, —C1-3 alkyl-unsubstituted heterocycloalkyl, —C1-3 alkyl-substituted heterocycloalkyl, —C1-3 alkyl-substituted spiroheterocyclic alkyl, —C1-3 alkyl-C(O)NR5R6, —C1-3 alkyl-NR7R8, —C1-4 alkyl-unsubstituted heteroaryl, and —C1-4 alkyl-substituted heteroaryl; herein, R5 and R6 may be independently hydrogen or C1-3 alkyl, or R5 and R6 together form unsubstituted C4-6 heterocycloalkyl, or R5 and R6 together form C4-6 substituted heterocycloalkyl; R7 and R8 may be independently hydrogen or C1-3 alkyl, or R7 and R8 together form unsubstituted C4-6 heterocycloalkyl, or R7 and R8 together form C4-6 substituted heterocycloalkyl; provided that when R4 is hydrogen, m is 0, and n is 1.
In another aspect, the present application provides a pharmaceutical composition, including the aforementioned piperidine derivative, or a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, and a pharmaceutically acceptable carrier.
In a third aspect, the present application provides bioactivity of the aforementioned piperidine derivative or a pharmaceutical composition thereof as a MOP receptor and NOP receptor (ORL-1 receptor) agonist.
In a fourth aspect, the present application provides a preparation method for the aforementioned piperidine derivative, the preparation method including the following steps:
The accompanying drawings are used to provide an understanding of the technical solutions of the present application, and constitute a part of the specification. They are used to explain the technical solutions of the present application together with the examples of the present application, and do not constitute a limitation to the technical solutions of the present application.
In some embodiments, the present application provides a compound shown in Formula (I), and a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof,
wherein,
n is 0 or 1;
m is 0 or 1;
p is 0, 1, or 2;
R1 and R2 are each independently selected from: hydrogen, halogen, C1-3 alkyl, and C1-3 alkoxy; provided that R1 and R2 are not hydrogen at the same time;
R3 is selected from unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, substituted heteroaryl, unsubstituted C3-8 cycloalkyl, substituted C3-8 cycloalkyl, unsubstituted C4-6 heterocycloalkyl, and substituted C4-6heterocycloalkyl; herein, substituted aryl, substituted heteroaryl, substituted C3-8 cycloalkyl, or substituted C4-6 heterocycloalkyl is substituted by 1-3 substituents independently selected from the following groups: halogen, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, halogenated C1-4 alkoxy, aryl, and heteroaryl;
R4 is selected from hydrogen, —C1-3 alkyl-unsubstituted heterocycloalkyl, —C1-3 alkyl-substituted heterocycloalkyl, —C1-3 alkyl-substituted spiroheterocyclic alkyl, —C1-3 alkyl-C(O)NR5R6, —C1-3 alkyl-NR7R8, —C1-4 alkyl unsubstituted-heteroaryl, and —C1-4 alkyl-substituted heteroaryl; herein, R5 and R6 may be independently hydrogen or C1-3 alkyl, or R5 and R6 together form C4-6 unsubstituted heterocycloalkyl, or R5 and R6 together form C4-6 substituted heterocycloalkyl; R7 and R8 may be independently hydrogen or C1-3 alkyl, or R7 and R8 together form C4-6 unsubstituted heterocycloalkyl, or R7 and R8 together form C4-6 substituted heterocycloalkyl; provided that when R4 is hydrogen, m is 0, and n is 1.
In some embodiments, in Formula (I), n is 0;
m is 0;
p is 0, 1, or 2.
In some embodiments, in Formula (I), n is 1;
m is 0;
p is 0, 1, or 2.
In some embodiments, in Formula (I), n is 0;
m is 1;
p is 0, 1, or 2.
In some embodiments, in Formula (I), R1 and R2 are each independently selected from: hydrogen, halogen, C1-3 alkyl; provided that R1 and R2 are not hydrogen at the same time.
In some embodiments, in Formula (I), R1 and R2 are each independently selected from: hydrogen, chlorine, fluorine, C1-3 alkyl; provided that R1 and R2 are not hydrogen at the same time.
In some embodiments, in Formula (I), R1 and R2 are each independently selected from: hydrogen, chlorine, fluorine, methyl; provided that R1 and R2 are not hydrogen at the same time; optionally, R1 and R2 are both chlorine, or R1 is chlorine and R2 is fluorine, or R1 is fluorine and R2 is chlorine, or R1 and R2 are both fluorine, or R1 is fluorine, methyl, or chlorine and R2 is hydrogen, or R1 is hydrogen and R2 is chlorine or fluorine.
In some embodiments, in Formula (I), R3 is selected from unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl, herein unsubstituted aryl is phenyl or naphthyl; unsubstituted heteroaryl is furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, or tetrazolyl; substituted aryl is phenyl or naphthyl substituted by 1-3 groups independently selected from the following: halogen, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, halogenated C1-4 alkoxy, aryl, and heteroaryl; substituted heteroaryl is furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, or tetrazolyl substituted by 1-2 groups independently selected from the following: halogen, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, halogenated C1-4 alkoxy, aryl, and heteroaryl.
In some embodiments, in Formula (I), R3 is phenyl, or phenyl substituted by 1-3 groups independently selected from the following: halogen, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, and halogenated C1-4 alkoxy; or phenyl substituted by 1-3 groups independently selected from the following: fluorine, chlorine, bromine, iodine, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, and halogenated C1-4 alkoxy.
In some embodiments, in Formula (I), R3 is phenyl, 3,4-dichlorophenyl, 3,4-difluorophenyl, 2-chloro-4-fluorophenyl, 2-fluoro-4-chlorophenyl, 4-trifluoromethylphenyl, 4-trifluoromethoxyphenyl, 2,4-dichlorophenyl, 2,6-dichlorophenyl, 2,4-difluorophenyl, 2-chloro-4-methylphenyl, 2-methyl-4-fluorophenyl, 2-methyl-4-chlorophenyl, 2-methoxy-4-chlorophenyl, 2,4-dimethylphenyl, 2,6-dimethylphenyl, 2,4,6-trimethylphenyl, 4-tert-butylphenyl, 2-chlorophenyl, 2-methylphenyl, 2-fluorophenyl, 2-methoxyphenyl, 4-chlorophenyl, 4-fluorophenyl, or 4-methoxyphenyl.
In some embodiments, in Formula (I), R3 is furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, or tetrazolyl, optionally substituted by 1-2 groups independently selected from the following: fluorine, chlorine, bromine, iodine, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, and halogenated C1-4 alkoxy; preferably, R3 is 5-trifluoromethylpyridin-2-yl, or 5-chlorothiophen-2-yl.
In some embodiments, in Formula (I), R3 is unsubstituted C3-8 cycloalkyl, or substituted C3-8 cycloalkyl, herein, C3-8 cycloalkyl in unsubstituted C3-8 cycloalkyl, substituted C3-8 cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl; substituted C3-8 cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl substituted by 1-3 groups independently selected from the following: fluorine, chlorine, bromine, iodine, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, halogenated C1-4 alkoxy, and phenyl; preferably, substituted C3-8 cycloalkyl is 4-tert-butylcyclohexyl, 4-isopropylcyclohexyl, 4-ethylcyclohexyl, 4-methylcyclohexyl, 4-trifluoromethylcyclohexyl, 2,3-dihydro-1H-inden-2-yl, or 2-chlorocyclohexyl.
In some embodiments, in Formula (I), R3 is cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, 4-tert-butylcyclohexyl, 4-isopropylcyclohexyl, 4-ethylcyclohexyl, 4-methylcyclohexyl, 4-trifluoromethylcyclohexyl, 2,3-dihydro-1H-inden-2-yl, or 2-chlorocyclohexyl.
In some embodiments, in Formula (I), R3 is unsubstituted C4-6 heterocycloalkyl or substituted C4-6 heterocycloalkyl, herein, C4-6 heterocycloalkyl in unsubstituted C4-6 heterocycloalkyl or substituted C4-6 heterocycloalkyl is tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydrothienyl, piperidinyl, morpholinyl, or piperazinyl; substituted C4-6 heterocycloalkyl is tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydrothienyl, piperidinyl, morpholinyl, or piperazinyl substituted by 1-3 groups independently selected from the following: fluorine, chlorine, bromine, iodine, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, halogenated C1-4 alkoxy, and phenyl; preferably, substituted C4-6 heterocycloalkyl is N-isopropylpiperidin-4-yl.
In some embodiments, in Formula (I), R4 is selected from: hydrogen, —C1-3 alkyl-unsubstituted heterocycloalkyl selected from at least one of oxa and thia, —C1-3 alkyl-substituted heterocycloalkyl selected from at least one of oxa and thia, —C1-3 alkyl-substituted spiroheterocyclic alkyl, —C1-3 alkyl-C(O)NR5R6, —C1-3 alkyl-NR7R8, —C1-4 alkyl-unsubstituted heteroaryl selected from at least one of aza, oxa, and thia, and —C1-4 alkyl-substituted heteroaryl selected from at least one of aza, oxa, and thia; herein, R5 and R6 may be independently hydrogen or C1-3 alkyl, or R5 and R6 and N connected thereto together form C4-6 unsubstituted azacycloalkyl, or R5 and R6 and N connected thereto together form C4-6 substituted azacycloalkyl; R7 and R8 may be independently hydrogen or C1-3 alkyl, or R7 and R8 and N connected thereto together form C4-6 unsubstituted azacycloalkyl, or R7 and R8 and N connected thereto together form C4-6 substituted azacycloalkyl; provided that when R4 is hydrogen, m is 0 and n is 1; the substitution means substitution by one or more of the following groups: C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 alkanoyl, C1-C4 alkanoyloxy, hydroxyl, nitro, halogen, oxo and cyano.
In some embodiments, in Formula (I), R4 is selected from hydrogen, m is 0, and n is 1.
In some embodiments, in Formula (I), R4 is selected from: —C1-3 alkyl-unsubstituted C2-6 heterocycloalkyl selected from at least one of oxa and thia, —C1-3 alkyl-substituted C2-6 heterocycloalkyl selected from at least one of oxa and thia, —C1-3 alkyl-substituted spiroheterocyclic alkyl, —C1-3 alkyl-C(O)NR5R6, —C1-3 alkyl-NR7R8, —C1-4 alkyl-unsubstituted C3-6 heteroaryl selected from at least one of aza, oxa, and thia, and —C1-4 alkyl-substituted C3-6 heteroaryl selected from at least one of aza, oxa and thia; herein, R5 and R6 may be independently hydrogen or C1-3 alkyl, or R5 and R6 and N connected thereto together form C4-6 unsubstituted azacycloalkyl, or R5 and R6 and N connected thereto together form C4-6 substituted azacycloalkyl; R7 and R8 may be independently hydrogen or C1-3 alkyl, or R7 and R8 and N connected thereto together form C4-6 unsubstituted azacycloalkyl, or R7 and R8 and N connected thereto together form C4-6 substituted azacycloalkyl; the substitution means substitution by one or more of the following groups: C1-4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 alkanoyl, C1-C4 alkanoyloxy, hydroxyl, nitro, halogen, oxo and cyano.
In some embodiments, in Formula (I), R4 is hydrogen, 2-(morpholinyl)ethyl, 2-(1,1-dioxothiomorpholine)ethyl, 2-(4-methylpiperazin-1-yl)ethyl, 2-(4-acetylpiperazin-1-yl)ethyl, 2-(3-oxopiperazin-1-yl)ethyl, 2-(pyrrolidin-1-yl)ethyl, 2-(piperidin-1-yl)ethyl, 2-(N,N′-dimethylamino)ethyl, 2-(2-oxopyrrolidin-1-yl)ethyl, N,N′-dimethylacetamide, oxiran-2-ylmethyl, or 2-(2-oxa-6-azaspiro[3.3]heptan-6-yl)ethyl; provided that when R4 is hydrogen, m is 0, and n is 1.
In some embodiments, in Formula (I), R4 is —C1-3 alkyl-substituted spiroheterocyclic alkyl, and the substituted spiroheterocyclic alkyl may be 0 or 1 oxygenated azaspiro[3.3]heptane, azaspiro[3.5]nonane, azaspiro[3.4]octane, azaspiro[5.5]undecane, or azaspiro[4.5]decane.
Further, the compound of Formula (I) is a compound shown below, and a stereoisomer, pharmaceutically acceptable salt, solvate, deuterate, metabolite, or prodrug thereof:
In some embodiments, the present application further discloses a pharmaceutical composition, including: an effective dose of the piperidine derivative shown in General Formula (I) of the present application, and a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, or further including one or more other therapeutic agents and a pharmaceutically acceptable carrier or excipient.
The pharmaceutically acceptable excipient(s) may, for example, be selected from carriers (e.g., solid, liquid, or semi-solid carriers), adjuvants, diluents (e.g., solid diluents such as fillers or filling agents; and liquid diluents such as solvents and co-solvents), granulants, binders, flow aids, coating agents, controlled release agents (e.g., lagging or delayed release polymers or waxes), disintegrants, buffers, lubricants, preservatives, antifungal agents and antibacterial agents, antioxidants, tension regulators, thickeners, flavor enhancers, sweeteners, pigments, plasticizers, taste masking agents, stabilizers, or any other excipients commonly used in pharmaceutical compositions.
As used herein, the term “pharmaceutically acceptable” means that the compounds, materials, compositions and/or dosage forms are suitable, within sound medical judgment, for exposure to the tissues of a subject (e.g., a human subject) without undue toxicity, irritation, allergy, or other problems or complications, commensurate with reasonable benefit/risk ratio. Each excipient must also be “acceptable” in the sense of compatibility with other components of the formulation.
Pharmaceutical compositions comprising compounds having Chemical Formula (I) may be formulated according to known techniques, referring to, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.
The pharmaceutical composition may be in any form suitable for oral, parenteral, topical, intranasal, endobronchial, sublingual, ophthalmic, otic, rectal, intravaginal, or transdermal administration.
Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard shell or soft shell), caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, or patches such as buccal patches.
In some embodiments, the pharmaceutical composition further includes one or more other therapeutic agents.
In some embodiments, the present application provides the piperidine derivative shown in General Formula (I) of the present application, and a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, for use as a medicine.
In some embodiments of the present application, the present application provides the piperidine derivative shown in General Formula (I) of the present application, and a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, for modulating a μ-opioid peptide receptor (MOPR) and a κ-opioid peptide receptor (KOPR), or a μ-opioid peptide receptor (MOPR) and a nociceptin opioid peptide receptor (NOPR)/opioid receptor like-1 (ORL-1) receptor.
In some embodiments of the present application, the present application provides the piperidine derivative shown in General Formula (I) of the present application, and a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, for the treatment of pain, anxiety, depression, alcohol addiction, substance abuse/dependence.
In some embodiments, the present application further provides a method of using a piperidine derivative shown in General Formula (I), or a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, or the composition of the present application in the treatment of pain, anxiety, depression, alcohol addiction, substance abuse/dependence. Pain may be selected from: acute pain, chronic pain, bone pain, joint pain, postoperative pain, muscle pain, toothache, headache, inflammatory pain, neuropathic pain, and Crohn's disease-related abdominal pain.
In some embodiments of the present application, the present application provides use of the piperidine derivative shown in General Formula (I) of the present application, or a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, or a pharmaceutical composition thereof in the preparation of drugs for the treatment of pain, anxiety, depression, alcohol addiction, substance abuse/dependence.
In some embodiments, the present application provides the above use, and the pain may be selected from: acute pain, chronic pain, bone pain, joint pain, postoperative pain, muscle pain, toothache, headache, inflammatory pain, neuropathic pain, and Crohn's disease-related abdominal pain.
In some embodiments, the present application provides a method for treating disorders in a patient co-mediated by a μ-opioid peptide receptor (MOPR) and a κ-opioid peptide receptor (KOPR), or co-mediated by a μ-opioid peptide receptor (MOPR) and a nociceptin opioid peptide receptor (NOPR)/opioid receptor like-1 (ORL-1) receptor, the method including administering the piperidine derivative shown in General Formula (I) of the present application, or a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, or a pharmaceutical composition thereof to a patient in need thereof.
In some embodiments, the present application provides a method for treating or preventing pain, anxiety, depression, alcohol addiction, substance abuse/dependence in a patient, the method including administering the piperidine derivative shown in General Formula (I) of the present application, or a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, or the pharmaceutical composition to a patient in need thereof.
The pain may be selected from: acute pain, chronic pain, bone pain, joint pain, postoperative pain, muscle pain, toothache, headache, inflammatory pain, neuropathic pain, and Crohn's disease-related abdominal pain.
In some embodiments, the present application provides a method for modulating a μ-opioid peptide receptor (MOPR) and a κ-opioid peptide receptor (KOPR), or a μ-opioid peptide receptor (MOPR) and a nociceptin opioid peptide receptor (NOPR)/opioid receptor like-1 (ORL-1) receptor, the method including administering the piperidine derivative shown in General Formula (I) of the present application, or a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, or the pharmaceutical composition.
In some embodiments, the present application provides a method for modulating a μ-opioid peptide receptor (MOPR) and a κ-opioid peptide receptor (KOPR), or a μ-opioid peptide receptor (MOPR) and a nociceptin opioid peptide receptor (NOPR)/opioid receptor like-1 (ORL-1) receptor to treat or prevent pain, anxiety, depression, alcohol addiction, substance abuse/dependence in a patient, the method including administering the piperidine derivative shown in General Formula (I) of the present application, or a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, or the pharmaceutical composition to a patient in need thereof. The pain may be selected from: acute pain, chronic pain, bone pain, joint pain, postoperative pain, muscle pain, toothache, headache, inflammatory pain, neuropathic pain, and Crohn's disease-related abdominal pain.
In some embodiments, the present application provides a preparation method for the aforementioned piperidine derivative, or a stereoisomer, a pharmaceutically acceptable salt, a solvate, a deuterate, a metabolite, or a prodrug thereof, including the steps of:
In the above preparation method, in Formulas (I-2) and (I-5), X1 and X2 each represents a leaving group, such as bromine, chlorine, or sulfonate group, or the compound of Formula (I-5) is aldehyde, ketone, or boric acid; in formulas (I-1) and (I-3), Pr1 represents an amino protection group, such as tert-butyl oxycarbonyl, benzyloxycarbonyl CBz, or fluorenomethoxycarbonyl Fmoc, etc.; and the other groups in Formulas (I-1) to (I-7) and Formula (I) are defined as above.
The compound of the present application has good opioid receptor agonistic activity, is suitable for medicinal use, and has clinical application value. Moreover, the compound of the present application can be synthesized in simple steps, and thus has good economic utilization value.
Unless otherwise stated, the definitions of groups and terms set forth in the specification and claims of the present application include their definitions as examples, exemplary definitions, preferred definitions, definitions set forth in tables, definitions of specific compounds in examples, etc., and may be arbitrarily combined with one another. The so-combined group definitions and compound structures should fall within the scope described in the specification of the present application.
Carbon, hydrogen, oxygen, sulfur, nitrogen, or halogen involved in the groups and compounds of the present application all includes their isotopes, and carbon, hydrogen, oxygen, sulfur, nitrogen, or halogen involved in the groups and compounds of the present application are optionally further replaced by one or more of their corresponding isotopes, wherein isotopes of carbon include 12C, 13C, and 14C, isotopes of hydrogen include protium (H), deuterium (D, also known as heavy hydrogen), tritium (T, also known as superheavy hydrogen), isotopes of oxygen include 16O, 17O, and 18O, isotopes of sulfur include 32S, 33S, 34S, and 36S, isotopes of nitrogen include 14N and 15N, isotopes of fluorine include 19F, isotopes of chlorine include 35Cl and 37Cl, and isotopes of bromine include 79Br and 81Br.
“Alkyl” refers to linear and branched monovalent saturated hydrocarbon groups, with the main chain including 1-10 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.; and alkyl may be further substituted with any substituent.
“Cycloalkyl” refers to monovalent saturated carbocyclic hydrocarbon groups, monocyclic, typically having 3-10 carbon atoms. Non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. Cycloalkyl may optionally be further substituted with any substituent.
“Heterocyclic alkane” refers to saturated cyclic hydrocarbon groups containing at least one heteroatom, monocyclic, heteroatom being N, O, S, P, and their oxidized forms. Non-limiting examples include aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, etc. Heterocyclic alkane may optionally be further substituted with any substituent.
“Aryl” refers to 6 to 14-membered all-carbon monocyclic or fused polycyclic (i.e. a ring sharing an adjacent pair of carbon atoms) groups having a conjugated R-electron system, preferably 6 to 10-membered, e.g., phenyl and naphthyl. The aryl ring includes the aryl ring described above fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring. Aryl may be substituted or unsubstituted, when it is substituted, the substituent may be substituted at any usable connection site, and the substituent is preferably independently and optionally substituted with one or more substituents selected from hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.
“Heteroaryl” refers to a heteroaromatic system including 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl is preferably 5 to 10-membered (e.g., 5, 6, 7, 8, 9, or 10-membered), more preferably 5 or 6-membered, e.g., furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, etc. The heteroaryl ring includes heteroaryl described above fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring.
“Pharmaceutically acceptable salts” refer to those salts which maintain the biological effectiveness and properties of free acids or free bases, and where the free acids are obtained by reaction with non-toxic inorganic or organic bases, or where the free acids are obtained by reaction with non-toxic inorganic or organic acids.
“Carriers” refer to vehicles or diluents that do not cause significant irritation to an organism and do not eliminate bioactivity and properties of a given compound.
“Excipients” refer to inert substances added to pharmaceutical compositions to further depend on compounds for administration. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and different types of starch, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulants, lubricants, binders, disintegrants, etc.
“Prodrugs” refer to compounds that can be converted to biologically active compounds of the present application under physiological conditions or under the action of a solvent. The prodrug of the present application is prepared by modifying the phenolic group in the compound, and the modification may be removed by conventional operations or in vivo to give the parent compound. When the prodrug of the present application is administered to a mammalian individual, the prodrug is fragmented to form free hydroxyl groups. Examples of prodrugs include, but are not limited to, phenolic hydroxyl and phosphate sodium salt derivatives of the compounds of the present application.
“Effective dose” refers to an amount of a compound that causes physiological or medical response in a tissue, system or subject, and this amount is a desirable amount, including an amount of the compound that, when administered to a subject, is sufficient to prevent or mitigate to some extent one or more symptoms of the disease or condition under treatment.
“Solvates” refer to the compounds of the present application or salts thereof, and they also include stoichiometric or non-stoichiometric solvents that bind by intermolecular non-covalent forces. When the solvent is water, it is hydrate.
“Optional” or “optionally” means that the subsequently described event or environment may, but not necessarily occur, and the description includes occasions where the event or environment occurs or does not occur. For example, “alkyl optionally substituted with F” means that alkyl may, but does not have to, be substituted with F, and the description includes cases where alkyl is substituted with F and cases where alkyl is not substituted with F.
Additional features and advantages of the present application will be set forth in the description which follows, and in part will become apparent from the description, or may be learned by practice of the present application. The objects and other advantages of the present application may be realized and obtained by the structure particularly indicated in the description, the claims, and the accompanying drawings.
To further clarify the purpose, technical solutions, and advantages of the present application, examples of the present application will be described in detail below. It should be noted that the examples of the present application and the features of the examples may be arbitrarily combined with each other provided that there is no conflict.
The compound of the general formula of the present application, and the preparation method and use thereof will be described in further detail below with reference to specific examples. The following examples are intended to illustrate and explain the present application only and should not be construed as limiting the protection scope of the present application. All techniques implemented based on the foregoing contents of the present application are covered within the scope of the present application which is intended to be protected.
Unless otherwise stated, the raw materials and reagents used in the following examples are all commercially available, or may be prepared by known methods. The following abbreviations are used in the present application: equiv. represents equivalent ratio; sat. represents saturation; M represents mol/L; r.t represents room temperature; EtOH represents ethanol; MeOH represents methanol; DCM represents dichloromethane; DCE represents 1,2-dichloroethane; DMSO represents dimethyl sulfoxide; DMF represents N,N-dimethylformamide; EA represents ethyl acetate; PE represents petroleum ether; THE represents tetrahydrofuran; TEA represents triethylamine; TFA represents trifluoroacetic acid; AcOH represents acetic acid; CDI represents carbonyl diimidazole; Boc represents tert-butoxycarbonyl (an amino protection group); NaBH (OAc)3 represents sodium triacetoxy borohydride; Boc2O represents di-tert-butyl dicarbonate; AIBN represents azobisisobutyronitrile; HMPA represents hexamethylphosphoryl triamine; MsCl represents methyl sulfonyl chloride; NBS represents N-bromosuccinimide; and TMS represents trimethylsilane.
Compounds are named according to conventional nomenclature in the art or using ChemDraw® software (PerkinElmer), and commercial reagents adopt the names in supplier catalogs.
1H NMR data is collected and recorded at 400 MHz by Bruker Ultrashield 400 NMR instrument, and chemical shift δ value (ppm) is reported using CDCl3, CD3OD, D2O, or DMSO-d6 as a solvent and TMS (δ=0) as internal standard. Mass spectrum is collected and recorded by Shimadzu LCMS-2020 liquid chromatography-mass spectrometry system, and detected by Shimadzu shim-pack VP-ODS (150 L*2.0, 4.6 μM) chromatographic column. Mobile phase A is an aqueous solution of 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile. High Performance Liquid Chromatography (HPLC) is measured by Agilent Technologies 1260 Infinity liquid chromatograph, and detected using YMC Triart C18 EXRS (4.6*150 mm, 3 μm) chromatographic column. Conditions of gradient elution: at a flow rate of 1.0 mL/min, 30-10% solvent A1 and 70-90% solvent B1, and then 90% B1 and 10% A1, maintaining for 0.5 min, with the percentage being the volume percentage of a given solvent based on the total solvent volume. Solvent A1: an aqueous solution of 0.1% ammonia water; and solvent B1: acetonitrile. Percentage is the volume percentage of a solute in a solution; injection volume: determined by the concentration of reaction solution, for a general sample with the concentration of 0.2 mg/mL, 2 μL is injected; detection wavelength: 254/220 nm; and the temperature of chromatographic column: 30° C.
Synthesis of intermediate 1c: 4,5-dichloro-2-fluoronitrobenzene (la) (10.00 g, 47.62 mmol), N-Boc-4-aminopiperidine (9.54 g, 47.62 mmol) and solvent DMF (120 mL) were added into a clean dry single-neck flask (250 mL), and anhydrous K2CO3 (9.87 g, 71.43 mmol) was added to the flask. The mixture was heated to 60° C. and stirred for reaction for 2 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. After the completion of the reaction, the reaction system was cooled to room temperature, and the reaction solution was slowly poured into an ice-water mixture (500 mL) under stirring condition. A large amount of yellow solid was precipitated and filtered, and the filter cake was washed with a small amount of water, and dried to obtain 17.58 g yellow solid product, with a yield of 94.59%. 1H NMR (400 MHz, chloroform-d) δ 8.30 (s, 1H), 8.01 (d, J=7.4 Hz, 1H), 6.98 (s, 1H), 4.03 (d, J=13.3 Hz, 2H), 3.68-3.53 (m, 1H), 3.06 (t, J=12.3 Hz, 2H), 2.05 (d, J=13.0 Hz, 2H), 1.59-1.48 (m, 2H), 1.48 (s, 9H).
Synthesis of intermediate 1d: tert-butyl 4-((4,5-dichloro-2-nitrophenyl) amino)piperidin-1-carboxylate (1c) (17.50 g, 44.84 mmol) was added into a single-neck flask (1 L), and anhydrous ethanol (450 mL) was added to the reaction flask. Iron powder (15.03 g, 269.05 mmol) and saturated NH4Cl aqueous solution (45 mL) were added to the reaction system under stirring at room temperature. The mixture was heated to 80° C., and stirring was continued for reaction for 1.5 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. Filtration was performed using diatomite, the filter cake was washed with a small amount of anhydrous ethanol, and the filtrate was evaporated under reduced pressure using a rotary evaporator to remove the solvent to obtain a white solid. The solid was dissolved in ethyl acetate (350 mL), and after washing with water and saturated NaCl aqueous solution, the organic phase was separated therefrom, which was dried with anhydrous Na2SO4 and then filtered. The filtrate was evaporated under reduced pressure using a rotary evaporator to remove the solvent to obtain a gray-white solid crude product. Petroleum ether (200 mL) was added to the crude product, followed by dispersing, slurrying, filtering, and drying to obtain 14.23 g orange solid product, with a yield of 88.08%. 1H NMR (400 MHz, chloroform-d) δ 6.78 (s, 1H), 6.66 (s, 1H), 4.04 (s, 2H), 3.52-3.16 (m, 4H), 2.95 (t, J=12.5 Hz, 2H), 2.08-1.96 (m, 2H), 1.47 (d, J=1.5 Hz, 9H), 1.43-1.30 (m, 2H).
Synthesis of intermediate 1e: tert-butyl 4-((2-amino-4,5-dichlorophenyl)amino) piperidin-1-carboxylate (1d) (14.20 g, 39.41 mmol) and anhydrous tetrahydrofuran (300 mL) were added into a clean dry three-necked flask (500 mL), under argon displacement protection, the flask was cooled to 0° C. in an ice water bath, a dichloromethane solution (50 mL) of N,N′-carbonyldiimidazole (8.31 g, 51.24 mmol) was added dropwise, thereafter, the ice water bath was removed, the flask was stirred at room temperature for 1 hour, and heated to 35° C., stirring was performed for reaction for 18 hours, and the reaction of the raw materials was detected, by TLC plate, to be substantially complete. Saturated NaHCO3 aqueous solution (150 mL) was slowly added to the reaction system under stirring condition, followed by extraction with ethyl acetate (250 mL×3). The organic phase was dried through anhydrous Na2SO4 and then filtered. The filtrate was evaporated under reduced pressure using a rotary evaporator to remove the solvent to obtain a gray-white solid crude product. Petroleum ether (250 mL) and ethyl acetate (2.5 mL) were added to the crude product, followed by dispersing, slurrying, filtering, and drying to obtain 14.50 g gray-white solid product, with a yield of 95.22%. 1H NMR (400 MHz, chloroform-d) δ 10.66 (s, 1H), 7.23 (s, 1H), 7.20 (s, 1H), 4.50-4.22 (m, 3H), 2.97-2.77 (m, 2H), 2.35-2.17 (m, 2H), 1.88-1.76 (m, 2H), 1.53 (s, 9H).
Synthesis of intermediate 1g: tert-butyl 4-(4,5-dichloro-2-carbonyl-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-carboxylate (1e) (5.00 g, 12.94 mmol) was added into a dry three-necked round-bottom flask (250 mL), anhydrous tetrahydrofuran (130 mL) was added thereto, under argon displacement protection, the flask was cooled to 0° C. in an ice water bath, NaH (2.59 g, 64.72 mmol, 60% in mineral oil) was added in batches to the reaction mixture, thereafter, stirring was performed for reaction for 30 minutes, 4-(2-bromoethyl)morphinoline hydrobromide (4.81 g, 17.47 mmol) was slowly added to the reaction mixture, thereafter, stirring was continued for reaction for 1 hour, the ice water bath was removed, the flask was heated slowly to 35° C., and stirred for reaction for 18 hours, and the reaction of the raw materials was detected, by TLC plate, to be substantially complete. Under the conditions of cooling in an ice water bath and stirring, ice water was added dropwise to the reaction system for quenching the reaction, followed by extraction with ethyl acetate (150 mL×3). The organic phase was dried through anhydrous Na2SO4 and then filtered. The filtrate was evaporated under reduced pressure using a rotary evaporator to remove the solvent to obtain a gray-white solid crude product. Petroleum ether (150 mL) was added to the crude product, followed by dispersing, stirring, filtering, and drying to obtain 5.85 g white solid product, with a yield of 90.49%. 1H NMR (400 MHz, chloroform-d) δ 7.17 (s, 1H), 7.14 (s, 1H), 4.47-4.19 (m, 3H), 3.95 (t, J=6.6 Hz, 2H), 3.74-3.60 (m, 4H), 2.85 (t, J=12.5 Hz, 2H), 2.65 (t, J=6.6 Hz, 2H), 2.59-2.44 (m, 4H), 2.24 (qd, J=12.8, 4.5 Hz, 2H), 1.80 (d, J=12.1 Hz, 2H), 1.51 (s, 9H).
Tert-butyl 4-(5,6-dichloro-3-(2-morpholinoethyl)-2-carbonyl-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-carboxylate (1f) (5.85 g, 11.71 mmol) was added into a dry single neck flask (100 mL), anhydrous dichloromethane (60 mL) was added thereto, while the flask was cooled in an ice water bath, trifluoroacetic acid (15 mL) was added dropwise thereto, thereafter, the ice water bath was removed, stirring was performed at room temperature for reaction for 3 hours, and the reaction of the raw materials was detected, by TLC plate, to be substantially complete. The solvent was removed by evaporation under reduced pressure with a rotary evaporator to obtain a light brown oil crude product. Ether (150 mL) was added to the crude product, followed by dispersing, stirring, filtering, and drying to obtain 6.01 g white solid product, with a yield of 99.95%. The white solid did not need further purification, and could be directly used in the next experiment.
Synthesis of compound EX1: 5,6-dichloro-1-(2-morpholinoethyl)-3-(piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one trifluoroacetate (1g) (100 mg, 195 μM) and anhydrous 1,2-dichloroethane (2 mL) were added into a dry Schlenk tube, dry triethylamine (19.7 mg, 195 μmol, 27 μL) was added thereto, and stirred for 5 minutes, and 3,4-dichlorobenzaldehyde (51 mg, 292 μmol), acetic acid (14 mg, 234 μmol, 14 μL), NaBH(OAc)3 (1.17 mmol, 248 mg), and 4 Å molecular sieve (100 mg) were added to the reaction tube. Under argon protection, stirring was performed at room temperature for reaction for 24 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. Saturated sodium bicarbonate aqueous solution was added to the reaction flask for quenching the reaction, followed by extraction with dichloromethane for three times, combining the organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, and evaporation under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by silica gel chromatography column (mobile phase was dichloromethane and methanol, in a ratio of 150:1 to 70:1) to obtain 55 mg white solid product, with a yield of 50.6%. LC-MS(ESI) m/z: 557.10 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.48 (s, 1H), 7.41 (d, J=8.2 Hz, 1H), 7.30 (s, 1H), 7.22 (d, J=8.0 Hz, 1H), 7.12 (s, 1H), 4.34-4.22 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.51 (s, 2H), 3.01 (d, J=11.2 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.39 (q, J=11.1 Hz, 2H), 2.17 (t, J=11.7 Hz, 2H), 1.78 (d, J=11.7 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 3,4-difluorobenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 525.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.30 (s, 1H), 7.26-7.18 (m, 1H), 7.16-7.10 (m, 2H), 7.10-7.03 (m, 1H), 4.36-4.23 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.51 (s, 2H), 3.01 (d, J=11.3 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.38 (q, J=11.0, 10.5 Hz, 2H), 2.16 (t, J=11.5 Hz, 2H), 1.78 (d, J=11.7 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 2-chloro-4-fluorobenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 541.10 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.52 (t, J=7.3 Hz, 1H), 7.30 (s, 1H), 7.15-7.09 (m, 2H), 7.04-6.98 (m, 1H), 4.37-4.23 (m, 1H), 3.95 (t, J=6.6 Hz, 2H), 3.73-3.57 (m, 6H), 3.05 (d, J=11.0 Hz, 2H), 2.65 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.7 Hz, 4H), 2.47-2.33 (m, 2H), 2.28 (t, J=11.6 Hz, 2H), 1.79 (d, J=11.6 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 4-trifluoromethyl benzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 557.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.60 (d, J=7.9 Hz, 2H), 7.50 (d, J=7.9 Hz, 2H), 7.30 (s, 1H), 7.13 (s, 1H), 4.36-4.24 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.67 (t, J=4.7 Hz, 4H), 3.62 (s, 2H), 3.02 (d, J=11.3 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.40 (q, J=11.5 Hz, 2H), 2.19 (t, J=11.5 Hz, 2H), 1.78 (d, J=11.8 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 4-trifluoromethoxybenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 573.20; 1H NMR (400 MHz, chloroform-d) δ 7.40 (d, J=8.1 Hz, 2H), 7.31 (s, 1H), 7.19 (d, J=8.1 Hz, 2H), 7.12 (s, 1H), 4.36-4.23 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.56 (s, 2H), 3.03 (d, J=11.2 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.38 (q, J=10.3 Hz, 2H), 2.17 (t, J=11.3 Hz, 2H), 1.78 (d, J=10.6 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 2,4-dichlorobenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 557.10 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.50 (d, J=8.3 Hz, 1H), 7.39 (d, J=2.1 Hz, 1H), 7.31-7.26 (m, 2H), 7.12 (s, 1H), 4.37-4.22 (m, 1H), 3.95 (t, J=6.6 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.64 (s, 2H), 3.04 (d, J=10.9 Hz, 2H), 2.65 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.40 (q, J=11.9 Hz, 2H), 2.28 (t, J=11.2 Hz, 2H), 1.78 (d, J=10.9 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 4-tert-butylbenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 545.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.39-7.32 (m, 3H), 7.29 (d, J=8.1 Hz, 2H), 7.11 (s, 1H), 4.37-4.24 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.66 (t, J=4.6 Hz, 4H), 3.55 (s, 2H), 3.06 (d, J=11.2 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.45-2.28 (m, 2H), 2.15 (t, J=11.1 Hz, 2H), 1.76 (d, J=10.9 Hz, 2H), 1.33 (s, 9H).
It was synthesized from the raw materials of intermediate 1g and 4-tert-butyl cyclohexanone, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 537.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.26 (s, 1H), 7.12 (s, 1H), 4.31-4.18 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.26 (d, J=11.4 Hz, 2H), 2.65 (t, J=6.7 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.40-2.21 (m, 3H), 2.08-1.89 (m, 4H), 1.78 (d, J=10.7 Hz, 2H), 1.51-1.28 (m, 6H), 1.17-1.05 (m, 1H), 0.89 (s, 9H).
It was synthesized from the raw materials of intermediate 1g and cyclohexanone, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 481.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.39 (s, 1H), 7.11 (s, 1H), 4.38-4.21 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.06 (d, J=10.9 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.48-2.20 (m, 5H), 1.95-1.73 (m, 6H), 1.65 (d, J=12.7 Hz, 1H), 1.34-1.18 (m, 4H), 1.19-1.03 (m, 1H).
It was synthesized from the raw materials of intermediate 1g and cycloheptanone, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 495.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.38 (s, 1H), 7.11 (s, 1H), 4.35-4.19 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 2.94 (d, J=11.0 Hz, 2H), 2.70-2.58 (m, 3H), 2.52 (t, J=4.6 Hz, 4H), 2.44 (t, J=11.3 Hz, 2H), 2.28 (q, J=11.6, 10.5 Hz, 2H), 1.92-1.82 (m, 2H), 1.78 (d, J=10.0 Hz, 2H), 1.75-1.65 (m, 2H), 1.62-1.37 (m, 8H).
It was synthesized from the raw materials of intermediate 1g and cyclohexyl formaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 495.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.32 (s, 1H), 7.11 (s, 1H), 4.35-4.20 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.02 (d, J=11.3 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.34 (q, J=11.6, 10.0 Hz, 2H), 2.17 (d, J=7.0 Hz, 2H), 2.06 (t, J=11.2 Hz, 2H), 1.88-1.63 (m, 7H), 1.49 (bs, 1H), 1.32-1.11 (m, 3H), 0.90 (q, J=10.9, 10.2 Hz, 2H).
Synthesis of compound EX12: 5,6-dichloro-1-(2-morpholinoethyl)-3-(piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one trifluoroacetate (1g) (100 mg, 195 μM) and anhydrous 1,2-dichloroethane (2 mL) were added into a dry Schlenk tube, dry triethylamine (23 mg, 233 μmol, 32 μL) was added thereto, and stirred for 5 minutes, and 4-isopropylcyclohexanone (55 mg, 390 μmol), acetic acid (15 mg, 253 μmol, 14 μL), and NaBH(OAc)3 (165 mg, 779 μmol) were added to the reaction tube. Under argon protection, the tube was heated to 40° C. and stirred for reaction for 72 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. Saturated sodium bicarbonate aqueous solution was added to the reaction flask for quenching the reaction, followed by extraction with dichloromethane for three times, combining the organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, and evaporation under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by silica gel chromatography column (mobile phase was dichloromethane and methanol, in a ratio of 150:1 to 70:1) to obtain 86 mg white solid product, with a yield of 84.3%.
Compound EX12 in Example 12 was subjected to separation and purification by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 minutes) to obtain a trans product, 15 mg white solid, with a yield of 14.71%. LC-MS (ESI) m/z: 523.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.36 (s, 1H), 7.11 (s, 1H), 4.33-4.20 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.67 (d, J=4.7 Hz, 4H), 3.15 (d, J=11.1 Hz, 2H), 2.64 (t, J=6.7 Hz, 2H), 2.52 (d, J=4.6 Hz, 4H), 2.38-2.17 (m, 5H), 1.85-1.68 (m, 6H), 1.65-1.59 (m, 2H), 1.57-1.49 (m, 2H), 1.44-1.33 (m, 2H), 1.19-1.09 (m, 1H), 0.91 (s, 3H), 0.89 (s, 3H).
Compound EX12 in Example 12 was subjected to separation and purification by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 minutes) to obtain a cis product, 44 mg white solid, with a yield of 43.14%. LC-MS (ESI) m/z: 523.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.36 (s, 1H), 7.11 (s, 1H), 4.32-4.21 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.67 (d, J=4.7 Hz, 4H), 3.15 (d, J=11.1 Hz, 2H), 2.64 (t, J=6.7 Hz, 2H), 2.52 (d, J=4.7 Hz, 4H), 2.36-2.17 (m, 5H), 1.86-1.67 (m, 6H), 1.65-1.52 (m, 4H), 1.43-1.33 (m, 2H), 1.18-1.08 (m, 1H), 0.90 (s, 3H), 0.88 (s, 3H).
Intermediate 1g (100 mg, 194 μmol) and anhydrous dichloromethane (2 mL) were added into a dry Schlenk tube, and triethylamine (39 mg, 389 μmol, 54 μL) was added thereto, and stirred for 5 minutes. 4-chlorophenylboronic acid (61 mg, 389 μmol), Cu(OAc)2 (35 mg, 194 mol) and 4 Å molecular sieve (100 mg) were added to the reaction tube. The mixture was stirred at room temperature for reaction for 24 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. Dichloromethane (10 mL) was added into the reaction tube, followed by filtration with diatomite. The filtrate was evaporated under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by silica gel chromatography column (mobile phase was dichloromethane/methanol, in a ratio of 110:1 to 100:1) to obtain 40 mg white solid product, with a yield of 40.3%. LC-MS (ESI) m/z: 509.00 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.26-7.20 (m, 3H), 7.15 (s, 1H), 6.91 (d, J=9.0 Hz, 2H), 4.49-4.37 (m, 1H), 3.97 (s, 2H), 3.78 (d, J=12.5 Hz, 2H), 3.68 (s, 4H), 2.88 (t, J=11.7 Hz, 2H), 2.68 (bs, 2H), 2.62-2.37 (m, 6H), 1.92 (d, J=10.7 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 4-tert-butylphenylboronic acid, according to the synthesis method in Example 13. LC-MS (ESI) m/z: 531.10 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.32 (d, J=8.7 Hz, 2H), 7.27 (s, 1H), 7.14 (s, 1H), 6.94 (d, J=8.7 Hz, 2H), 4.50-4.37 (m, 1H), 3.96 (t, J=6.7 Hz, 2H), 3.81 (d, J=11.9 Hz, 2H), 3.72-3.63 (m, 4H), 2.87 (t, J=11.6 Hz, 2H), 2.66 (t, J=6.6 Hz, 2H), 2.59-2.43 (m, 6H), 1.90 (d, J=10.5 Hz, 2H), 1.31 (s, 9H).
Intermediate 1g (100 mg, 194 μmol) and anhydrous DMF (2 mL) were added into a dry Schlenk tube, and 2-chloro-5-trifluoromethylpyridine (35 mg, 194 μmol) and K2CO3 (81 mg, 584 μmol) were added thereto. The mixture was heated to 60° C., and stirred for reaction for 5 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. After the reaction mixture was cooled to room temperature, a small amount of water was added, followed by extraction with ethyl acetate (10 mL×3). The organic phase was washed with water and saturated saline, dried with anhydrous sodium sulfate, and then filtered. The filtrate was evaporated under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by a silica gel chromatography column (the mobile phase was petroleum ether/ethyl acetate, in a ratio of 5:1 to 1:1) to obtain 45 mg white solid product, with a yield of 42.4%. LC-MS (ESI) m/z: 544.01 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 8.43 (s, 1H), 7.67 (dd, J=9.0, 2.5 Hz, 1H), 7.14 (s, 1H), 7.12 (s, 1H), 6.73 (d, J=9.0 Hz, 1H), 4.70-4.60 (m, 2H), 4.58-4.46 (m, 1H), 3.95 (t, J=6.5 Hz, 2H), 3.73-3.61 (m, 4H), 3.04 (td, J=13.1, 2.6 Hz, 2H), 2.65 (t, J=6.5 Hz, 2H), 2.59-2.45 (m, 4H), 2.37 (qd, J=12.7, 4.3 Hz, 2H), 1.99-1.89 (m, 2H).
It was synthesized from the raw materials of intermediate 1g and 2-fluoro-4-chlorobenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 541.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.40 (t, J=8.1 Hz, 1H), 7.30 (s, 1H), 7.15 (dd, J=8.3, 2.0 Hz, 1H), 7.12 (s, 1H), 7.09 (dd, J=9.6, 2.1 Hz, 1H), 4.33-4.21 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.66 (t, J=4.6 Hz, 4H), 3.61 (s, 2H), 3.04 (d, J=11.1 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.46-2.29 (m, 2H), 2.23 (t, J=11.7 Hz, 2H), 1.78 (d, J=10.4 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 2,4-difluorobenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 525.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.47-7.35 (m, 1H), 7.31 (s, 1H), 7.12 (s, 1H), 6.89 (td, J=8.4, 2.5 Hz, 1H), 6.81 (td, J=9.4, 2.5 Hz, 1H), 4.34-4.21 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.66 (t, J=4.6 Hz, 4H), 3.61 (s, 2H), 3.04 (d, J=11.1 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.45-2.30 (m, 2H), 2.22 (t, J=11.7 Hz, 2H), 1.78 (d, J=11.3 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 2-chloro-4-methylbenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 538.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.40 (d, J=7.6 Hz, 1H), 7.31 (s, 1H), 7.19 (s, 1H), 7.11 (s, 1H), 7.08 (d, J=7.8 Hz, 1H), 4.37-4.23 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.73-3.59 (m, 6H), 3.07 (d, J=11.0 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.7 Hz, 4H), 2.46-2.34 (m, 2H), 2.33 (s, 3H), 2.31-2.20 (m, 2H), 1.77 (d, J=11.2 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 4-fluoro-2-methylbenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 521.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.28-7.21 (m, 2H), 7.12 (s, 1H), 6.93-6.81 (m, 2H), 4.36-4.22 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.73-3.61 (m, 4H), 3.46 (s, 2H), 3.01 (d, J=11.1 Hz, 2H), 2.64 (t, J=6.8 Hz, 2H), 2.58-2.44 (m, 4H), 2.40 (s, 3H), 2.37-2.25 (m, 2H), 2.16 (t, J=11.7 Hz, 2H), 1.76 (d, J=12.7 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 4-ethylcyclohexanone, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 509.30; (cis-trans isomer mixture)1H NMR (400 MHz, chloroform-d) δ 7.38 (s, 1H), 7.11 (s, 1H), 4.39-4.20 (m, 1H), 3.95 (t, J=6.7 Hz, 2H), 3.73-3.61 (m, 4H), 3.21-3.00 (m, 2H), 2.64 (t, J=6.8 Hz, 2H), 2.59-2.47 (m, 4H), 2.47-2.15 (m, 5H), 2.00-1.72 (m, 4H), 1.68-1.53 (m, 4H), 1.51-1.41 (m, 2H), 1.40-1.29 (m, 2H), 1.28-1.15 (m, 1H), 0.88 (t, J=7.4 Hz, 3H).
It was synthesized from the raw materials of intermediate 1g and 4-methylcyclohexanone, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 495.30 (M+H)+; (cis-trans isomer mixture, main product: by-product=2:1) main product: 1H NMR (400 MHz, chloroform-d) δ 7.39 (d, J=9.8 Hz, 1H), 7.12 (s, 1H), 4.38-4.21 (m, 1H), 3.95 (t, J=6.7 Hz, 2H), 3.73-3.62 (m, 4H), 3.13 (d, J=7.7 Hz, 1H), 2.64 (t, J=6.7 Hz, 2H), 2.60-2.46 (m, 4H), 2.45-2.21 (m, 5H), 1.91-1.73 (m, 4H), 1.67-1.43 (m, 6H), 1.30 (q, J=12.1 Hz, 1H), 0.96 (d, J=6.9 Hz, 3H).
It was synthesized from the raw materials of intermediate 1g and 4-trifluoromethyl cyclohexanone, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 549.30 (M+H)+; (cis-trans isomer mixture, main product: by-product=2:1) main product: 1H NMR (400 MHz, chloroform-d) δ 7.31 (s, 1H), 7.12 (s, 1H), 4.38-4.19 (m, 1H), 3.95 (t, J=6.8 Hz, 2H), 3.75-3.59 (m, 4H), 3.17 (d, J=11.2 Hz, 2H), 2.65 (t, J=6.8 Hz, 2H), 2.59-2.48 (m, 4H), 2.44-2.28 (m, 3H), 2.25-2.12 (m, 2H), 2.11-1.99 (m, 2H), 1.98-1.86 (m, 3H), 1.81 (d, J=12.1 Hz, 2H), 1.66-1.51 (m, 2H), 1.45-1.21 (m, 2H).
It was synthesized from the raw materials of intermediate 1g and N-isopropyl-4-piperidinone, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 524.40 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.37 (s, 1H), 7.11 (s, 1H), 4.29 (t, J=12.7 Hz, 1H), 3.94 (t, J=6.8 Hz, 2H), 3.79-3.58 (m, 5H), 3.12-2.93 (m, 4H), 2.76 (br s, 1H), 2.64 (t, J=6.8 Hz, 2H), 2.57-2.48 (m, 4H), 2.48-2.23 (m, 6H), 2.17 (br s, 2H), 1.89-1.76 (m, 4H), 1.67 (br s, 2H), 1.07 (d, J=6.3 Hz, 6H).
It was synthesized from the raw materials of intermediate 1g and 4-fluorobenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 507.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.38-7.28 (m, 3H), 7.12 (s, 1H), 7.03 (t, J=8.6 Hz, 2H), 4.38-4.23 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.53 (s, 2H), 3.02 (d, J=11.2 Hz, 2H), 2.64 (t, J=6.7 Hz, 2H), 2.52 (t, J=4.7 Hz, 4H), 2.36 (q, J=22.4, 10.0 Hz, 2H), 2.14 (t, J=11.9 Hz, 2H), 1.77 (d, J=11.0 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 4-chlorobenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 523.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.32 (s, 5H), 7.12 (s, 1H), 4.38-4.23 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.73-3.62 (m, 4H), 3.55 (s, 2H), 3.13-2.92 (m, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.59-2.47 (m, 4H), 2.38 (br s, 2H), 2.26-2.07 (m, 2H), 1.78 (d, J=12.3 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 2-indanone, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 515.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.39 (s, 1H), 7.24-7.17 (m, 2H), 7.17-7.09 (m, 3H), 4.43-4.28 (m, 1H), 3.95 (t, J=6.5 Hz, 2H), 3.73-3.61 (m, 4H), 3.35-3.23 (m, 1H), 3.23-3.04 (m, 4H), 2.95 (dd, J=15.5, 8.5 Hz, 2H), 2.64 (t, J=6.7 Hz, 2H), 2.58-2.47 (m, 4H), 2.39 (q, J=12.5 Hz, 2H), 2.24 (t, J=11.8 Hz, 2H), 1.84 (d, J=11.9 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 2-chlorocyclohexanone, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 515.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.37 (s, 1H), 7.11 (s, 1H), 4.62 (s, 1H), 4.39-4.25 (m, 1H), 3.95 (t, J=6.7 Hz, 2H), 3.67 (t, J=4.4 Hz, 4H), 3.28 (d, J=9.9 Hz, 2H), 2.64 (t, J=6.7 Hz, 2H), 2.57-2.43 (m, 6H), 2.41-2.21 (m, 3H), 2.07 (d, J=10.7 Hz, 1H), 1.91-1.66 (m, 8H), 1.56-1.45 (m, 1H), 1.32 (br s, 1H).
It was synthesized from the raw materials of intermediate 1g and 4-chloro-2-methylbenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 537.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.24 (d, J=8.8 Hz, 2H), 7.19-7.09 (m, 3H), 4.35-4.22 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.46 (s, 2H), 3.01 (d, J=11.1 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.42-2.26 (m, 5H), 2.17 (t, J=11.7 Hz, 2H), 1.76 (d, J=11.6 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 4-chloro-2-methoxybenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 553.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.35 (d, J=7.1 Hz, 2H), 7.12 (s, 1H), 6.96 (d, J=8.1 Hz, 1H), 6.87 (s, 1H), 4.36-4.23 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.83 (s, 3H), 3.66 (t, J=4.6 Hz, 4H), 3.59 (s, 2H), 3.06 (d, J=11.3 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.47-2.30 (m, 2H), 2.23 (t, J=11.9 Hz, 2H), 1.77 (d, J=11.3 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 2,4-dimethylbenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 517.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.33-7.26 (m, 1H), 7.22-7.14 (m, 1H), 7.11 (s, 1H), 7.04-6.94 (m, 2H), 4.38-4.22 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.73-3.60 (m, 4H), 3.48 (s, 2H), 3.04 (d, J=11.0 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.58-2.46 (m, 4H), 2.45-2.25 (m, 8H), 2.24-2.07 (m, 2H), 1.75 (d, J=11.4 Hz, 2H).
It was synthesized from the raw materials of intermediate 1g and 2,4,6-trimethylbenzaldehyde, according to the synthesis method in Example 1. LC-MS (ESI) m/z: 531.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.20 (s, 1H), 7.10 (s, 1H), 6.86 (s, 2H), 4.36-4.20 (m, 1H), 3.93 (t, J=6.7 Hz, 2H), 3.70-3.62 (m, 4H), 3.49 (s, 2H), 3.03-2.90 (m, 2H), 2.63 (t, J=6.6 Hz, 2H), 2.56-2.48 (m, 4H), 2.39 (s, 6H), 2.32-2.18 (m, 7H), 1.78-1.66 (m, 2H).
Synthesis of intermediate 32a: 5-chloro-2-fluoronitrobenzene and 1-N-Boc-4-aminopiperidine were used as raw materials for synthesis according to the synthesis method of intermediate 1f to obtain 835 mg colorless waxy product, with a yield of 63.2%. 1H NMR (400 MHz, chloroform-d) δ 7.05 (s, 1H), 7.02 (s, 2H), 4.50-4.38 (m, 1H), 4.31 (br s, 2H), 3.97 (t, J=6.4 Hz, 2H), 3.69 (t, J=4.4 Hz, 4H), 2.85 (t, J=13.4 Hz, 2H), 2.68 (d, J=6.8 Hz, 2H), 2.55 (t, J=4.5 Hz, 4H), 2.26 (qd, J=12.5, 4.2 Hz, 2H), 1.80 (d, J=12.8 Hz, 2H), 1.50 (s, 9H).
Synthesis of intermediate 32b: intermediate 32a was subjected to the action of trifluoroacetic acid at room temperature to remove Boc protection group to obtain 720 mg white trifluoroacetate solid, with a yield of 99.9%. The solid was directly used in the next reaction without purification.
Synthesis of compound EX32: intermediate 32b and 2-chlorobenzyl bromide were used as raw materials in anhydrous dichloromethane, and triethylamine was added thereto for a substitution reaction to produce the target compound, which was separated and purified by column chromatography (mobile phase was dichloromethane/methanol, with the methanol volume ratio being 1%) to obtain a white solid product (250 mg, with a yield of 97.8%). LC-MS (ESI) m/z: 490.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.53 (d, J=7.5 Hz, 1H), 7.36 (d, J=7.9 Hz, 1H), 7.30-7.24 (m, 1H), 7.21 (d, J=7.6 Hz, 1H), 7.16 (d, J=9.3 Hz, 1H), 7.06-6.99 (m, 2H), 4.42-4.29 (m, 1H), 3.96 (t, J=6.9 Hz, 2H), 3.74-3.61 (m, 6H), 3.06 (d, J=11.1 Hz, 2H), 2.66 (t, J=6.9 Hz, 2H), 2.59-2.49 (m, 4H), 2.43 (q, J=12.5 Hz, 2H), 2.28 (t, J=11.8 Hz, 2H), 1.78 (d, J=12.0 Hz, 2H).
Synthesis of intermediate 33a: 5-chloro-2,4-difluoronitrobenzene was used as the raw material for synthesis according to the synthesis method of intermediate 1e in Example 1, to obtain 3.24 g white solid, with a yield of 72.80%. 1H NMR (400 MHz, chloroform-d) δ 9.88 (s, 1H), 7.13 (d, J=6.3 Hz, 1H), 6.96 (d, J=9.2 Hz, 1H), 4.46-4.28 (m, 3H), 2.85 (t, J=13.1 Hz, 2H), 2.25 (qd, J=12.5, 4.6 Hz, 2H), 1.86-1.78 (m, 2H), 1.52 (s, 9H).
Synthesis of intermediate 33b: 33a was used as the raw material for synthesis according to the synthesis method of intermediate 1f in Example 1, to obtain intermediate 33b, 1.29 g colorless oily product, with a yield of 98.78%. 1H NMR (400 MHz, chloroform-d) δ 7.05 (d, J=6.3 Hz, 1H), 6.94 (d, J=9.1 Hz, 1H), 4.45-4.23 (m, 3H), 3.95 (t, J=6.7 Hz, 2H), 3.73-3.63 (m, 4H), 2.90-2.77 (m, 2H), 2.65 (t, J=6.7 Hz, 2H), 2.59-2.46 (m, 4H), 2.22 (qd, J=12.7, 4.6 Hz, 2H), 1.86-1.74 (m, 2H), 1.50 (s, 9H).
Synthesis of intermediate 33c: intermediate 33b (1.30 g, 2.69 mmol) was added into a dry single neck flask (50 mL), anhydrous dioxane (5 mL) was added thereto, HCl/dioxane solution (4.0 M) was added dropwise thereto under cooling in an ice water bath, thereafter, the ice water bath was removed, and stirring was continued at room temperature for reaction for 3 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. The solvent was removed by evaporation under reduced pressure by a rotary evaporator to obtain a light brown oil crude product. Methyl tert-butyl ether (50 mL) was added to the crude product, followed by dispersing, stirring, filtering, and drying to obtain 1.13 g white solid product (intermediate 33c), with a yield of 100%. The white solid did not need further purification, and could be directly used in the next experiment.
Synthesis of compound EX33: intermediate 33c and 2-chloro-4-fluorobenzaldehyde were used as raw materials for synthesis according to the synthesis method in Example 1, to obtain a white solid product (58 mg, with a yield of 23.14%). LC-MS (ESI) m/z: 525.35 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.52 (t, J=7.5 Hz, 1H), 7.16-7.06 (m, 2H), 7.06-6.97 (m, 2H), 4.39-4.25 (m, 1H), 3.95 (t, J=6.7 Hz, 2H), 3.72-3.65 (m, 4H), 3.63 (s, 2H), 3.04 (d, J=10.9 Hz, 2H), 2.65 (t, J=6.7 Hz, 2H), 2.60-2.47 (m, 4H), 2.46-2.19 (m, 4H), 1.79 (d, J=11.6 Hz, 2H).
Synthesis of compound EX34: intermediate 33c and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain a white solid product (156 mg, with a yield of 64.4%).
Synthesis of compound EX34A: intermediate 33c and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX34, which was subjected to preparation and separation by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 min) to obtain a trans product, a white solid product (16 mg, with a yield of 6.61%). LC-MS (ESI) m/z: 507.38 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.16 (d, J=9.4 Hz, 1H), 7.02 (d, J=6.2 Hz, 1H), 4.36-4.23 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.67 (t, J=4.7 Hz, 4H), 3.05 (d, J=11.4 Hz, 2H), 2.64 (t, J=6.7 Hz, 2H), 2.53 (t, J=4.6 Hz, 4H), 2.44-2.20 (m, 5H), 1.91 (d, J=11.7 Hz, 2H), 1.84-1.74 (m, 4H), 1.48-1.35 (m, 1H), 1.26 (q, J=12.3 Hz, 2H), 1.08-0.95 (m, 3H), 0.87 (s, 3H), 0.85 (s, 3H).
Synthesis of compound EX34B: intermediate 33c and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX34, which was subjected to preparation and separation by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 min) to obtain a cis product, a white solid product (48 mg, with a yield of 19.84%). LC-MS (ESI) m/z: 507.38 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.11 (d, J=9.3 Hz, 1H), 7.03 (d, J=6.2 Hz, 1H), 4.34-4.22 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.68 (t, J=4.7 Hz, 4H), 3.15 (d, J=10.7 Hz, 2H), 2.64 (t, J=6.7 Hz, 2H), 2.53 (t, J=4.7 Hz, 4H), 2.36-2.13 (m, 5H), 1.83-1.75 (m, 2H), 1.73-1.59 (m, 5H), 1.57-1.45 (m, 2H), 1.44-1.32 (m, 2H), 1.18-1.08 (m, 1H), 0.90 (s, 3H), 0.89 (s, 3H).
Synthesis of intermediate 35a: 4-chloro-2,5-difluoronitrobenzene was used as raw material for synthesis according to the synthesis method of intermediate 1f in Example 1 to obtain a product (1.12 g, with a yield of 85.8%). 1H NMR (400 MHz, chloroform-d) δ 7.09 (d, J=5.5 Hz, 1H), 6.89 (d, J=8.4 Hz, 1H), 4.48-4.17 (m, 3H), 3.95 (t, J=6.8 Hz, 2H), 3.67 (t, J=4.5 Hz, 4H), 2.88-2.76 (m, 2H), 2.65 (t, J=6.8 Hz, 2H), 2.53 (t, J=4.5 Hz, 4H), 2.25 (q, J=12.3, 11.0 Hz, 2H), 1.80 (d, J=12.1 Hz, 2H), 1.51 (s, 9H).
Synthesis of intermediate 35b: 35a was used as raw material to obtain a white solid product (1.13 g, with a yield of 100%) according to the synthesis of intermediate 33c in Example 33. 1H NMR (400 MHz, Methanol-d4) δ 7.47 (d, J=6.2 Hz, 1H), 7.30 (d, J=9.0 Hz, 1H), 4.59-4.48 (m, 1H), 4.30 (t, J=6.0 Hz, 2H), 3.90 (br s, 4H), 3.66-3.50 (m, 5H), 3.45 (br s, 3H), 3.20 (td, J=13.2, 3.0 Hz, 2H), 2.69 (qd, J=13.5, 4.3 Hz, 2H), 2.16-2.03 (m, 2H).
Synthesis of compound EX35: intermediate 35b and 2-chloro-4-fluorobenzaldehyde were used as raw materials for synthesis according to the synthesis method in Example 1 to obtain a white solid product (110 mg, with a yield of 43.89%). LC-MS (ESI) m/z: 525.36 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.53 (t, J=7.5 Hz, 1H), 7.23 (d, J=6.1 Hz, 1H), 7.15-7.08 (m, 1H), 7.05-6.96 (m, 1H), 6.88 (d, J=8.6 Hz, 1H), 4.39-4.24 (m, 1H), 3.95 (t, J=6.7 Hz, 2H), 3.72-3.58 (m, 6H), 3.05 (d, J=11.0 Hz, 2H), 2.65 (t, J=6.7 Hz, 2H), 2.60-2.47 (m, 4H), 2.40 (qd, J=12.3, 3.4 Hz, 2H), 2.28 (t, J=11.7 Hz, 2H), 1.79 (d, J=11.6 Hz, 2H).
Synthesis of compound EX36: intermediate 35b and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain a white solid product (126 mg, with a yield of 52.1%).
Synthesis of compound EX36A: intermediate 35b and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX36, which was subjected to preparation and separation by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 min) to obtain a trans product, a white solid product (16 mg, with a yield of 6.61%). LC-MS (ESI) m/z: 507.54 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.31 (d, J=6.2 Hz, 1H), 6.85 (d, J=8.7 Hz, 1H), 4.36-4.24 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.66 (t, J=4.6 Hz, 4H), 3.06 (d, J=11.1 Hz, 2H), 2.64 (t, J=6.7 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.45-2.22 (m, 5H), 1.92 (d, J=12.1 Hz, 2H), 1.86-1.73 (m, 4H), 1.49-1.35 (m, 1H), 1.27 (q, J=12.4, 11.7 Hz, 2H), 1.09-0.94 (m, 3H), 0.87 (s, 3H), 0.86 (s, 3H).
Synthesis of compound EX36B: intermediate 35b and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX36, which was subjected to preparation and separation by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 min) to obtain a cis product, a white solid product (46 mg, with a yield of 19.02%). LC-MS (ESI) m/z: 507.52 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.27 (d, J=6.1 Hz, 2H), 6.86 (d, J=8.7 Hz, 1H), 4.34-4.21 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.15 (d, J=11.5 Hz, 2H), 2.64 (t, J=6.7 Hz, 2H), 2.52 (t, J=4.5 Hz, 4H), 2.38-2.16 (m, 5H), 1.84-1.76 (m, 2H), 1.76-1.68 (m, 2H), 1.68-1.57 (m, 3H), 1.57-1.48 (m, 2H), 1.44-1.32 (m, 2H), 1.19-1.09 (m, 1H), 0.91 (s, 3H), 0.89 (s, 3H).
Synthesis of intermediate 37a: 2-chloro-4,5-difluoronitrobenzene was used as raw material for synthesis according to the synthesis method of intermediate 1f in Example 1 to obtain a product (1.30 g, with a yield of 98.5%). 1H NMR (400 MHz, chloroform-d) δ 6.95 (dd, J=10.2, 6.7 Hz, 1H), 6.89 (dd, J=9.7, 6.9 Hz, 1H), 4.46-4.21 (m, 3H), 3.94 (t, J=6.7 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 2.93-2.75 (m, 2H), 2.65 (t, J=6.7 Hz, 2H), 2.53 (t, J=4.6 Hz, 4H), 2.22 (qd, J=12.6, 4.6 Hz, 2H), 1.86-1.74 (m, 2H), 1.50 (s, 9H).
Synthesis of intermediate 37b: intermediate 37a was used as raw material for synthesis according to the synthesis method of intermediate 1g in Example 1 to obtain a white product (1.00 g, with a yield of 100%). 1H NMR (400 MHz, Deuterium Oxide) δ 7.22 (dd, J=10.6, 6.8 Hz, 1H), 7.14 (dd, J=10.1, 6.8 Hz, 1H), 4.47-4.32 (m, 1H), 4.22 (t, J=5.9 Hz, 2H), 4.14-3.63 (m, 5H), 3.57-3.40 (m, 5H), 3.39-3.16 (m, 2H), 3.10 (td, J=13.3, 3.0 Hz, 2H), 2.48 (qd, J=13.5, 4.3 Hz, 2H), 2.03 (d, J=13.7 Hz, 2H).
Synthesis of compound EX37: intermediate 37b and 2-chloro-4-fluorobenzaldehyde were used as raw materials for synthesis according to the synthesis method in Example 1 to obtain a white solid product (106 mg, with a yield of 48.26%). LC-MS (ESI) m/z: 509.38 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.52 (t, J=7.6 Hz, 1H), 7.16-7.05 (m, 2H), 7.05-6.96 (m, 1H), 6.93-6.83 (m, 1H), 4.40-4.24 (m, 1H), 3.95 (t, J=6.7 Hz, 2H), 3.72-3.64 (m, 4H), 3.63 (s, 2H), 3.04 (d, J=10.8 Hz, 2H), 2.65 (t, J=6.8 Hz, 2H), 2.59-2.46 (m, 4H), 2.46-2.15 (m, 4H), 1.79 (d, J=11.6 Hz, 2H).
Synthesis of compound EX38: intermediate 37b and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain a white solid product (132 mg, with a yield of 54.5%).
Synthesis of compound EX38A: intermediate 37b and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX38, which was subjected to preparation and separation by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 min) to obtain a trans product, a white solid product (15 mg, with a yield of 6.16%). LC-MS (ESI) m/z: 491.52 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.18 (dd, J=10.5, 6.8 Hz, 1H), 6.86 (dd, J=9.9, 6.8 Hz, 1H), 4.35-4.24 (m, 1H), 3.94 (t, J=6.8 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.05 (d, J=11.0 Hz, 2H), 2.64 (t, J=6.8 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.45-2.16 (m, 6H), 1.91 (d, J=11.2 Hz, 2H), 1.86-1.72 (m, 4H), 1.45-1.38 (m, 1H), 1.33-1.19 (m, 2H), 1.09-1.00 (m, 2H), 0.87 (s, 3H), 0.86 (s, 3H).
Synthesis of compound EX38B: intermediate 37b and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX38, which was subjected to preparation and separation by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 min) to obtain a cis product, a white solid product (45 mg, with a yield of 18.47%). LC-MS (ESI) m/z: 491.52 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.14 (dd, J=10.4, 6.8 Hz, 1H), 6.86 (dd, J=9.9, 6.8 Hz, 1H), 4.33-4.23 (m, 1H), 3.94 (t, J=6.8 Hz, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.15 (d, J=11.4 Hz, 2H), 2.64 (t, J=6.8 Hz, 2H), 2.52 (t, J=4.5 Hz, 4H), 2.37-2.12 (m, 5H), 1.84-1.75 (m, 3H), 1.75-1.57 (m, 5H), 1.57-1.47 (m, 2H), 1.44-1.33 (m, 2H), 1.19-1.07 (m, 1H), 0.90 (s, 3H), 0.89 (s, 3H).
Synthesis of intermediate 39c: 4-(2-hydroxyethyl)thiomorpholine-1,1-dioxide (2.00 g, 11.16 mmol) and anhydrous dichloromethane (120 mL) were added into a dry single neck flask (200 mL). Triethylamine (1.69 g, 2.33 mL, 16.74 mmol) was added to the reaction flask, under argon displacement protection, the flask was cooled to 0° C. in an ice water bath, methylsulfonyl chloride (1.53 g, 1.04 mL, 13.39 mmol) was added to the reaction system, and thereafter, stirring was continued at room temperature for reaction for 2 hours. The reaction of the raw materials was detected, by TLC, to be substantially complete. Saturated NaHCO3 aqueous solution was added for quenching the reaction, followed by liquid separation. The organic phase was washed by saturated NaCl aqueous solution, followed by drying with anhydrous MgSO4, filtering, and rotary-drying to obtain light brown oily crude product 39b (2.15 g, with a yield of 74.88%). The crude product was directly used in the next reaction without further purification.
Tert-butyl 4-(5,6-dichloro-2-carbonyl-2,3-dihydro-1Hbenzo[d]imidazol-1-yl) piperidin-1-carboxylate (1e) (500 mg, 1.29 mmol) and anhydrous DMF (8 mL) were added into a dry double-necked flask (25 mL), under argon displacement protection, the flask was cooled to 0° C. in an ice water bath. NaH (207 mg, 5.18 mmol, 60% in mineral oil) was added to the reaction flask. Stirring was continued in an ice water bath for reaction for 30 minutes, and 2-(1,1-dioxothiomorpholine)ethyl mesylate (39b) (833 mg, 3.24 mmol) anhydrous DMF (5 mL) solution was added dropwise to the reaction mixture, with the temperature being held at 0° C., stirring was performed for reaction for 1 hour, and the temperature was gradually raised to 45 for reaction for 12 hours. The reaction of the raw materials was detected, by TLC, to be substantially complete. After the completion of the reaction, the reaction mixture solution was cooled to room temperature, and slowly poured into ice water, and white solid was precipitated. After the solid was filtered out, washing with water, and drying were performed, and the crude product was dispersed in petroleum ether, followed by slurrying, filtering, and drying to obtain a white solid product (632 mg, with a yield of 89.2%). 1H NMR (400 MHz, chloroform-d) δ 7.19 (s, 1H), 7.03 (s, 1H), 4.45-4.24 (m, 3H), 3.95 (t, J=6.2 Hz, 2H), 3.11-3.03 (m, 4H), 3.03-2.96 (m, 4H), 2.88-2.74 (m, 4H), 2.24 (qd, J=12.6, 4.6 Hz, 2H), 1.85-1.73 (m, 2H), 1.51 (s, 9H).
Synthesis of intermediate 39d: Synthesis was performed according to the synthesis method of intermediate 1f in Example 1 to obtain a white solid product (205 mg, with a yield of 99.9%). The solid was directly used in the next reaction without purification.
Synthesis of compound EX39: intermediate 39d (200 mg, 356 μmol) and anhydrous dichloromethane (3.5 mL) were added into a dry round-bottom flask (50 mL), triethylamine (216 mg, 2.14 mmol) was added to the reaction solution, which was stirred for 5 minutes and cooled to 0° C. in an ice water bath, under argon protection, 2-chloro-4-fluorobenzyl bromide (87.6 mg, 392 μmol) dichloromethane (1.5 mL) solution was added dropwise to the reaction mixture solution. Thereafter, the temperature was gradually raised to room temperature and stirring was continued for reaction for 12 hours. The reaction was detected, by TLC plate, to be completed. Saturated sodium bicarbonate aqueous solution was added to the reaction mixture solution for quenching the reaction, followed by extraction with dichloromethane (25 mL×3), washing with saturated saline, drying with anhydrous magnesium sulfate, and filtering. The filtrate was evaporated by a rotary evaporator to remove the solvent to obtain a light brown crude product, which was subjected to purification and separation with silica gel column chromatography (the mobile phase was DCM/MeOH, in a ratio of 150:1 to 100:1) to obtain a white solid product (165 mg, with a yield of 78.51%). LC-MS (ESI) m/z: 589.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.51 (t, J=7.4 Hz, 1H), 7.32 (s, 1H), 7.13 (dd, J=8.6, 2.6 Hz, 1H), 7.07-6.95 (m, 2H), 4.35-4.19 (m, 1H), 3.95 (t, J=6.2 Hz, 2H), 3.64 (s, 2H), 3.18-3.02 (m, 6H), 3.02-2.91 (m, 4H), 2.82 (t, J=6.2 Hz, 2H), 2.48-2.33 (m, 2H), 2.33-2.19 (m, 2H), 1.86-1.62 (m, 3H).
Synthesis of intermediate 40a: tert-butyl 4-(5,6-dichloro-2-carbonyl-2, 3-dihydro-1H-benzo[d]imidazol-1-yl]piperidin-1-carboxylate (3.00 g, 7.77 mmol), hexamethylphosphoryl triamine (HMPA) (2.78 g, 15.53 mmol), and anhydrous tetrahydrofuran (80 mL) were added into a clean dry double-mouth flask (200 mL). Under argon displacement protection, the flask was cooled to 0° C. in an ice water bath. NaH (932 mg, 23.30 mmol, 60% in mineral oil) was added to the reaction mixture in batches. After stirring for half an hour, 1-chloro-2bromoethane (4.46 g, 31.07 mmol, 2.68 mL) was slowly added to the reaction system. Thereafter, the ice water bath was removed, stirring was continued at room temperature for one hour, and the temperature was gradually raised to 80° C. for reflux reaction for 8 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. After the completion of the reaction, the reaction system was cooled to room temperature, and under stirring condition, ice water was added dropwise to the reaction system for quenching the reaction, followed by extraction with ethyl acetate (150 mL×3). The organic phase was washed with saturated NH4Cl aqueous solution and saturated NaCl aqueous solution respectively, dried with anhydrous Na2SO4, and then filtered and evaporated with a rotary evaporator to remove the solvent, to obtain a light brown solid crude product. The crude product was dispersed in a petroleum ether/ethyl acetate (1% ethyl acetate) mixed solvent, stirred for 30 minutes, and then filtered to obtain a white solid product (2.91 g, with a yield of 83.5%). 1H NMR (400 MHz, chloroform-d) δ 7.19 (s, 1H), 7.17 (s, 1H), 4.47-4.22 (m, 3H), 4.16 (t, J=6.1 Hz, 2H), 3.82 (t, J=6.1 Hz, 2H), 2.96-2.73 (m, 2H), 2.24 (qd, J=12.7, 4.6 Hz, 2H), 1.88-1.72 (m, 2H), 1.51 (s, 9H).
Synthesis of intermediate 40b: tert-butyl 4-(5,6-dichloro-3-(2-chloroethyl)-2-carbonyl-2,3-dihydro-1H-benzo[d]imidazol-1-yl) piperidin-1-carboxylate (40a) (325 mg, 724 μmol), and Cs2CO3 (708 mg, 2.17 mmol) were added into a clean dry Schlenk tube (25 mL), anhydrous dioxane (7.2 mL) was added thereto, under stirring at room temperature, N-methylpiperazine (290 mg, 321 μL, 2.90 mmol) was added, and under argon displacement protection, the tube was heated to 105° C. for reflux reaction for 12 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. After the completion of the reaction, the reaction flask was cooled to room temperature, an appropriate amount of water was added thereto, followed by extraction with ethyl acetate (15 mL×3). The organic phase was washed with saturated NaCl aqueous solution, and dried with anhydrous Na2SO4, and then filtered and evaporated with a rotary evaporator to remove the solvent, to obtain a light brown oily crude product, which was subjected to separation by silica gel column chromatography (the mobile phase was DCM/MeOH, with 0.5%-2% MeOH) to obtain a light yellow waxy product (301 mg, with a yield of 81.10%). 1H NMR (400 MHz, chloroform-d) δ 7.16 (d, J=0.8 Hz, 2H), 4.48-4.19 (m, 3H), 3.94 (t, J=6.7 Hz, 2H), 2.93-2.76 (m, 2H), 2.73-2.50 (m, 6H), 2.50-2.30 (m, 4H), 2.28 (s, 4H), 2.28-2.16 (m, 3H), 1.85-1.75 (m, 2H), 1.51 (s, 9H).
Synthesis of intermediate 40c: 40b was used as raw material for synthesis according to the synthesis method of intermediate 1g in Example 1 to obtain a white solid product (273 mg, with a yield of 100%). The solid was directly used in the next reaction without purification.
Synthesis of compound EX40: 40c and 2-chloro-4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (198 mg, with a yield of 69.05%). LC-MS (ESI) m/z: 554.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.52 (t, J=7.4 Hz, 1H), 7.30 (s, 1H), 7.17-7.09 (m, 2H), 7.01 (t, J=8.3 Hz, 1H), 4.37-4.22 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.63 (s, 2H), 3.05 (d, J=11.1 Hz, 2H), 2.72-2.54 (m, 6H), 2.55-2.32 (m, 6H), 2.33-2.20 (m, 5H), 1.78 (d, J=10.8 Hz, 2H).
Synthesis of intermediate 41a: 1-acetylpiperazine and 40a were used as raw materials for synthesis according to the synthesis method of intermediate 40b in Example 40 to obtain a light yellow waxy product (266 mg, with a yield of 73.62%). 1H NMR (400 MHz, chloroform-d) δ 7.16 (d, J=0.8 Hz, 2H), 4.48-4.19 (m, 3H), 3.94 (t, J=6.7 Hz, 2H), 2.93-2.76 (m, 2H), 2.73-2.50 (m, 6H), 2.50-2.30 (m, 4H), 2.28 (s, 4H), 2.28-2.16 (m, 3H), 1.85-1.75 (m, 2H), 1.51 (s, 9H).
Synthesis of compound EX41: 41a was used as raw material for synthesis according to the synthesis method of intermediate 40c in Example 40 to obtain intermediate 41b (235 mg, with a yield of 99.6%). Intermediate 41b and 2-chloro-4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (116 mg, with a yield of 46.75%). LC-MS (ESI) m/z: 584.20 (M+3H)+; 1H NMR (400 MHz, chloroform-d) δ 7.52 (t, J=7.5 Hz, 1H), 7.30 (s, 1H), 7.15-7.07 (m, 2H), 7.05-6.96 (m, 1H), 4.35-4.23 (m, 1H), 3.96 (t, J=6.4 Hz, 2H), 3.64 (s, 2H), 3.57 (t, J=5.0 Hz, 2H), 3.42 (t, J=5.0 Hz, 2H), 3.05 (d, J=11.0 Hz, 2H), 2.67 (t, J=6.4 Hz, 2H), 2.53 (t, J=5.0 Hz, 2H), 2.48 (t, J=5.0 Hz, 2H), 2.39 (qd, J=12.4, 3.4 Hz, 2H), 2.27 (t, J=11.7 Hz, 2H), 2.07 (s, 3H), 1.78 (d, J=11.2 Hz, 2H).
Synthesis of intermediate 42a: 2-piperazinone and 40a were used as raw materials for synthesis according to the synthesis method of intermediate 40b in Example 40. to obtain a colorless waxy product (167 mg, with a yield of 48.8%). 1H NMR (400 MHz, chloroform-d) δ 7.18 (s, 1H), 7.07 (s, 1H), 6.43 (s, 1H), 4.47-4.19 (m, 3H), 3.97 (t, J=6.4 Hz, 2H), 3.38-3.29 (m, 2H), 3.19 (s, 2H), 2.95-2.80 (m, 2H), 2.79-2.68 (m, 4H), 2.24 (qd, J=12.6, 4.5 Hz, 2H), 1.87-1.73 (m, 2H), 1.51 (s, 9H).
Synthesis of compound EX42: 42a was used as raw material for synthesis according to the synthesis method of intermediate 40c in Example 40 to obtain intermediate 42b (150 mg, with a yield of 100%). Intermediate 42b and 2-chloro-4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (88 mg, with a yield of 56.02%). LC-MS (ESI) m/z: 554.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.51 (t, J=7.3 Hz, 1H), 7.30 (s, 1H), 7.12 (d, J=8.5 Hz, 1H), 7.06 (s, 1H), 7.01 (t, J=8.4 Hz, 1H), 6.42 (s, 1H), 4.34-4.22 (m, 1H), 3.96 (t, J=6.5 Hz, 2H), 3.63 (s, 2H), 3.38-3.27 (m, 2H), 3.19 (s, 2H), 3.04 (d, J=11.1 Hz, 2H), 2.81-2.66 (m, 4H), 2.38 (qd, J=12.1, 3.5 Hz, 2H), 2.27 (t, J=11.6 Hz, 2H), 1.78 (d, J=11.4 Hz, 2H).
Synthesis of intermediate 43a: pyrrole and 40a were used as raw materials for synthesis according to the synthesis method of intermediate 40b in Example 40 to obtain a colorless oily product (485 mg, with a yield of 90.1%). 1H NMR (400 MHz, chloroform-d) δ 7.16 (s, 1H), 7.13 (s, 1H), 4.41 (tt, J=12.5, 4.2 Hz, 1H), 4.31 (s, 2H), 3.97 (t, J=7.2 Hz, 2H), 2.86 (d, J=13.8 Hz, 2H), 2.78 (t, J=7.2 Hz, 2H), 2.65-2.55 (m, 4H), 2.23 (qd, J=12.7, 4.6 Hz, 2H), 1.82-1.76 (m, 6H), 1.51 (s, 9H).
Synthesis of compound EX43: 43a was used as raw material for synthesis according to the synthesis method of intermediate 40c in Example 40 to obtain intermediate 43b (333 mg, with a yield of 90.5%). Intermediate 43b and 2-chloro-4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (182 mg, with a yield of 51.40%). LC-MS (ESI) m/z: 525.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.52 (t, J=7.5 Hz, 1H), 7.29 (s, 1H), 7.16-7.07 (m, 2H), 7.05-6.96 (m, 1H), 4.39-4.23 (m, 1H), 3.97 (t, J=7.2 Hz, 2H), 3.63 (s, 2H), 3.04 (d, J=10.9 Hz, 2H), 2.79 (t, J=7.3 Hz, 2H), 2.69-2.51 (m, 4H), 2.38 (qd, J=12.3, 3.3 Hz, 2H), 2.26 (t, J=11.6 Hz, 2H), 1.86-1.72 (m, 6H).
Synthesis of intermediate 44a: piperidine and 40a were used as raw materials for synthesis according to the synthesis method of intermediate 40b in Example 40 to obtain a colorless oily product (461 mg, with a yield of 83.2%). 1H NMR (400 MHz, chloroform-d) δ 7.19 (s, 1H), 7.15 (s, 1H), 4.40 (tt, J=12.5, 4.1 Hz, 1H), 4.31 (s, 2H), 3.93 (t, J=6.9 Hz, 2H), 2.84 (t, J=13.4 Hz, 2H), 2.59 (t, J=6.9 Hz, 2H), 2.53-2.37 (m, 4H), 2.23 (qd, J=12.7, 4.6 Hz, 2H), 1.79 (d, J=11.8 Hz, 2H), 1.60-1.53 (m, 4H), 1.51 (s, 9H), 1.47-1.39 (m, 2H).
Synthesis of compound EX44: 44a was used as raw material for synthesis according to the synthesis method of intermediate 40c in Example 40 to obtain intermediate 44b (303 mg, with a yield of 96.2%). Intermediate 44b and 2-chloro-4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (110 mg, with a yield of 27.1%). LC-MS (ESI) m/z: 539.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.52 (t, J=7.5 Hz, 1H), 7.28 (d, J=1.9 Hz, 1H), 7.19 (s, 1H), 7.15-7.09 (m, 1H), 7.05-6.96 (m, 1H), 4.37-4.25 (m, 1H), 3.95 (t, J=6.9 Hz, 2H), 3.63 (s, 2H), 3.04 (d, J=11.0 Hz, 2H), 2.60 (t, J=7.0 Hz, 2H), 2.54-2.43 (m, 4H), 2.43-2.31 (m, 2H), 2.27 (t, J=11.6 Hz, 2H), 1.78 (d, J=11.6 Hz, 2H), 1.64-1.51 (m, 4H), 1.50-1.38 (m, 2H).
Intermediate 1e and bromomethyl cyclopropane were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (88 mg, with a yield of 50.0%). LC-MS (ESI) m/z: 453.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.42 (s, 1H), 7.12 (s, 1H), 4.42-4.27 (m, 1H), 3.95 (t, J=6.6 Hz, 2H), 3.73-3.60 (m, 4H), 3.34-3.18 (m, 2H), 2.65 (t, J=6.7 Hz, 2H), 2.59-2.46 (m, 4H), 2.47-2.26 (m, 4H), 2.24-2.10 (m, 2H), 1.87-1.75 (m, 2H), 0.99-0.84 (m, 1H), 0.62-0.49 (m, 2H), 0.21-0.06 (m, 2H).
Synthesis of intermediate 46a: N,N-dimethyl bromide ethyl hydrobromide and intermediate 1e were used as raw materials for synthesis according to the synthesis method of intermediate 1f to obtain a pink solid product (1.1 g, with a yield of 93.2%). 1H NMR (400 MHz, chloroform-d) δ 7.16 (s, 1H), 7.09 (s, 1H), 4.44-4.31 (m, 3H), 3.93-3.90 (t, J=6.8 Hz, 2H), 2.87-2.81 (t, J=12.4 Hz, 2H), 2.62-2.58 (t, J=6.8 Hz, 2H), 2.31 (s, 6H), 2.28-2.17 (m, 2H), 1.81-1.78 (d, J=10.4 Hz, 2H), 1.50 (s, 9H).
Synthesis of compound EX46: 46a was used as raw material for synthesis according to the synthesis method of intermediate 40c in Example 40 to obtain intermediate 46b (1.1 g, with a yield of 98.9%). Intermediate 46b and 2-chloro-4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (300 mg, with a yield of 56.6%). LC-MS (ESI) m/z: 499.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.53 (t, J=7.5 Hz, 1H), 7.31 (s, 1H), 7.15-7.08 (m, 2H), 7.04-6.97 (m, 1H), 4.38-4.25 (m, 1H), 3.96 (t, J=7.0 Hz, 2H), 3.64 (s, 2H), 3.05 (d, J=10.9 Hz, 2H), 2.66 (t, J=6.9 Hz, 2H), 2.46-2.32 (m, 8H), 2.28 (t, J=11.7 Hz, 2H), 1.79 (d, J=11.7 Hz, 2H).
Synthesis of intermediate 47a: 4,5-dichloro-2-nitrotoluene (5 g, 23.06 mmol) was added into a clean dry single neck flask (500 mL), and solvents carbon tetrachloride (230 mL), N-bromosuccinimide (NBS) (4.51 g, 25.36 mmol), and azobisisobutyronitrile (AIBN) (379 mg, 2.31 mmol) were added to the reaction flask. The flask was heated to 80° C. for reflux reaction for 18 hours. The reaction of the raw materials was detected, by TLC, to be substantially complete. The flask was cooled to room temperature, and saturated NaHCO3 aqueous solution was added to the reaction system for quenching the reaction, followed by extraction with dichloromethane (100 mL×3), washing with saturated NaCl aqueous solution, drying with anhydrous MgSO4, filtering, and evaporation under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by silica gel column chromatography (the mobile phase was petroleum ether) to obtain 3.02 g light yellow solid product, with a yield of 45.97%. The product was directly used in the next reaction.
Synthesis of intermediate 47b: N-BOC-4-aminopiperidine (2.32 g, 11.58 mmol) and anhydrous dichloromethane (60 mL) were added into a clean dry round-bottom flask (250 mL), and triethylamine (1.60 g, 2.20 mL, 15.79 mmol) was added thereto. Under stirring at room temperature, 1-(bromomethyl)-4,5-dichloro-2-nitrobenzene (47a) (3.00 g, 10.53 mmol) dichloromethane (40 mL) solution was added dropwise to the reaction system. Thereafter, stirring was continued at room temperature for reaction for 12 hours. The reaction of the raw materials was detected, by TLC, to be completed. After the completion of the reaction, the solvent was removed by evaporation under reduced pressure with a rotary evaporator, and the residue was separated and purified by silica gel column chromatography (the mobile phase was petroleum ether and ethyl acetate, with the ratio gradient being 20:1-8:1) to obtain 2.77 g light yellow oil product, with a yield of 65.07%. 1H NMR (400 MHz, chloroform-d) δ 8.08 (s, 1H), 7.87 (s, 1H), 4.07 (s, 2H), 4.01 (s, 2H), 2.90-2.74 (m, 2H), 2.70-2.57 (m, 1H), 1.93-1.78 (m, 2H), 1.46 (s, 9H), 1.34-1.25 (m, 2H).
Synthesis of intermediate 47c: tert-butyl 4-((4,5-dichloro-2-nitrophenylmethyl) amino)piperidin-1-carboxylate (47b) (2.50 g, 6.18 mmol) and anhydrous ethanol (60 mL) were added into a single-neck flask (250 mL). Under stirring at room temperature, iron powder (2.07 g, 37.10 mmol) and saturated NH4Cl aqueous solution (10 mL) were slowly added to the reaction system. The flask was heated to 80° C., and stirring was continued for reaction for 2 hours. The reaction of the raw materials was detected, by TLC plate, to be complete. After the completion of the reaction, the reaction mixture solution was cooled to room temperature, and filtered with diatomite. The filter cake was washed with anhydrous ethanol, and the filtrate was collected. The solution was removed by evaporation under reduced pressure with a rotary evaporator to obtain a white solid. The solid was dissolved in ethyl acetate (100 mL), and added with a small amount of water (25 mL) to precipitate a large amount of white solid. After filtration and drying, 2.00 g white solid product was obtained, with a yield of 86.41%. The product was directly used in the next reaction.
Synthesis of intermediate 47d: intermediate 47c was used as raw material for synthesis according to the synthesis method of intermediate 1e in Example 1 to obtain a white solid product (590 mg, with a yield of 64.52%). 1H NMR (400 MHz, chloroform-d) δ 7.78 (s, 1H), 7.13 (s, 1H), 6.84 (s, 1H), 4.59-4.45 (m, 1H), 4.26 (s, 4H), 2.84 (s, 2H), 1.76-1.62 (m, 4H), 1.48 (s, 9H).
Synthesis of intermediate 47e: intermediate 47d was used as raw material for synthesis according to the synthesis method of intermediate 1f in Example 1 to obtain a crude product, which was separated and purified by alkaline alumina column chromatography (the mobile phase was petroleum ether and ethyl acetate, with the ratio gradient being 5:1-0:1), to obtain a white solid product (627 mg, with a yield of 85.76%). 1H NMR (400 MHz, chloroform-d) δ 7.13 (s, 2H), 4.51-4.38 (m, 1H), 4.24 (s, 2H), 4.16 (s, 2H), 3.96 (t, J=7.1 Hz, 2H), 3.80-3.63 (m, 4H), 2.90-2.71 (m, 2H), 2.69-2.42 (m, 6H), 1.73-1.60 (m, 4H), 1.47 (s, 9H).
Synthesis of compound EX47: 47e was used as raw material for synthesis according to the synthesis method of intermediate 1g in Example 1 to obtain intermediate 47f, a white solid product (616 mg, with a yield of 99.99%). The product was directly used in the next reaction. 1H NMR (400 MHz, Methanol-d4) δ 7.42 (s, 1H), 7.25 (s, 1H), 4.54-4.42 (m, 1H), 4.40 (s, 2H), 4.30 (t, J=5.8 Hz, 2H), 4.23-3.63 (m, 6H), 3.61-3.43 (m, 5H), 3.30-3.05 (m, 4H), 2.17 (qd, J=13.2, 4.1 Hz, 2H), 2.05-1.91 (m, 2H).
Intermediate 47f and 2-chloro-4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (123 mg, with a yield of 46.67%). LC-MS (ESI) m/z: 555.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.45 (t, J=7.6 Hz, 1H), 7.17-7.05 (m, 3H), 6.97 (t, J=8.2 Hz, 2H), 4.40-4.27 (m, 1H), 4.20 (s, 2H), 3.95 (t, J=7.1 Hz, 2H), 3.77-3.67 (m, 4H), 3.58 (s, 2H), 2.98 (d, J=11.0 Hz, 2H), 2.61 (t, J=7.1 Hz, 2H), 2.59-2.50 (m, 4H), 2.24 (t, J=11.7 Hz, 2H), 1.91-1.75 (m, 2H), 1.71-1.62 (m, 2H), 1.24 (t, J=7.0 Hz, 1H).
Synthesis of compound EX48: intermediate 46b and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain a white solid product (1.53 g, with a yield of 62.5%).
Synthesis of compound EX48A: intermediate 46b and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX48, which was subjected to preparation and separation by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 min) to obtain a white solid product (169 mg, with a yield of 6.91%). LC-MS (ESI) m/z: 481.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.41 (s, 1H), 7.08 (s, 1H), 4.36-4.26 (m, 1H), 3.92 (t, J=7.0 Hz, 2H), 3.06 (d, J=11.3 Hz, 2H), 2.60 (t, J=6.9 Hz, 2H), 2.44-2.35 (m, 2H), 2.35-2.23 (m, 9H), 1.92 (d, J=11.3 Hz, 2H), 1.84-1.76 (m, 4H), 1.74-1.69 (m, 2H), 1.47-1.37 (m, 1H), 1.26 (q, J=11.9 Hz, 2H), 1.08-1.02 (m, 1H), 0.87 (s, 3H), 0.86 (s, 3H).
Synthesis of compound EX48B: intermediate 46b and 4-isopropylcyclohexanone were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX48, which was subjected to preparation and separation by high performance liquid chromatography (Waters high performance liquid chromatography, chromatographic column: YMC-Triart-C18 EXRS 20 mm×100 mm×5 μm; mobile phase: A=water+0.1 vol % ammonia (25%), B=acetonitrile; gradient 70%-90% B, 10 min) to obtain a white solid product (411 mg, with a yield of 16.80%). LC-MS (ESI) m/z: 481.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.35 (s, 1H), 7.08 (s, 1H), 4.35-4.22 (m, 1H), 3.92 (t, J=7.0 Hz, 2H), 3.20-3.09 (m, 2H), 2.60 (t, J=7.0 Hz, 2H), 2.36-2.26 (m, 8H), 2.26-2.16 (m, 2H), 1.85-1.67 (m, 5H), 1.67-1.57 (m, 3H), 1.57-1.48 (m, 2H), 1.43-1.33 (m, 2H), 1.18-1.10 (m, 1H), 0.91 (s, 3H), 0.89 (s, 3H).
Synthesis of intermediate 49a: tert-butyl 4-(5,6-dichloro-3-(2-chloroethyl)-2-carbonyl-2,3-dihydro-1H-benzo[d]imidazol-1-yl) piperidin-1-carboxylate (40a) (250 mg, 557 μmol) and Cs2CO3 (544 mg, 1.67 mmol) were added into a clean dry Schlenk tube (25 mL). Anhydrous dioxane (5.5 mL) was added to the tube. Under stirring at room temperature, 2-oxa-6-aza-spiro[3,3]heptane (138 mg, 1.39 mmol) was added. Under argon displacement protection, the tube was heated to 105° C. for reflux reaction for 12 hours. The reaction of the raw materials was detected, by TLC plate, to be substantially complete. After the completion of the reaction, the reaction flask was cooled to room temperature, an appropriate amount of water was added thereto, followed by extraction with ethyl acetate (15 mL×3). The organic phase was washed with saturated NaCl aqueous solution, and dried with anhydrous Na2SO4, and then filtered and evaporated with a rotary evaporator to remove the solvent, to obtain a light brown oily crude product, which was subjected to separation by silica gel column chromatography (the mobile phase was DCM/MeOH, with 0.2%-1% MeOH) to obtain 186 mg white solid product, with a yield of 65.28%. LC-MS (ESI) m/z: 454.10 (M−56+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.16 (s, 1H), 7.09 (s, 1H), 4.71 (s, 4H), 4.47-4.20 (m, 3H), 3.78 (t, J=6.6 Hz, 2H), 3.38 (s, 4H), 2.83 (t, J=13.5 Hz, 2H), 2.70 (t, J=6.5 Hz, 2H), 2.23 (qd, J=12.7, 4.4 Hz, 2H), 1.79 (d, J=12.1 Hz, 2H), 1.51 (s, 9H).
Synthesis of intermediate 49b: 49a (150 mg, 293 μmol) was used as raw material for synthesis according to the synthesis method of intermediate 1g to obtain a white solid product (154 mg, with a yield of 99.95%). The solid was directly used in the next reaction without purification.
Synthesis of compound EX49: 49b and 2-chloro-4fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39, followed by separation by silica gel column chromatography (the mobile phase was DCM/MeOH, with 0.2%-1% MeOH) to obtain a white solid product (86 mg, with a yield of 54.38%). LC-MS (ESI) m/z: 554.12 (M+H)+; 1H NMR (400 MHz, Methanol-d4) δ 7.66 (s, 1H), 7.59 (dd, J=8.6, 6.3 Hz, 1H), 7.40 (s, 1H), 7.26 (dd, J=8.7, 2.6 Hz, 1H), 7.13 (td, J=8.4, 2.5 Hz, 1H), 4.33 (tt, J=12.5, 4.4 Hz, 1H), 3.92 (t, J=5.9 Hz, 2H), 3.71 (s, 2H), 3.63 (s, 4H), 3.22 (s, 4H), 3.12 (d, J=11.1 Hz, 2H), 2.91 (t, J=5.8 Hz, 2H), 2.48 (qd, J=12.5, 3.6 Hz, 2H), 2.34 (t, J=11.8 Hz, 2H), 1.79 (d, J=11.4 Hz, 2H).
Intermediate 1g in Example 1 and 5-chlorothiophene-2-formaldehyde were used as raw materials for synthesis according to the synthesis method in Example 1 to obtain a light yellow solid product (289 mg, with a yield of 93.3%). LC-MS (ESI) m/z: 530.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.31 (s, 1H), 7.13 (s, 1H), 6.75 (d, J=3.1 Hz, 1H), 6.70 (d, J=3.8 Hz, 1H), 4.36-4.24 (m, 1H), 3.95 (t, J=6.6 Hz, 2H), 3.73-3.61 (m, 6H), 3.10 (d, J=11.0 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.59-2.46 (m, 4H), 2.37 (qd, J=12.5, 3.8 Hz, 2H), 2.20 (t, J=11.7 Hz, 2H), 1.78 (d, J=11.7 Hz, 2H).
Synthesis of intermediate 51a: 1-(bromomethyl)-4,5-dichloro-2-nitrobenzene (47a) (5.33 g, 18.71 mmol) and phthalimide potassium salt (3.30 g, 17.82 mmol) were added into a clean dry single neck flask (100 mL), and dry DMF (30 mL) was added thereto. The flask was heated to 125° C., and stirred for reaction for 12 hours. The reaction of the raw materials was detected, by TLC, to be complete. After the completion of the reaction, the reaction mixture solution was cooled to room temperature. Under stirring condition, the reaction mixture solution was slowly poured into an ice-water mixture (120 mL). A large amount of yellow solid was precipitated. After stirring evenly, filtration was performed, the filter cake was washed with a small amount of water. After drying, a yellow solid product (5.93 g, with a yield of 94.8%) was obtained. 1H NMR (400 MHz, chloroform-d) δ 8.25 (s, 1H), 7.97-7.89 (m, 2H), 7.84-7.77 (m, 2H), 7.32 (s, 1H), 5.25 (s, 2H).
Synthesis of intermediate 51b: 2-(4,5-dichloro-2-nitrophenylmethyl) isodihydroindole−1,3-dione (51a) (2.5 g, 7.12 mmol) and anhydrous ethanol (71 mL) were added into a single-neck flask (250 mL). Under stirring at room temperature, iron powder (2.78 g, 49.84 mmol) and saturated NH4Cl aqueous solution (10 mL) were slowly added to the reaction system. The flask was heated to 55° C., and stirring was continued for reaction for 1 hour. The reaction of the raw materials was detected, by TLC plate, to be complete. After the completion of the reaction, the reaction mixture solution was cooled to room temperature, and filtered with diatomite. The filter cake was washed with anhydrous ethanol, and the filtrate was collected. The solution was removed by evaporation under reduced pressure with a rotary evaporator to obtain a yellow solid. The solid was dissolved in ethyl acetate (100 mL), and added with a small amount of water (25 mL). After filtering out insoluble matter, liquid separation was performed to collect the organic phase, and anhydrous Na2SO4 was added for drying, followed by filtering, and evaporation under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by silica gel column chromatography (the mobile phase was petroleum ether and ethyl acetate, with the ratio gradient being 10:1-3:1) to obtain a light yellow solid product (1.86 g, with a yield of 81.4%). 1H NMR (400 MHz, chloroform-d) δ 7.88-7.81 (m, 2H), 7.77-7.70 (m, 2H), 7.51 (s, 1H), 6.74 (s, 1H), 4.68 (s, 2H), 4.56 (s, 2H).
Synthesis of intermediate 51c: 2-(2-amino-4,5-dichlorophenylmethyl) isodihydroindole-1,3-dione (51b) (1.86 g, 5.79 mmol), 4-tert-butylpiperidone (1.38 g, 6.95 mmol), and anhydrous 1,2-dichloroethane (60 mL) were added into a clean dry round-bottom flask (100 mL). Under stirring at room temperature, acetic acid (348 mg, 5.79 mmol, 331 μL) and NaBH(OAc)3 (3.07 g, 14.48 mmol) were added to the reaction system. Under argon displacement protection, stirring was performed at room temperature for reaction for 48 hours. The reaction was detected, by TLC plate, to be complete. After the completion of the reaction, saturated NaHCO3 aqueous solution was added to the reaction system for quenching the reaction, followed by extraction with dichloromethane (50 mL×3), washing with saturated NaCl aqueous solution, drying with anhydrous MgSO4, filtering, and evaporation under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by silica gel column chromatography (the mobile phase was petroleum ether and ethyl acetate, with the ratio gradient being 15:1-4:1) to obtain a light yellow solid product (925 mg, with a yield of 31.7%). 1H NMR (400 MHz, chloroform-d) δ 7.89-7.80 (m, 2H), 7.78-7.69 (m, 2H), 7.54 (s, 1H), 6.63 (s, 1H), 5.38 (d, J=6.1 Hz, 1H), 4.64 (s, 2H), 4.14-3.97 (m, 2H), 3.43-3.29 (m, 1H), 3.09-2.90 (m, 2H), 2.07-1.94 (m, 2H), 1.57-1.40 (m, 12H).
Synthesis of intermediate 51e: tert-butyl 4-((4,5-dichloro-2-((1,3-dicarbonyl isodihydroindole-2-yl)methyl)phenyl)amino)piperidin-1-carboxylate (51c) (800 mg, 1.59 mmol) and solvent ethanol (16 mL) were added into a clean dry single-neck flask (50 mL). Under stirring, hydrazine hydrate (467 mg, 7.93 mmol, 453 μL, 85%) was added to the reaction system. The flask was heated to 80° C. for reflux reaction for 2 hours to precipitate a large amount of solid. The reaction was detected, by TLC, to be complete. After the completion of the reaction, the reaction system was cooled to room temperature, and filtered. The filtrate was collected, and evaporated under reduced pressure by a rotary evaporator to remove the solvent to obtain a light yellow oily crude product tert-butyl 4-((2-(aminomethyl)-4,5-dichlorophenyl) amino)piperidin-1-carboxylate (51d) (590 mg, with a yield of 99.38%). The crude product (590 mg, 1.58 mmol) was dissolved in dry tetrahydrofuran (15 mL), under argon displacement protection, N,N′-carbonyldiimidazole (358 mg, 2.21 mmol) dichloromethane (6 mL) solution was added dropwise to the reaction system, and stirring was performed at room temperature for reaction for 18 hours. The reaction was detected, by TLC plate, to be substantially complete. After the completion of the reaction, saturated NaHCO3 solution was added dropwise to the reaction system for quenching the reaction, followed by extraction with ethyl acetate three times (25 mL×3). The organic phase was washed with saturated NaCl solution, added with anhydrous Na2SO4 for drying, filtered, and evaporated under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by alkaline alumina column chromatography (the mobile phase was petroleum ether and ethyl acetate, with the ratio gradient being 5:1-1:1) to obtain a white solid product (528 mg, with a yield of 83.7%). 1H NMR (400 MHz, chloroform-d) δ 7.14 (s, 1H), 7.07 (s, 1H), 5.62 (s, 1H), 4.41-4.17 (m, 4H), 4.09-3.95 (m, 1H), 2.91-2.68 (m, 2H), 2.64-2.45 (m, 2H), 1.83-1.70 (m, 2H), 1.49 (s, 9H).
Synthesis of intermediate 51f: tert-butyl 4-(6,7-dichloro-2-carbonyl-3,4-dihydroquinazolin-1(2H)-yl) piperidin-1-carboxylate (51e) (220 mg, 549 μmol) and the solvent, dry tetrahydrofuran (5.5 mL), were added into a dry clean three-necked flask (50 mL), and under argon displacement protection, the flask was cooled in an ice water bath to 0° C. NaH (40 mg, 1.65 mmol, 60% in mineral oil) was added to the reaction system, and stirred for reaction for 30 minutes. 4-(2-bromoethyl) morpholine hydrobromide (212 mg, 769 μmol) was added to the reaction system, stirring was continued for 30 minutes, then the ice water bath was removed, the flask was slowly heated to 35° C., and stirring was continued for reaction for 18 hours. The reaction was detected, by TLC plate, to be substantially complete. After the completion of the reaction, under cooling in an ice water bath, ice water was added dropwise to the reaction system for quenching the reaction, followed by extraction with ethyl acetate three times (25 mL×3). The organic phase was washed with saturated NaCl solution, added with anhydrous Na2SO4 for drying, filtered, and evaporated under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by alkaline alumina column chromatography (the mobile phase was petroleum ether and ethyl acetate, with the ratio gradient being 5:1-1:1) to obtain a white solid product (192 mg, with a yield of 68.0%). 1H NMR (400 MHz, chloroform-d) δ 7.14 (s, 1H), 7.03 (s, 1H), 4.41-4.15 (m, 4H), 4.04-3.92 (m, 1H), 3.73-3.62 (m, 4H), 3.50 (t, J=6.6 Hz, 2H), 2.90-2.69 (m, 2H), 2.61-2.43 (m, 8H), 1.83-1.72 (m, 2H), 1.48 (s, 9H).
Synthesis of intermediate 51g: tert-butyl 4-(6,7-dichloro-3-(2-morpholinoethyl)-2-carbonyl-3,4-dihydroquinazoline-1(2H)-yl) piperidin-1-carboxylate (51f) (185 mg, 360 μmol) and the solvent dichloromethane (5 mL) were added into a dry clean round-bottom flask (25 mL). Under stirring at room temperature, trifluoroacetic acid (1 mL) was added dropwise for reaction for 2 hours. The reaction was detected, by TLC plate, to be substantially complete. The reaction mixture solution was evaporated under reduced pressure by a rotary evaporator to remove the solvent. Ether (10 mL) was added to the residue, followed by dispersing, crushing, and filtering to obtain a white solid product (190 mg, with a yield of 99.99%). The product was directly used in the next reaction.
Synthesis of compound EX51: 6,7-dichloro-3-(2-morpholinoethyl)-1-(piperidin-4-yl)-3,4-dihydroquinazolin-2(1H)-ketotrifluoroacetate (51g) (190 mg, 360 μmol) and dry dichloromethane (3.5 mL) were added into a clean dry Schlenk reaction flask (10 mL). Triethylamine (146 mg, 1.44 mmol, 200 μL) was added thereto, and stirred at room temperature for 5 minutes. 2-chloro-4fluorobenzyl bromide (97 mg, 433 μmol) was added to the reaction system, and stirred at room temperature for reaction for 12 hours. The reaction was detected, by TLC plate, to be substantially complete. After the completion of the reaction, the reaction mixture solution was evaporated under reduced pressure by a rotary evaporator to remove the solvent to obtain a crude product, which was separated and purified by alkaline alumina column chromatography (the mobile phase was petroleum ether and ethyl acetate, with the ratio gradient being 5:1-1:1) to obtain a white solid product (158 mg, with a yield of 78.9%). LC-MS (ESI) m/z: 556.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.56 (t, J=7.5 Hz, 1H), 7.17-7.05 (m, 3H), 6.98 (t, J=8.6 Hz, 1H), 4.25 (s, 2H), 3.94-3.81 (m, 1H), 3.74-3.64 (m, 4H), 3.61 (s, 2H), 3.52 (t, J=6.7 Hz, 2H), 3.01 (d, J=11.2 Hz, 2H), 2.70 (q, J=11.6, 10.9 Hz, 2H), 2.56 (t, J=5.9 Hz, 2H), 2.53-2.44 (m, 4H), 2.24 (t, J=11.8 Hz, 2H), 1.75 (d, J=12.2 Hz, 2H).
Synthesis of compound EX52: intermediate 51e was used as raw material and dichloromethane was used as solvent to obtain 218 mg white solid 52a, with a yield of 100%, under the action of trifluoroacetic acid. The solid was directly used in the next reaction without further purification.
52a and 2-chlorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (74 mg, with a yield of 72.2%). LC-MS (ESI) m/z: 425.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.57 (d, J=6.8 Hz, 1H), 7.35 (dd, J=7.9, 1.4 Hz, 1H), 7.27 (t, J=7.0 Hz, 2H), 7.23-7.15 (m, 2H), 7.13 (s, 1H), 5.31 (s, 1H), 4.23 (s, 2H), 4.05-3.87 (m, 1H), 3.68 (s, 2H), 3.05 (d, J=10.5 Hz, 2H), 2.71 (q, J=10.7 Hz, 2H), 2.25 (t, J=11.1 Hz, 2H), 1.75 (d, J=11.2 Hz, 2H).
Synthesis of intermediate 53b: 1-(2-hydroxypyrrole)-2-one was used as raw material for synthesis of 53a according to the synthesis method of 39b to obtain 1.8 g light yellow oily crude product, with a yield of 82.4%. The product was directly used in the next reaction without purification.
53a and 1e were used as raw materials for synthesis of 53b according to the synthesis method of 39c to obtain 231 mg white solid product, with a yield of 35.88%. 1H NMR (400 MHz, chloroform-d) δ 7.16 (s, 1H), 7.15 (s, 1H), 4.40-4.27 (m, 3H), 4.00 (t, J=6.0 Hz, 2H), 3.58 (t, J=6.0 Hz, 2H), 3.36 (t, J=7.0 Hz, 2H), 2.84 (t, J=12.7 Hz, 2H), 2.32-2.18 (m, 4H), 1.99-1.89 (m, 2H), 1.83-1.74 (m, 2H), 1.51 (s, 9H).
Synthesis of intermediate 53c: 53b was used as raw material for synthesis according to the synthesis method of intermediate 1f in Example 1 to obtain 215 mg white solid product, with a yield of 90.5%. The product was directly used in the next reaction without purification.
Synthesis of compound EX53: 53c and 2-chlorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (68 mg, with a yield of 78.4%). LC-MS (ESI) m/z: 522.30 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.55 (d, J=7.7 Hz, 1H), 7.37 (d, J=7.8 Hz, 1H), 7.31 (s, 1H), 7.28 (d, J=6.5 Hz, 1H), 7.21 (t, J=7.6 Hz, 1H), 7.16 (s, 1H), 4.34-4.22 (m, 1H), 4.00 (t, J=6.0 Hz, 2H), 3.69 (s, 2H), 3.58 (t, J=6.0 Hz, 2H), 3.35 (t, J=7.0 Hz, 2H), 3.08 (d, J=11.1 Hz, 2H), 2.40 (qd, J=12.0, 3.6 Hz, 2H), 2.33-2.20 (m, 4H), 1.99-1.87 (m, 2H), 1.77 (d, J=10.0 Hz, 2H).
Synthesis of intermediate 54a: intermediate 1e and 2-chloro-N,N-dimethyl acetamide were used as raw materials for synthesis according to the synthesis method of intermediate 1f in Example 1 to obtain a colorless oily product (610 mg, with a yield of 99.9%). 1H NMR (400 MHz, chloroform-d) δ 7.17 (s, 1H), 7.05 (s, 1H), 4.62 (s, 2H), 4.44-4.34 (m, 1H), 4.31 (d, J=13.6 Hz, 2H), 3.14 (s, 3H), 2.99 (s, 3H), 2.84 (t, J=12.2 Hz, 2H), 2.25 (qd, J=12.7, 4.6 Hz, 2H), 1.82 (d, J=10.4 Hz, 2H), 1.51 (s, 9H).
Synthesis of intermediate 54b: intermediate 54a was used as raw material for synthesis according to the synthesis method of intermediate 1g in Example 1 to obtain a white solid product (628 mg, with a yield of 100%). The product was directly used in the next experiment.
Synthesis of compound EX54: 54b and 2-chloro-4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (299 mg, with a yield of 94.1%). LC-MS (ESI) m/z: 514.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.52 (t, J=7.5 Hz, 1H), 7.30 (s, 1H), 7.12 (d, J=8.2 Hz, 1H), 7.05 (s, 1H), 7.01 (t, J=8.7 Hz, 1H), 4.62 (s, 2H), 4.38-4.24 (m, 1H), 3.63 (s, 2H), 3.14 (s, 3H), 3.04 (d, J=10.5 Hz, 2H), 2.99 (s, 3H), 2.39 (q, J=12.6, 11.8 Hz, 2H), 2.27 (t, J=11.7 Hz, 2H), 1.81 (d, J=11.5 Hz, 2H).
54b and 4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (238 mg, with a yield of 60.2%). LC-MS (ESI) m/z: 480.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.37-7.28 (m, 3H), 7.07-6.98 (m, 3H), 4.61 (s, 2H), 4.35-4.23 (m, 1H), 3.52 (s, 2H), 3.13 (s, 3H), 3.01 (d, J=12.0 Hz, 2H), 2.98 (s, 3H), 2.37 (qd, J=12.4, 3.8 Hz, 2H), 2.14 (t, J=11.7 Hz, 2H), 1.79 (d, J=11.4 Hz, 2H).
46b and 4-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (140 mg, with a yield of 41.8%). LC-MS (ESI) m/z: 466.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.36-7.28 (m, 3H), 7.09 (s, 1H), 7.02 (t, J=8.7 Hz, 2H), 4.37-4.25 (m, 1H), 3.92 (t, J=7.0 Hz, 2H), 3.52 (s, 2H), 3.02 (d, J=11.6 Hz, 2H), 2.60 (t, J=7.0 Hz, 2H), 2.42-2.32 (m, 2H), 2.31 (s, 6H), 2.14 (t, J=11.2 Hz, 2H), 1.77 (d, J=13.7 Hz, 2H).
Intermediate 1g and 2-methylbenzaldehyde were used as raw materials for synthesis according to the synthesis method in Example 1 to obtain a white solid product (120 mg, with a yield of 61.2%). LC-MS (ESI) m/z: 504.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.33-7.28 (m, 1H), 7.28-7.25 (m, 1H), 7.20-7.14 (m, 3H), 7.11 (s, 1H), 4.36-4.24 (m, 1H), 3.93 (t, J=6.6 Hz, 2H), 3.66 (t, J=4.6 Hz, 4H), 3.51 (s, 2H), 3.04 (d, J=11.1 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.51 (t, J=4.6 Hz, 4H), 2.41 (s, 3H), 2.39-2.27 (m, 2H), 2.18 (t, J=11.4 Hz, 2H), 1.76 (d, J=10.4 Hz, 2H).
Intermediate 1g and 2-chlorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (78 mg, with a yield of 50.9%). LC-MS (ESI) m/z: 524.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.54 (d, J=7.6 Hz, 1H), 7.36 (d, J=7.9 Hz, 1H), 7.31 (s, 1H), 7.27 (t, J=7.4 Hz, 1H), 7.20 (t, J=7.5 Hz, 1H), 7.12 (s, 1H), 4.36-4.24 (m, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.69 (s, 2H), 3.66 (t, J=4.6 Hz, 4H), 3.08 (d, J=11.0 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.52 (t, J=4.6 Hz, 4H), 2.41 (qd, J=12.3, 3.5 Hz, 2H), 2.29 (t, J=11.7 Hz, 2H), 1.78 (d, J=11.5 Hz, 2H).
1g and 2-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (106 mg, with a yield of 71.5%). LC-MS (ESI) m/z: 508.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.43 (td, J=7.5, 1.9 Hz, 1H), 7.33 (s, 1H), 7.30-7.22 (m, 1H), 7.15 (t, J=7.5 Hz, 1H), 7.12 (s, 1H), 7.05 (t, J=9.1 Hz, 1H), 4.36-4.22 (m, 1H), 3.94 (t, J=6.7 Hz, 2H), 3.73-3.56 (m, 6H), 3.07 (d, J=11.4 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.53 (t, J=4.6 Hz, 4H), 2.37 (qd, J=12.4, 3.8 Hz, 2H), 2.23 (t, J=11.3 Hz, 2H), 1.77 (d, J=11.6 Hz, 2H).
Synthesis of intermediate 60a: intermediate 1e was used as raw material and was subjected to the action of trifluoroacetic acid at room temperature to remove Boc protection group to obtain 520 mg white trifluoroacetate solid, with a yield of 99.8%. The solid was directly used in the next reaction without purification.
Synthesis of intermediate 60b: intermediate 60a and 2-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain 197 mg white solid intermediate 60b, with a yield of 99.9%. 1H NMR (400 MHz, chloroform-d) 610.67 (s, 1H), 7.43 (t, J=7.7 Hz, 1H), 7.35 (s, 1H), 7.31-7.25 (m, 1H), 7.20 (s, 1H), 7.15 (t, J=7.3 Hz, 1H), 7.06 (t, J=9.0 Hz, 1H), 4.35-4.20 (m, 1H), 3.70 (s, 2H), 3.11 (d, J=11.1 Hz, 2H), 2.41 (q, J=12.6 Hz, 2H), 2.26 (t, J=11.9 Hz, 2H), 1.80 (d, J=11.9 Hz, 2H).
Synthesis of compound EX60: intermediate 60b and bromomethyl ethylene oxide were used raw materials for synthesis according to the synthesis method of intermediate 1f to obtain a light yellow solid product (48 mg, with a yield of 42.0%). LC-MS (ESI) m/z: 451.10 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.43 (t, J=7.1 Hz, 1H), 7.34 (s, 1H), 7.30-7.23 (m, 2H), 7.15 (t, J=7.3 Hz, 1H), 7.06 (t, J=9.1 Hz, 1H), 4.41 (dd, J=15.2, 2.6 Hz, 1H), 4.36-4.24 (m, 1H), 3.67 (s, 2H), 3.62 (dd, J=15.3, 6.4 Hz, 1H), 3.27-3.17 (m, 1H), 3.08 (d, J=10.7 Hz, 2H), 2.87 (t, J=4.3 Hz, 1H), 2.65 (dd, J=4.6, 2.6 Hz, 1H), 2.38 (q, J=11.3, 10.8 Hz, 2H), 2.24 (t, J=11.4 Hz, 2H), 1.85-1.71 (m, 2H).
Synthesis of intermediate 61a: 2,4-difluoronitrobenzene and 1-N-Boc-4-aminopiperidine were used as raw materials for synthesis according to the synthesis method of intermediate 1f to obtain 1.07 g yellow waxy product, with a yield of 80.0%. 1H NMR (400 MHz, chloroform-d) δ 6.97-6.90 (m, 1H), 6.88 (d, J=9.1 Hz, 1H), 6.80 (t, J=8.9 Hz, 1H), 4.50-4.17 (m, 3H), 3.99 (t, J=6.9 Hz, 2H), 3.68 (t, J=4.4 Hz, 4H), 2.85 (s, 2H), 2.67 (t, J=7.2 Hz, 2H), 2.54 (t, J=4.5 Hz, 4H), 2.26 (qd, J=12.9, 4.3 Hz, 2H), 1.80 (d, J=12.6 Hz, 2H), 1.50 (s, 9H).
Synthesis of intermediate 61b: intermediate 61a was subjected to the action of trifluoroacetic acid at room temperature to remove Boc protection group to obtain 1.03 g white trifluoroacetate solid, with a yield of 99.9%. The solid was directly used in the next reaction without purification.
Synthesis of compound EX61: intermediate 61b and 2-chlorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain 226 mg light yellow solid product, with a yield of 88.4%. LC-MS (ESI) m/z: 474.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.56 (d, J=7.6 Hz, 1H), 7.35 (d, J=7.9 Hz, 1H), 7.31-7.23 (m, 1H), 7.19 (t, J=7.7 Hz, 1H), 7.04 (d, J=9.1 Hz, 1H), 6.95-6.88 (m, 1H), 6.79 (t, J=8.9 Hz, 1H), 4.42-4.29 (m, 1H), 3.98 (t, J=6.8 Hz, 2H), 3.74-3.59 (m, 6H), 3.07 (d, J=11.0 Hz, 2H), 2.66 (t, J=7.0 Hz, 2H), 2.59-2.50 (m, 4H), 2.44 (q, J=12.8 Hz, 2H), 2.29 (t, J=11.6 Hz, 2H), 1.79 (d, J=11.7 Hz, 2H).
Synthesis of intermediate 62a: 4-fluoro-3-nitrotoluene and 1-N-Boc-4-aminopiperidine were used as raw materials for synthesis according to the synthesis method of intermediate 1f to obtain 526 mg colorless waxy product, with a yield of 78.4%. 1H NMR (400 MHz, chloroform-d) δ 7.01 (d, J=8.0 Hz, 1H), 6.90-6.82 (m, 2H), 4.51-4.39 (m, 1H), 4.30 (br s, 2H), 3.99 (t, J=7.0 Hz, 2H), 3.69 (t, J=4.5 Hz, 4H), 2.95-2.76 (m, 2H), 2.67 (t, J=7.3 Hz, 2H), 2.55 (t, J=4.4 Hz, 4H), 2.40 (s, 3H), 2.29 (qd, J=12.4, 3.7 Hz, 2H), 1.80 (d, J=13.1 Hz, 2H), 1.50 (s, 9H).
Synthesis of intermediate 62b: intermediate 62a was subjected to the action of trifluoroacetic acid at room temperature to remove Boc protection group to obtain 459 mg white trifluoroacetate solid, with a yield of 99.8%. The solid was directly used in the next reaction without purification.
Synthesis of compound EX62: intermediate 62b and 2-chlorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (186 mg, with a yield of 90.9%). LC-MS (ESI) m/z: 470.20 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.54 (d, J=7.6 Hz, 1H), 7.35 (d, J=7.8 Hz, 1H), 7.30-7.23 (m, 1H), 7.20 (d, J=7.8 Hz, 1H), 7.15 (d, J=8.6 Hz, 1H), 6.91-6.81 (m, 2H), 4.42-4.29 (m, 1H), 3.98 (t, J=7.1 Hz, 2H), 3.76-3.60 (m, 6H), 3.05 (d, J=11.1 Hz, 2H), 2.67 (t, J=7.2 Hz, 2H), 2.55 (t, J=4.6 Hz, 4H), 2.51-2.43 (m, 2H), 2.40 (s, 3H), 2.28 (t, J=11.7 Hz, 2H), 1.78 (d, J=11.8 Hz, 2H).
52a and 2-fluorobromobenzyl were used as raw materials for synthesis according to the synthesis method in Example 39 to obtain a white solid product (56 mg, with a yield of 56.8%). LC-MS (ESI) m/z: 425.10 (M+H)+; 1H NMR (400 MHz, chloroform-d) δ 7.45 (t, J=7.5 Hz, 1H), 7.29-7.22 (m, 1H), 7.19-7.08 (m, 3H), 7.04 (t, J=9.1 Hz, 1H), 5.48 (s, 1H), 4.22 (s, 2H), 3.99-3.85 (m, 1H), 3.67 (s, 2H), 3.05 (d, J=10.9 Hz, 2H), 2.69 (q, J=11.7, 10.9 Hz, 2H), 2.20 (t, J=11.3 Hz, 2H), 1.74 (d, J=11.7 Hz, 2H).
Intermediate 1g and phenylacetaldehyde were used as raw materials for synthesis according to the synthesis method in Example 1 to obtain a white solid product (115 mg, with a yield of 59%). HRMS (ESI): m/z calcd for C26H33Cl2N4O2 ([M+H]+) 503.1975, found 503.1968; HPLC purity: 98.90%, tR=5.10 min. 1H NMR (400 MHz, chloroform-d) δ 7.38 (s, 1H), 7.33-7.28 (m, 2H), 7.25-7.18 (m, 3H), 7.12 (s, 1H), 4.34 (tt, J=12.5, 4.3 Hz, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.66 (t, J=4.6 Hz, 4H), 3.17 (d, J=11.2 Hz, 2H), 2.84 (dd, J=10.4, 6.0 Hz, 2H), 2.71-2.61 (m, 4H), 2.52 (t, J=4.6 Hz, 4H), 2.39 (qd, J=12.4, 3.8 Hz, 2H), 2.22 (t, J=11.5 Hz, 2H), 1.82 (d, J=10.9 Hz, 2H).
Intermediate 1g and 2,6-dichlorobenzaldehyde were used as raw materials for synthesis according to the synthesis method in Example 1 to obtain a white solid product (163 mg, with a yield of 75%). HRMS (ESI): m/z calcd for C25H29Cl4N4O2 ([M+H]+) 557.1039, found 557.1034; HPLC purity: 99.46%, tR=5.89 min. 1H NMR (400 MHz, chloroform-d) δ 7.32 (d, J=8.0 Hz, 2H), 7.26 (s, 1H), 7.15 (t, J=8.0 Hz, 1H), 7.10 (s, 1H), 4.30 (tt, J=12.3, 4.2 Hz, 1H), 3.94 (t, J=6.6 Hz, 2H), 3.80 (s, 2H), 3.66 (t, J=4.5 Hz, 4H), 3.07 (d, J=11.1 Hz, 2H), 2.64 (t, J=6.6 Hz, 2H), 2.51 (t, J=4.6 Hz, 4H), 2.41 (t, J=11.4 Hz, 2H), 2.31 (qd, J=12.1, 3.5 Hz, 2H), 1.74 (d, J=11.0 Hz, 2H).
Intermediate 1g and 2,6-dimethylbenzaldehyde were used as raw materials for synthesis according to the synthesis method in Example 1 to obtain a white solid product (163 mg, with a yield of 75%). HRMS (ESI): m/z calcd for C27H35Cl2N4O2 ([M+H]+) 517.2132, found 517.2118; HPLC purity: 100%, tR=6.60 min. 1H NMR (400 MHz, chloroform-d) δ 7.20 (s, 1H), 7.12-7.04 (m, 2H), 7.04-6.99 (m, 2H), 4.35-4.22 (m, 1H), 3.92 (t, J=6.6 Hz, 2H), 3.65 (t, J=4.6 Hz, 4H), 3.53 (s, 2H), 2.97 (d, J=8.2 Hz, 2H), 2.63 (t, J=6.6 Hz, 2H), 2.51 (t, J=4.6 Hz, 4H), 2.42 (s, 6H), 2.35-2.19 (m, 4H), 1.73 (d, J=9.2 Hz, 2H).
Intermediate 51g and 4-isopropylcyclohexanone were used as raw materials for synthesis of compound EX67 (345 mg white solid, with a yield of 46%) according to the synthesis method of compound EX12. The compound was a mixture of cis-trans isomers, in a ratio of about 5:2. HRMS (ESI): m/z calcd for C28H43Cl2N4O2 ([M+H]+) 537.2758, found 537.2741; HPLC purity (mixture): 28.09%, tR1=7.48 min; 71.91%, tR2=8.61 min. Main products: 1H NMR (400 MHz, chloroform-d) δ 7.26 (s, 1H), 7.12 (s, 1H), 4.24 (s, 2H), 4.05 (m, 1H), 3.68 (t, J=4.6 Hz, 4H), 3.51 (t, J=6.7 Hz, 2H), 3.25 (d, J=11.4 Hz, 2H), 2.77-2.61 (m, 2H), 2.60-2.44 (m, 7H), 2.34 (t, J=11.3 Hz, 2H), 1.86-1.69 (m, 4H), 1.69-1.52 (m, 4H), 1.45-1.31 (m, 3H), 1.19-1.08 (m, 1H), 0.89 (d, J=6.6 Hz, 6H). Secondary products: 1H NMR (400 MHz, chloroform-d) δ 7.35 (s, 1H), 7.12 (s, 1H), 4.24 (s, 2H), 4.21-4.12 (m, 1H), 3.68 (t, J=4.6 Hz, 4H), 3.51 (t, J=6.7 Hz, 2H), 3.25 (d, J=11.4 Hz, 2H), 2.77-2.61 (m, 2H), 2.60-2.44 (m, 7H), 2.01 (d, J=11.9 Hz, 2H), 1.86-1.69 (m, 4H), 1.69-1.52 (m, 4H), 1.19-1.08 (m, 1H), 1.08-0.95 (m, 3H), 0.86 (d, J=7.0 Hz, 6H).
Intermediate 47f and 4-isopropylcyclohexyl were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX68, 78 mg white solid, with a yield of 38%. The compound was a mixture of cis-trans isomers, in a ratio of about 3:1. HRMS (ESI): m/z calcd for C28H43Cl2N4O2 ([M+H]+) 537.2758, found 537.2741; HPLC purity (220 nm): 23.07%, tR1=9.12 min; 74.25%, tR1=10.33 min. Main products: 1H NMR (400 MHz, chloroform-d) δ 7.13 (s, 1H), 7.11 (s, 1H), 4.30 (tt, J=12.1, 4.1 Hz, 1H), 4.22 (s, 2H), 3.96 (t, J=7.1 Hz, 2H), 3.73 (t, J=4.6 Hz, 4H), 3.11 (d, J=11.3 Hz, 2H), 2.62 (t, J=7.1 Hz, 2H), 2.56 (t, J=4.7 Hz, 4H), 2.33-2.25 (m, 1H), 2.25-2.15 (m, 2H), 1.97-1.77 (m, 3H), 1.77-1.65 (m, 4H), 1.65-1.49 (m, 4H), 1.46-1.32 (m, 2H), 1.18-1.08 (m, 1H), 0.89 (d, J=6.6 Hz, 6H). Secondary products: 1H NMR (400 MHz, chloroform-d) δ 7.11 (s, 1H), 7.10 (s, 1H), 4.30 (tt, J=12.1, 4.1 Hz, 1H), 4.22 (s, 2H), 3.96 (t, J=7.1 Hz, 2H), 3.73 (t, J=4.6 Hz, 4H), 3.04 (d, J=11.7 Hz, 2H), 2.62 (t, J=7.1 Hz, 2H), 2.56 (t, J=4.7 Hz, 4H), 2.42-2.33 (m, 2H), 2.33-2.25 (m, 1H), 1.97-1.77 (m, 3H), 1.77-1.65 (m, 4H), 1.65-1.49 (m, 4H), 1.46-1.32 (m, 2H), 1.18-1.08 (m, 1H), 0.86 (d, J=6.8 Hz, 6H).
Intermediate 62b and 4-isopropylcyclohexyl were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX69, which was subjected to separation by high performance liquid chromatography to obtain cis-product EX69B, 96 mg white solid, with a separation yield of 19%. HRMS (ESI): m/z calcd for C28H45N4O2 ([M+H]+) 469.3537, found 469.3542; HPLC purity (220 nM): 96.12%, tR=4.87 min. 1H NMR (400 MHz, chloroform-d) δ 7.19 (d, J=7.5 Hz, 1H), 6.87 (d, J=8.4 Hz, 1H), 6.84 (s, 1H), 4.39-4.25 (m, 1H), 3.98 (t, J=7.2 Hz, 2H), 3.70 (t, J=4.6 Hz, 4H), 3.15 (d, J=9.2 Hz, 2H), 2.67 (t, J=7.2 Hz, 2H), 2.56 (t, J=4.6 Hz, 4H), 2.48-2.29 (m, 6H), 2.29-2.15 (m, 2H), 1.85-1.76 (m, 2H), 1.76-1.68 (m, 2H), 1.68-1.58 (m, 3H), 1.58-1.47 (m, 2H), 1.44-1.32 (m, 2H), 1.18-1.09 (m, 1H), 0.89 (d, J=6.6 Hz, 6H).
Intermediate 32b and 4-isopropylcyclohexyl were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX70, which was subjected to separation by high performance liquid chromatography to obtain cis-product EX70B, 106 mg white solid, with a separation yield of 17%. HRMS (ESI): m/z calcd for C27H42ClN4O2 ([M+H]+) 489.2991, found 489.2996; HPLC purity: 96.24%, tR=5.53 min. 1H NMR (400 MHz, chloroform-d) δ 7.21 (d, J=7.8 Hz, 1H), 7.09-6.96 (m, 2H), 4.32 (tt, J=13.0, 4.3 Hz, 1H), 3.97 (t, J=6.9 Hz, 2H), 3.69 (t, J=4.6 Hz, 4H), 3.16 (d, J=11.2 Hz, 2H), 2.66 (t, J=6.8 Hz, 2H), 2.54 (t, J=4.6 Hz, 4H), 2.47-2.30 (m, 3H), 2.30-2.17 (m, 2H), 1.87-1.76 (m, 2H), 1.76-1.66 (m, 2H), 1.66-1.58 (m, 3H), 1.58-1.48 (m, 2H), 1.45-1.33 (m, 2H), 1.19-1.09 (m, 1H), 0.90 (d, J=6.6 Hz, 6H).
Intermediate 61b and 4-isopropylcyclohexyl were used as raw materials for synthesis according to the synthesis method of compound EX12 to obtain compound EX71, which was subjected to separation by high performance liquid chromatography to obtain cis-product EX71B, 76 mg light yellow solid, with a separation yield of 12%. HRMS (ESI): m/z calcd for C27H42FN4O2 ([M+H]+) 473.3286, found 473.3297; HPLC purity: >99%, tR=4.18 min. 1H NMR (400 MHz, chloroform-d) δ 7.12-7.02 (m, 1H), 6.91 (dd, J=8.6, 4.5 Hz, 1H), 6.78 (td, J=9.1, 2.4 Hz, 1H), 4.31 (tt, J=12.4, 4.3 Hz, 1H), 3.98 (t, J=7.0 Hz, 2H), 3.68 (t, J=4.6 Hz, 4H), 3.16 (d, J=11.0 Hz, 2H), 2.66 (t, J=7.0 Hz, 2H), 2.53 (t, J=4.7 Hz, 4H), 2.44-2.27 (m, 3H), 2.22 (t, J=11.7 Hz, 2H), 1.85-1.76 (m, 2H), 1.76-1.58 (m, 5H), 1.58-1.46 (m, 2H), 1.44-1.33 (m, 2H), 1.18-1.08 (m, 1H), 0.90 (d, J=6.6 Hz, 6H).
The compounds were dissolved in 100% DMSO solution (10 mM) as a stock solution for later use. The compounds were diluted in a 384-well round bottom plate. The initial screening concentration of the compounds was: 1 μM; 0.1 μm, repeated twice. The starting concentration for compound rescreening was 1 μM (MOP receptor) or 10 μM (NOP receptor) or 100 μM (KOP receptor and DOP receptor), 3-fold dilution, 10 concentration gradients, repeated twice; positive controls were: DAMGO (MEC) (starting concentration was 1 μM, 3-fold dilution, 10 concentration gradients, repeated three times, MOP receptor); Nociceptin (N/O FQ) (Sigma) (starting concentration was 1 μM, 3-fold dilution, 10 concentration gradients, repeated three times, NOP receptor); Naltrexone hydrochloride (Sigma) (starting concentration was 0.1 μM, 3-fold dilution, 10 concentration gradients, repeated three times, KOP receptor); Naltrexone hydrochloride (starting concentration was 10 μM, 3-fold dilution, 10 concentration gradients, repeated three times, DOP receptor). The working solution was 50 mM Tris pH 7.4, 5 mM MgCl2, the washing buffer was 50 mM Tris pH 7.4, stored at 4° C., 0.5% BSA solution (0.5 mL BSA added in 100 mL double distilled water, stored at 4° C.).
The reaction system was 500 μL, and 100 μL of working solution and 5 μL of solution of the compounds (1% DMSO, 99% water) were added to a 96-well deep-well plate, and shaken well (5 min, 500 rpm). 299 μL of reaction solution containing 1 μL CHO-K1 MOP receptor cell membrane (PerkinElmer) (or HEK293-NOP receptor cell membrane, or CHO-K1 DOP receptor cell membrane, PerkinElmer) was added to each well (298 μL of reaction solution containing 2 μL CHO-K1 KOP receptor cell membrane, PerkinElmer, was added in KOP receptor test) and shaken well (5 min, 500 rpm). 100 μL of deuterated ligand was added to each well: [3H]-DAMGO (PerkinElmer) (Fianl conc. 1 nM) (MOP receptor), [3H]-Nociceptin (PerkinElmer) (Fianl conc. 0.5 nM) (NOP receptor), [3H]-U69593 (PerkinElmer) (Fianl conc. 1.5 nM) (KOP receptor), [3H]-DADLE (PerkinElmer) (Fianl conc. 1 nM) (DOP receptor), and shaken well (5 min, 500 rpm). The plate was subjected to incubation at 27° C. for 1 hour. UNIFILTER-96 GF/C filter plate (0.5% PEI) was pre-incubated at 4° C. for 1 hour. The UNIFILTER-96 GF/C filter plate (0.5% PEI) was washed twice (per well) with 1 mL of washing buffer. The mixture solution containing cell membrane was transferred to the UNIFILTER-96 GF/C filter plate (0.5% PEI), which was washed four times with washing buffer, 50 mL each time, followed by drying (55° C., 10 minutes). Then, 40 μL of scintillation cocktail was added to each well, and scintillation counts were read using TopCount to determine the radioactivity bound to the membrane.
Each test compound was tested for binding rate (% Inhibition) at 10 concentrations, its IC50 (the concentration at which 50% of binding is inhibited) was determined by the graph in which the X axis is logarithm of concentration and the Y axis is response count, and the data was processed using Xl-fit 5.3.1 software. Nonlinear regression equation:
For the binding ability of representative compounds of the present application to receptor cell membranes, radiolabeled [3H]-DAMGO (PerkinElmer), [3H]-Nociceptin (PerkinElmer), [3H]-U69,593 (PerkinElmer), [3H]-DADLE (PerkinElmer) were used as interchangeable ligands, Ki value is determined by the equation Ki=IC50/(1+L/Kd), wherein Kd is binding affinity of [3H]-radioligand, and L is the concentration of [3H]-radioligand used.
The determined affinity Ki values of the compounds of the present application are shown in the following table, wherein “/” means no test data:
In the present application, the compounds represented by EX3, EX6, EX16, EX17, EX54, EX58, and EX67 show high selectivity and high affinity for MOP receptor and KOP receptor, and such bifunctional agonists, which act on both MOP receptor and KOP receptor, have been reported in existing literatures, to have the potential to reduce the side effects of single-selective MOP receptor agonists, such as addiction, and balance the side effects such as irritability and aversion produced by single-selective KOP receptor agonists through the euphoriant effect produced by agonizing MOP receptor, making it possible to achieve good analgesic effect with fewer side effects; and the compounds represented by EX12B and EX48B show high selectivity and high affinity for MOP receptor and NOP receptor, and such MOPr/NOPr bifunctional agonists can enhance the analgesic effect caused by activating MOP receptor and inhibit dopamine release to reduce addiction, by agonizing NOP receptor, making it possible to develop novel analgesic drugs without addiction.
Test of Agonistic Function of Compounds for MOP Receptor, NOP Receptor (ORL-1 Receptor), KOP Receptor, and DOP Receptor (cAMP Assay):
Opioid receptor is a G protein-coupled receptor, which is mainly coupled with Gi protein. When it is bound to a ligand to be activated, it can inhibit adenylate cyclase activity via Gi protein, thereby reducing intracellular cAMP level.
A cAMP kit (LANCE Ultra cAMP kit, PE) was used to test the agonistic or inhibitory effects of the compounds on four opioid receptors (MOPr, KOPr, DOPr, and NOPr (ORL-1)). cAMP assay is a competitive immunoassay to detect intracellular cAMP accumulation, and the measured signal value is negatively correlated with the concentration of cAMP.
A standard was prepared according to the instructions of the kit. A 384-well cell culture plate was used, and each well was added with 5 μL standard, 5 μL experimental buffer, 5 μL 4×Eu-cAMP tracer working solution, and 5 μL 4×ULight-anti-cAMP working solution. The plate was incubated at room temperature for 1 h. Readings were taken with microplate reader. The excitation wavelength was 330 nm, and the emission wavelengths were 620 nm and 665 nm. The signal ratio of 665 nm to 620 nm was calculated, and a standard curve was made by the ratio and cAMP concentration.
Cell lines (CHO-hMOPr, CHO-hNOPr, HEK293-hKOPr, HEK293-hDOPr) of opioid receptors expressed in a stable transfected manner, cells were digested, centrifuged, re-suspended in an experimental buffer containing 0.5 mM IBMX, counted, and inoculated into a 384-well cell culture plate at a density of 5 μl, about 3000 cells, per well. The compound was diluted with experimental buffer to prepare 4× solution, and 4× compound was added at 2.5 μl per well, and incubated at 37° C. for 10 min. 2.5 μl 4×Forskolin was further added, followed by incubation at 37° C. for 30 min. Then 5 μl 4×Eu-cAMP tracer working solution and 5 μl 4×ULight-anti-cAMP working solution were added sequentially, and incubated at room temperature for 1 h. Readings were taken with microplate reader. The excitation wavelength was 330 nm, and the emission wavelengths were 620 nm and 665 nm. The signal ratio of 665 nm to 620 nm, i.e., Ratio (665/620), was calculated.
Endomorphin 1 was selected for MOPr positive compound, Nociceptin for NOPr (ORL-1) positive compound, Dynorphin A 1-10 for KOPr positive compound, and DADLE for DOPr positive compound. The test compound was detected starting from the concentration of 10 μM, in 3-fold dilution, with 10 concentrations, and in a manner of double double-well detection.
The graph was made by Ratio (665/620) and the concentration of compounds, and curve fitting and EC50 calculation were performed using the GraphPad Prism 8.0 (GraphPad Software, San Diego, California USA) software nonlinear regression method.
According to the above method, the compounds of the present application were tested, and “NA” means no test data as listed in the table below.
The results of the testing of cAMP agonistic function of preferred compounds showed that the compounds that were bi-selective for MOP receptor and KOP receptor also showed good agonistic bioactivity for MOP receptor and KOP receptor, and were bifunctional agonists for MOP receptor/KOP receptor. Similarly, the compounds that were bi-selective for MOP receptor and NOP receptor also have good agonistic bioactivity for the two opioid receptors.
The animal models of formalin-induced inflammatory pain were established by Dubuisson and Dennis in 1977. It has been experimentally verified over time that injecting formalin diluted by a certain multiple subcutaneously into the hindfoot paw of rats using a microsyringe can create a sustained injurious stimulus that results in a spontaneous behavioral response of pain in the animals.
Experimental animals: SPF grade male SD rats, 6-7 weeks old, body weight ranged from 160 g to 180 g; using as few animals as possible on the premise of meeting research purposes and scientific standards.
MPE % (Maximum Possible Effect, in percentage) was calculated.
The measurement data was expressed by mean±standard deviation, all data statistics were carried out by SPSS13.0 statistical software, all data were subjected to homogeneity test for variance, data with homogeneity of variance (p>0.05) were subjected to one-way analysis of variance, and data with differences (p≤0.05) were subjected to LSD multiple comparative analysis, with p≤0.05 meaning having statistically significant differences; data with heterogeneity of variance (p≤0.05) were subjected to Kruskal-Wallis non-parametric test, data differences (p≤0.05) were subjected to Mann-Whitney pairwise analysis and comparison, with p≤0.05 meaning having statistically significant differences. Graph was made using GraphPad Prism 8.0 (GraphPad Software, San Diego, California USA) software.
As can be seen from the time-effect curve (FIG. TA), the time-effect curve of EX3, at a dose of 10 mg/kg, within the 0-40 min time interval substantially coincides with the effect curve of the morphine group (mor), and then the time-effect curve gradually approaches that of the model group (model), which suggests that compound EX3 (10 mg/kg) has good analgesic effect within the 0-40 min time interval, comparable to the analgesic effect of morphine (3 mg/kg). As can be seen from the maximum possible analgesic effect curve (
Spared Nerve Injury (SNI) animal models are developed to study the pharmacological mechanism of neuropathic pain. The model is established by artificial surgery, which can produce sustained and regenerative pain hypersensitivity on the surgical side to simulate typical symptoms of clinical neuropathic pain disorders. Creation of SNI models: on Day 0 of the experiment, the rat was anesthetized with an anesthesia machine fed with isoflurane, and fixed prone on the operating table, with the lateral gluteal region fully exposed, the left hind limb was shaved and sterilized with alcohol wipe, the skin was cut using surgical scissors in the femur in a direction parallel to the sciatic nerve, the gluteal muscles and biceps femoris muscle were bluntly separated to expose the sciatic nerve trunk, and the surrounding adhesion tissues and fascia were carefully separated. This proceeded until three branches were exposed, i.e., tibial nerve, common peroneal nerve, and sural nerve. For the rats in the surgery group, the tibial nerve and common peroneal nerve were ligated with silk thread, the tibial nerve and common peroneal nerve were cut off respectively, the sural nerve was ensured to be intact, and the muscles and epidermis were sutured layer by layer. After surgery, 60,000 units of penicillin were injected subcutaneously into each rat. The rats were reared in cages, and the administration experiment was carried out on Day 10 after operation. Main test indexes and method: body weight, all animals were weighed once a week and the weights were recorded in detail; measurement of mechanical pain threshold, the animals were placed in a customized multi-unit metal mesh cage for pain testing, the animals were allowed to acclimatize for 10 min, after the animals' grooming and exploratory activities were completed and they acclimatized to the testing environment, electronic Von Frey Hairs (produced by Bioseb, France) were used to stimulate the paw on the pinkie side of the hind limb on the SNI surgical side of the rats, pressure was continuously increased until the rats showed an obvious foot contraction response, the value of Von Frey Hairs at this time was recorded, which was the threshold of mechanical pain response, taking “grams” as the test unit, the test was repeated twice, and then the mean value was taken as the final test index; foot weight-bearing measurement, the weights borne by the two hind limb feet of the rats in the SNI model group were unbalanced, a foot support force measuring instrument (Dynamic Weight Bearing Test, BIO-DWB-DUAL, BIOSEB) was used to measure the weight bearing difference (left foot-right foot) between the two hind limb feet of the animals, measurement was repeated twice, and the mean value was taken as the balance value of the weight bearing difference between the two hind limb feet of the animals. The time points for the mechanical pain threshold and foot weight-bearing testing were before modeling, before administration, and 1, 2, and 4 hours after administration, a total of 5 test points. In the experiment of the present application, rats were randomly divided into 5 groups (8 rats per group) according to the body weight of the animals, and the mechanical pain threshold and base value of foot weight bearing before administration, which were solvent control group (5% DMSO+10% Solutol HS15+85% normal saline), positive control group (Gabapentin, 100 mg/kg, the dose was calculated through conversion based on clinical dose of Gabapentin), and test compound high, medium, and low dose groups (10, 3, and 1 mg/kg). The positive control group was administered orally, which was the same as the administration in clinical practice, the other experimental groups were all administered by subcutaneous injection into the abdomen, and all experimental groups were administered in a single dose. The experimental results were reported by mechanical pain threshold Von Frey Hairs and foot weight bearing difference, and the corresponding analgesic rates were calculated. Relative analgesic rate (%) of Von Frey Hairs=(test value−mean value of control group)/(SNI preoperative base value−mean value of control group)×100; and relative analgesic rate (%) of foot weight bearing=(mean value of control group−test value)/(mean value of control group−SNI preoperative base value)×100. Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, San Diego, California USA), and graph was made.
As could be seen from the experimental results (A and C in
The weights borne by the two hind feet of SNI model rats were unbalanced. In order to further verify the analgesic effect of compound EX3 for SNI neuropathic pain in rats, the weight bearing difference (left foot-right foot) of two hind feet of the animals was measured in this experiment, and the mean value was taken as the balance value of the weight bearing difference of two hind feet of the animals. The test time points were the same as those in Von Frey Hairs test. As could be seen from the experimental results (B and D in
From the evaluation of SNI neuropathic pain models, it could be seen that compound EX3 could effectively inhibit neuropathic pain in rats, and had good dose dependence, and the analgesic effect was consistent with the metabolic cycle of the drug, and the drug exerted its maximum efficacy in the first hour. Compared with the positive control Gabapentin (100 mg/kg), the high dose group (10 mg/kg) had certain analgesic advantages, had a longer duration of action, and still had good analgesic effect in the fourth hour. Compound EX3 exhibited superior analgesic effect to the positive control Gabapentin at a low dose.
The above results show that the compounds shown in the present application are bi-selective for MOP receptor and KOP receptor, and exert good analgesic effect through this bifunctional action on MOP receptor and KOP receptor, and the inhibition for neuropathic pain is especially far superior to that of the clinical drug Gabapentin. According to the reports in numerous existing literatures, this effect cannot be achieved by single selective MOP receptor agonists.
The compounds disclosed in the present application, preferably some of them, have been evaluated for pharmacodynamics in vivo using the above methods, and the results show that the compounds disclosed in the present application have good efficacy in treating inflammatory pain and neuropathic pain. By continuous observation of the phenomena during the experiment and analysis of data, the compounds disclosed in the present application have fewer side effects, such as constipation and itching, than the positive control.
Although the embodiments disclosed in the present application are as above, the contents described are only the embodiments adopted for the convenience of understanding the present application, and are not used to limit the present application. Any person skilled in the field to which the present application belongs may make any modifications and changes in the form and details of implementation without departing from the spirit and scope disclosed in the present application, but the scope of protection of the present application shall still be subject to the scope defined in the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110797657.5 | Jul 2021 | CN | national |
The present application is a U.S. National Phase Entry of International PCT Application No. PCT/CN2022/105776, having an international filing date of Jul. 14, 2022, which claims priority to Chinese invention patent application No. 202110797657.5, filed on Jul. 14, 2021 and titled “Piperidine Derivative, and Pharmaceutical Composition Thereof, Preparation Method Therefor, and Use Thereof”. The above-identified applications are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/105776 | 7/14/2022 | WO |
