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WO2015062555A1 - 具有更高蛋白激酶g抑制活性的化合物及其制备方法 - Google Patents

具有更高蛋白激酶g抑制活性的化合物及其制备方法 Download PDF

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Publication number
WO2015062555A1
WO2015062555A1 PCT/CN2014/090226 CN2014090226W WO2015062555A1 WO 2015062555 A1 WO2015062555 A1 WO 2015062555A1 CN 2014090226 W CN2014090226 W CN 2014090226W WO 2015062555 A1 WO2015062555 A1 WO 2015062555A1
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compound
pharmaceutically acceptable
acceptable salt
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etoac
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English (en)
French (fr)
Inventor
冯泽旺
赵宣
王振国
刘岩
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Tianjin Jenkem Technology Co Ltd
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Tianjin Jenkem Technology Co Ltd
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Priority to AU2014344316A priority Critical patent/AU2014344316B2/en
Priority to BR112016009918-4A priority patent/BR112016009918B1/pt
Priority to JP2016550929A priority patent/JP6195100B2/ja
Priority to EP14857892.5A priority patent/EP3067351B1/en
Publication of WO2015062555A1 publication Critical patent/WO2015062555A1/zh
Priority to US15/146,588 priority patent/US9567319B2/en
Anticipated expiration legal-status Critical
Priority to US15/409,403 priority patent/US9925171B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
    • C07C69/94—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of polycyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of six-membered aromatic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/32—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing keto groups
    • C07C65/40—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing keto groups containing singly bound oxygen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
    • C07C69/734—Ethers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
    • C07C69/738—Esters of keto-carboxylic acids or aldehydo-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/14—Nitrogen atoms not forming part of a nitro radical

Definitions

  • the present invention relates to a novel class of compounds, processes for their preparation and intermediates, pharmaceutical compositions and uses.
  • the present invention relates to a compound having a higher protein kinase G (PKG) inhibitory activity, a process for the preparation thereof and an intermediate, a pharmaceutical composition comprising the same, and a compound and a pharmaceutical composition for treating pain, Especially for the treatment of chronic pain.
  • PKG protein kinase G
  • Pain stimuli generated by impacting specific pain receptors travel along primary nociceptive neurons in the dorsal root ganglia (DRG, peripheral nervous system) and then into the spinal cord (the central nervous system) to transmit signals to the spinal cord.
  • DRG dorsal root ganglia
  • Second-order neurons which spread to the opposite side of the spinal cord, finally transmit the signal to the higher-level center of the brain where it is perceived as pain.
  • Peripheral pain receptors that respond to mechanical, thermal, and chemical stimuli are located on the nerve endings of primary nociceptive neurons. Activation of these receptors can produce acute or chronic pain. Acute pain tends to be intense, non-proliferative, and usually propagates along the axons of the thin myelin sheath of delta sensory neurons. Chronic pain is usually inactivated, spread, and propagates along non-myelinated axons of C-type nociceptive neurons.
  • Pain perception may change at different stages of the pain pathway.
  • the use of local anesthetics for peripheral receptors can eliminate painful stimuli.
  • drugs such as papaverine block the central nervous system of the pain pathway, while non-steroidal anti-inflammatory drugs block the peripheral nervous system.
  • Chronic pain perception in non-primary spinal cord injury is usually associated not only with peripheral pain receptor sensitization, but also with excitatory changes in second-order neurons, with both peripheral and central nervous system components.
  • the peripheral and central components regulate the "primary” and "secondary” hyperalgesia, respectively.
  • secondary hyperalgesia second-order neuronal gene expression changes in the central nervous system, causing "central sensitization” or "spinal hyperalgesia".
  • N-methyl-D-aspartate (NMDA) receptors in the spinal cord This process plays an important role.
  • Spinal cord injury without activation of the peripheral nervous system can also produce spinal hyperalgesia, leading to central pain syndrome.
  • Central neuropathic pain is associated with phosphorylation of reactive binding protein (CREB) transcription factors.
  • CREB reactive binding protein
  • Chronic pain begins in the periphery and is caused by nerve damage (neurological pain) or inflammation. Pain caused by both causes is a major clinical problem that hinders effective treatment.
  • Pain caused by both causes is a major clinical problem that hinders effective treatment.
  • LTH long-term over-excitation
  • Pain caused by both causes is a major clinical problem that hinders effective treatment.
  • LTH long-term over-excitation
  • Pain caused by both causes is a major clinical problem that hinders effective treatment.
  • LTH long-term over-excitation
  • Long-term over-excitedness is manifested by the increasing sensitization of neuronal cell bodies and axonal electrical stimulation to painful lesions in the affected area.
  • U.S. Patent No. 6,476,007 relates to the mechanism of central nervous system inflammatory hyperalgesia without considering the effects of the peripheral nervous system.
  • Targeting the pain pathways of the central nervous system has many major drawbacks.
  • the neuronal circuits in the spinal cord are very complex, and it is predicted that drugs that relieve pain may have the opposite effect.
  • the blood-brain barrier isolates the neurons of the central nervous system from the rest of the body, a huge obstacle that often prevents a large number of therapeutic drugs from reaching their targets.
  • drugs that penetrate the blood-brain barrier enter the entire central nervous system, causing serious side effects.
  • the peripheral nervous system does not have these disorders, and the structural features of DRG show that it can be treated against specific populations of primary sensory neurons.
  • pain can only be perceived when signals from the periphery are transmitted to the brain's advanced center, and DRG neurons are the entrance to these signals.
  • nitric oxide synthase NOS
  • sGC soluble guanosine cyclase
  • cGMP cyclic guanosine monophosphate
  • the active PKG then travels back from the injury site along the axon to the neuronal cell body, mitogen-activated protein kinase-erk (MAPKerk). Then, active MAPKerk is transferred into the nucleus to regulate the expression of pain-related genes that regulate the appearance of LTH.
  • MAPKerk mitogen-activated protein kinase-erk
  • the compound 46 is structurally modified in the present invention, and the water-soluble solution is obtained by adding a low-molecular-weight polyethylene glycol fragment having a high water solubility. Significantly increased sex, and the analgesic effect was significantly improved.
  • the invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof,
  • R 1 and R 2 are the same or different and are each independently selected from the group consisting of halogen, C 1 -C 6 alkoxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl;
  • R 3 is selected from H, halogen, substituted or unsubstituted C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, aryl and a heteroaryl group; and n is an integer from 0-15.
  • the compound has higher water solubility and has a higher inhibitory activity against PKG, thereby providing a significantly increased analgesic effect.
  • the invention provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of pain.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.
  • the invention provides a method of treating pain, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention.
  • the invention provides a process for the preparation of a compound of formula I or a pharmaceutically acceptable salt thereof.
  • 1A and 1B show the mean value of the Von Frey cilia test for low dose treatment of each test compound.
  • 2A and 2B show the mean value of the Von Frey cilia test for high dose treatment of each test compound.
  • Figure 3 shows the dose dependency of the Von Frey cilia test of the compound JK-02H of the present invention.
  • Figures 4A and 4B show the average of the weight-bearing tests for low dose treatment of each test compound.
  • Figures 5A and 5B show the mean of the weight-bearing tests for high dose treatment of each test compound.
  • Figure 6 shows the dose dependency of the weight loading test of the compound JK-02H of the present invention.
  • the inventors of the present application found that after the compound 46 disclosed in US2008/0176920 and the 6-OCH 2 CH 2 CH 3 of the compound JK-02A having a similar structure were replaced with a low molecular polyethylene glycol fragment, the obtained compound was water-soluble. The increase in sex also showed higher inhibitory activity against PKG, indicating a significant increase in analgesic effect.
  • the inventors of the present application have also synthesized compounds having a similar structure to the above compounds, and the results show that the water solubility of these compounds is significantly improved compared with the parent compound, which will have a positive effect on the improvement of its pharmacological action.
  • the present invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof having higher PKG inhibitory activity for the treatment of pain,
  • R 1 and R 2 are the same or different and are each independently selected from halogen (such as F or Cl), C 1 -C 6 alkoxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl;
  • R 3 is selected from H, halogen, substituted or unsubstituted C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 Alkynyl, aryl and heteroaryl; and n is an integer from 0-15.
  • R 1 and R 2 are each independently selected from halogen and C 1 -C 6 alkoxy; more preferably, R 1 is F and R 2 is F or OCH 3 .
  • R 3 is selected from H and substituted or unsubstituted C 1 -C 6 alkyl; more preferably, R 3 is selected from H and substituted or unsubstituted C 1 -C 3 Alkyl; most preferably, R 3 is H, methyl or propyl.
  • n is an integer from 1 to 10; more preferably, n is an integer from 1 to 6.
  • R 1 is a fluorine atom and R 2 is a methoxy group, and thus a preferred compound has the structure of formula II
  • R 3 and n are as defined above.
  • both R 1 and R 2 are fluorine atoms, and thus preferred compounds have the structure of formula III
  • R 3 and n are as defined above.
  • the invention provides the following preferred compounds:
  • JK-02H is increased by about 5 times
  • JK-03M is increased by about 4 times
  • JK-06H is increased by about 10 times
  • JK-07M is increased by about 8 times.
  • the JK-02P is increased by about 3 times.
  • the compounds having a chiral center disclosed herein may exist in the form of optically active and racemic forms, and some compounds may exhibit a variety of crystalline forms.
  • the invention includes any racemate, optically active, polymorphic or stereoisomer of the disclosed compounds, or mixtures thereof.
  • the invention provides the following preferred compounds:
  • the compound can be used in the form of a salt.
  • Acceptable salts are acid addition salts with physiologically acceptable organic acids such as tosylate, mesylate, acetate, propionate, glycolate, pyruvate, oxalic acid Salt, malate, maleate, fumarate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, salicylate, citrate, C a diacid salt, a tartrate salt, a succinate salt, a benzoate salt, an ascorbate salt, an alpha-ketoglutarate salt, an alpha-glycerol phosphate salt, or the like, or an acid addition salt formed with a physiologically acceptable inorganic acid.
  • physiologically acceptable inorganic acid such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate, bicarbonate and carbonate.
  • Another aspect of the invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of pain.
  • the compounds of the invention are useful in the treatment of a variety of pains including, but not limited to, chronic pain, neuropathic pain, acute pain, cancer pain, rheumatoid arthritis pain, migraine, visceral pain, and the like.
  • the compounds of the present invention are useful as analgesics for use in general anesthesia and monitoring anesthesia care. Drugs of different nature are often used in combination to achieve the balance required to maintain anesthesia (eg, loss of memory, analgesia, muscle relaxation, and sedation). Combination medications include inhalation anesthetics, hypnotics, anxiolytics, neuromuscular blockers, and opioids.
  • the compounds of the invention may be administered at different dosages and at different frequencies depending on, for example, the health, age, weight and sex of the subject.
  • Another aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.
  • the carrier should be tolerated by the individual to whom it is administered and capable of delivering the compound to the affected area.
  • Formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacy. Typically, the method of preparation comprises the steps of combining the active ingredient with a carrier or one or more additional auxiliaries and, if necessary, packaging the product into the desired single or multiple dosage unit.
  • the pharmaceutical composition may further comprise one or more other pharmaceutically active substances in addition to the active ingredient.
  • the pharmaceutical composition can be used simultaneously or sequentially with one or more other pharmaceutically active substances.
  • compositions of this invention may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may contain ingredients other than the active ingredient such as dispersing agents, wetting agents or suspending agents and the like.
  • This sterile injectable preparation can be prepared using a non-toxic diluent or solution such as water or 1,3-butanediol.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, monoglyceride or diglyceride, and the like.
  • Controlled release or sustained release formulations of the pharmaceutical compositions can be prepared using conventional techniques. Controlled release of the active ingredient can be stimulated by various inducing factors such as pH, temperature, enzymes, water or other physiological conditions or compounds.
  • Suitable pharmaceutical compositions comprising a compound of the invention may be administered by conventional means, such as oral, topical, parenteral, buccal, nasal, vaginal or rectal or Administered by inhalation.
  • the compounds of the present invention can be prepared in various forms by methods known in the art, such as tablets, capsules, aqueous solutions or oil solutions, suspensions, emulsions, creams, ointments, Gels, nasal sprays, suppositories, very fine powders or aerosols for inhalation.
  • parenteral administration including intravenous, intramuscular or infusion
  • sterile water may be used.
  • an oil solution or suspension or sterilized emulsion may be used for parenteral administration.
  • Another aspect of the invention provides a method of treating pain, the method comprising administering to a subject in need of treatment an analgesic effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, or Formula I comprising an effective amount to ameliorate pain
  • an analgesic effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, or Formula I comprising an effective amount to ameliorate pain
  • a pharmaceutical composition of a compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier a pharmaceutically acceptable carrier.
  • the method of treatment using a compound of the present invention or a pharmaceutically acceptable salt thereof can be used for the prevention and treatment of pain in a mammal, which can be a human.
  • Another aspect of the invention provides a process for the preparation of a compound of formula I, the process comprising the steps of:
  • X is a halogen, preferably Cl or Br; and R 1 , R 2 , R 3 and n are as defined above.
  • the invention provides a process for the preparation of a compound of formula I, or a pharmaceutically acceptable salt thereof, comprising:
  • the nucleophilic substitution reaction is carried out in the presence of a base to obtain a compound 15,
  • X is a chlorine, bromine or iodine atom, and R 1 , R 2 , R 3 and n are as defined above;
  • the nucleophilic substitution reaction is carried out in an organic solvent selected from the group consisting of acetonitrile, propionitrile, dimethylformamide (DMF), dimethylacetamide, 1,3-dimethyl-2 -Imidazolinone (DMI), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide (HMPA), tetrahydrofuran, dioxane, acetone, toluene, xylene, diethyl ether, propyl ether, diisopropyl ether a combination of methyl tert-butyl ether, n-hexane, cyclohexane, methylcyclohexane and n-heptane, and any ratio of any one or more thereof;
  • an organic solvent selected from the group consisting of acetonitrile, propionitrile, dimethylformamide (DMF), dimethylacetamide, 1,3-dimethyl-2 -Imidazolinone (
  • the base is selected from the group consisting of potassium carbonate, sodium carbonate, barium carbonate, calcium carbonate, potassium hydroxide, sodium hydroxide, lithium hydroxide, calcium hydroxide, sodium hydride, potassium hydride, calcium hydride, sodium metal, potassium metal.
  • R 1 , R 2 , R 3 and n are as defined above;
  • the hydrolysis reaction is carried out in an organic solvent selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, tetrahydrofuran, acetonitrile, and C. a combination of nitrile, DMF, dimethylacetamide, DMI, DMSO, HMPA, tetrahydrofuran, dioxane, and acetone, and any ratio of any one or more thereof;
  • the base is selected from the group consisting of potassium hydroxide, sodium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide, copper hydroxide, aluminum trichloride, boron trichloride, aluminum tribromide, tribromination Boron, sodium cyanide, Potassium cyanide, cesium carbonate, copper carbonate, lithium iodide, sodium borohydride, sodium hydride, potassium hydride, calcium hydride, and combinations thereof;
  • R 1 , R 2 , R 3 and n are as defined above;
  • the amidation reaction is carried out in an organic solvent selected from the group consisting of toluene, xylene, chlorobenzene, acetonitrile, tetrahydrofuran, dioxane, dichloromethane, chloroform, carbon tetrachloride, a combination of diethyl ether, propyl ether, diisopropyl ether, methyl tert-butyl ether, n-hexane, cyclohexane, methylcyclohexane and n-heptane, and any ratio of any one or more thereof;
  • an organic solvent selected from the group consisting of toluene, xylene, chlorobenzene, acetonitrile, tetrahydrofuran, dioxane, dichloromethane, chloroform, carbon tetrachloride, a combination of diethyl ether, propyl ether, diisopropyl ether,
  • the condensing agent is selected from the group consisting of N-hydroxy-7-azobenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), 2-(7-azobenzotriazole) )-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU , bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (BOP-Cl), 1H-benzotriazol-1-oxytris(1-pyrrolidinyl)phosphonium hexafluorophosphate ( PyBOP), 1,3-dicyclohexylcarbodiimide (DCC), N,N'-carbonyldiimidazole (CDI), 1-ethyl-(3-dimethylaminopropyl)carbodiimide ( E)
  • Boc represents a tert-butoxycarbonyl protecting group, and R 1 , R 2 , R 3 and n are as defined above;
  • the amidation reaction is carried out in an organic solvent selected from the group consisting of toluene, xylene, chlorobenzene, acetonitrile, tetrahydrofuran, dioxane, dichloromethane, chloroform, carbon tetrachloride, a combination of diethyl ether, propyl ether, diisopropyl ether, methyl tert-butyl ether, n-hexane, cyclohexane, methylcyclohexane and n-heptane, and any ratio of any one or more thereof;
  • an organic solvent selected from the group consisting of toluene, xylene, chlorobenzene, acetonitrile, tetrahydrofuran, dioxane, dichloromethane, chloroform, carbon tetrachloride, a combination of diethyl ether, propyl ether, diisopropyl ether,
  • the condensing agent is selected from the group consisting of HOAt, HOBt, HATU, HBTU, BOP-Cl, PyBOP, DCC, CDI, EDC, (DIC), DMAP, and combinations thereof;
  • the deprotection reaction is carried out in an organic solvent selected from the group consisting of toluene, xylene, chlorobenzene, acetonitrile, tetrahydrofuran, dioxane, dichloromethane, chloroform, carbon tetrachloride, a combination of diethyl ether, propyl ether, diisopropyl ether, methyl tert-butyl ether, n-hexane, cyclohexane, methylcyclohexane and n-heptane, and any ratio of any one or more thereof;
  • the deprotecting agent is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetyl chloride, aluminum trichloride, and boron trifluoride.
  • the invention also provides novel intermediates for the preparation of compounds of formula I which are compounds of formula 15, formula 16, formula 17 or formula 18:
  • Boc, R 1 , R 2 , R 3 and n are as defined above.
  • analog refers to a compound that is structurally similar to another compound, but not necessarily to its isomer.
  • inhibiting refers to a compound that reduces or prevents the ability of the described function. force.
  • alkyl refers to a straight or branched alkyl group.
  • C 1 -C 6 alkyl denotes a straight or branched alkyl group having from 1 to 6 carbon atoms.
  • Typical C 1 -C 6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like.
  • alkenyl refers to an ethylenically unsaturated straight or branched chain hydrocarbon radical comprising at least one double bond.
  • C 2 -C 6 alkenyl denotes an ethylenically unsaturated straight or branched chain hydrocarbon radical having from 2 to 6 carbon atoms and at least one double bond.
  • Typical C 2 -C 6 alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl, and the like.
  • alkynyl refers to an unsaturated straight or branched chain hydrocarbon radical comprising at least one triple bond.
  • C 2 -C 6 alkynyl denotes an unsaturated straight or branched chain hydrocarbon radical having from 2 to 6 carbon atoms and at least one triple bond.
  • Typical C 2 -C 6 alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl and the like.
  • C 3 -C 6 cycloalkyl as used herein means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • aryl refers to aromatic rings having one or two monocyclic or bicyclic C 5 -C 10 carbocyclic ring system, including but not limited to: phenyl, benzyl, naphthyl, tetrahydronaphthyl Naphthyl, indanyl, fluorenyl and the like.
  • heteroaryl refers to a monocyclic or bicyclic ring system having one or two aromatic rings and containing from 1 to 3 heteroatoms in addition to a carbon atom, wherein the heteroatoms are selected. From nitrogen, oxygen and sulfur.
  • heteroaryl groups include, but are not limited to, furyl, thienyl, pyridyl, and the like.
  • heterocyclyl refers to a monocyclic or bicyclic ring system containing a carbon atom and 1 to 3 heteroatoms in the ring wherein the hetero atom is selected from the group consisting of nitrogen, oxygen and sulfur.
  • the intermediate is not fully structurally confirmed, and its purity is usually evaluated by mass spectrometry (MS) or nuclear magnetic (NMR) analysis.
  • MS mass spectrometry
  • NMR nuclear magnetic
  • HATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DIPEA N,N-diisopropylethylamine
  • 3,4-difluoroanisole, 3-amino-4-methylbenzoic acid, 3-fluoro-4-methoxyphenol, 3,4-dimethoxyphenol, 2-fluoro used in the examples -4-Methoxybenzaldehyde, m-toluidine, 2-butynoic acid were purchased from BEHRINGER TECHNOLOGY CO., Ltd., hexaethylene glycol, heptaethylene glycol monomethyl ether was purchased from Jiaxing Bomei Biotechnology Co., Ltd., p-formylbenzoic acid The methyl ester was purchased from Shanghai Bangcheng Chemical Co., Ltd., and the remaining reagents were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.
  • the intermediate ((3R, 4R)-4) (132.7 g, 0.6 mol) was slowly added to a solution of LiAlH 4 (61.2 g, 1.6 mol) in tetrahydrofuran (3.6 L) after cooling to 0 ° C under nitrogen. .
  • the reaction mixture was refluxed for 12 h, cooled to EtOAc.
  • Distilled water (61.2 mL), 5% NaOH (61.2 mL) and distilled water (183.6 mL) were added dropwise with vigorous stirring. Filter and filter cake was washed with hot tetrahydrofuran (2 x 1.2 L). The filtrate and the washings were combined, and the crystals were evaporated, evaporated, mjjjjjjjjjj 71.0%.
  • the intermediate ((3R,4R)-9) (30.4g, 0.12mol) was dissolved in anhydrous methanol (500mL), 10% Pd/C (12.8g, 12mmol) was added and reacted at room temperature with hydrogen (0.1MPa) 18h. The catalyst was removed by filtration, and the filtrate was evaporated to dryness crystals.
  • the ice salt bath was cooled to triethylene glycol monomethyl ether (49.2 g, 0.30 mol) and pyridine (35.5 g, 0.45 mol).
  • a solution of p-toluenesulfonyl chloride (85.7 g, 0.45 mol) in dichloromethane (125 mL) was added dropwise to the mixture, and the mixture was adjusted to a drop rate, and the reaction solution was allowed to stand below 20 ° C and stirred at room temperature overnight.
  • Distilled water 100 mL was added, the pH of the reaction mixture was adjusted to 4.0 with a 6N NaOH solution, and the organic layer was washed with distilled water (2 ⁇ 50 mL).
  • Distilled water (100 mL) was added to the organic layer, the pH was adjusted to 1.5 with 6N HCl solution, and the organic layer was washed with distilled water (2 ⁇ 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated.
  • the title compound was synthesized by the method of Example 1 using 1-methoxy-2-fluoro-4-benzyloxybenzene instead of 3,4-difluoroanisole as a starting material.
  • the title compound was synthesized by the method of Example 1 using the intermediate (1B) instead of the intermediate (1A) as a starting material.
  • the title compound was synthesized by the method of Example 5 using the intermediate (3B) instead of the intermediate (3A) as a starting material.
  • the title compound was synthesized by the method of Example 1 using 2-fluoro-4-methoxystyrene instead of 3,4-difluoroanisole as a starting material.
  • the title compound was synthesized by the method of Example 2 using the intermediate (3D) instead of the intermediate (3A) as a starting material.
  • 2-methyl-4-methoxyethyl iodide (24.8 g, 0.10 mol), 2-butynoic acid (11.7 g, 0.14 mol), cuprous iodide (0.38 g, 2.0 mmol), triphenyl Phosphines (1.05 g, 4.0 mmol) and potassium carbonate (41.5 g, 0.30 mol) were dissolved in dimethyl sulphoxide (1.0 L) and were then evaporated. The reaction solution was warmed to 100 ° C and stirred with stirring for 24 h. After cooling to room temperature, it was diluted with EtOAc (3 mL). 13.1 g, yield 81.8%.
  • the title compound was synthesized by the method of Example 1 using 1-(2-methyl-4-methoxy)phenyl-1-propyne in place of 3,4-difluoroanisole as a starting material.
  • the title compound was synthesized by the method of Example 3 using the intermediate (3E) instead of the intermediate (3A) as a starting material.
  • Example 15 Water solubility test of a compound
  • Example 16 Efficacy of Compound JK-02H, Compound 46 and JK-02A on Hyperalgesia Induced by Freund's Complete Adjuvant (CFA) in Rats
  • Rat age 5-7 weeks, 7-8 weeks after 7 days of adaptation period
  • Von Frey cilia tests were performed on day 7, including three time points before treatment, 15 minutes and 2 hours after administration. After the one-week washout period, on the 14th day after the CFA injection, four additional Von Frey cilia tests were performed after a single high-dose dose, including four before treatment, 15 minutes, 2 hours, and 5 hours after administration. Time point.
  • Rats were unevenly distributed on the injected CFA foot and contralateral foot and were measured by a weight balance meter (Bio-medical, USA).
  • the load on the hind foot is recorded by a force plate embedded in the floor to measure the load of the animal in the box.
  • the average load (in g) of the injected CFA foot and the contralateral foot was measured within 10 seconds. The time interval of this experimental test is consistent with the interval of the Von Frey cilia test.

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Abstract

本发明公开了一类具有更高蛋白激酶G（PKG）抑制活性的式I的化合物及其药学上可接受的盐，（式I），其中R1和R2相同或不同，各自独立地选自卤素、C1-C6烷氧基、C1-C6烷基、C2-C6烯基和C2-C6炔基；R3选自H、卤素、取代或未被取代的C1-C6烷基、C3-C6环烷基、C2-C6烯基、C2-C6炔基、芳基和杂芳基；且n为0-15的整数。本发明还公开了包含所述化合物的药物组合物；所述化合物在治疗疼痛、尤其是治疗慢性疼痛中的应用；以及所述化合物的制备方法及新的中间体。

Description

具有更高蛋白激酶G抑制活性的化合物及其制备方法 技术领域
本发明涉及一类新的化合物及其制备方法和中间体、药物组合物和应用。具体地，本发明涉及一类具有更高蛋白激酶G(PKG)抑制活性的化合物及其制备方法和中间体、包含所述化合物的药物组合物以及所述化合物和药物组合物用于治疗疼痛、尤其是治疗慢性疼痛的应用。
背景技术
疼痛是中枢和外周两大神经系统沟通交流的结果。虽然中枢和外周这两大神经系统共同作用产生人们的主观体验，但它们的生理结构和功能不同。
撞击特定疼痛受体产生的疼痛刺激沿着居于背根神经节(DRG，外周神经系统部分)的初级伤害性感觉神经元传播，然后进入脊髓(中枢神经系统部分)传播，在脊髓将信号转发到二阶神经元，传播到脊髓的对侧，最后将信号传播到脑的更高级中枢，在那里感知为疼痛。
对机械、热和化学刺激产生反应的外周疼痛受体位于初级伤害性感觉神经元的神经末梢上。这些受体的激活可产生急性或慢性疼痛。急性疼痛趋于剧烈、不扩散，通常沿δ感觉神经元的薄髓鞘的轴突传播。慢性疼痛通常钝化、扩散，沿C-型伤害性感觉神经元的非髓鞘轴突传播。
在疼痛通路的不同阶段，疼痛感知可能会改变。例如，给外周受体使用局部麻醉药可消除疼痛刺激。众所周知，如类罂粟碱的药物在疼痛通路的中枢神经系统阶段起阻碍作用，而非甾体抗炎药在外周神经系统阶段起阻碍作用。非原发性脊髓损伤中的慢性疼痛感知通常不仅与外周疼痛受体敏化有关，还跟二阶神经元的兴奋性改变有关，具有外周和中枢神经系统两种组件。外周和中枢两种组件分别调节“初级”和“次级”痛觉过敏。在次级痛觉过敏中，中枢神经系统中的二阶神经元基因表达改变，引起“中枢致敏”或“脊髓痛觉过敏”现象。脊髓的N-甲基-D-天冬氨酸(NMDA)受体在 此过程中起重要作用。无外周神经系统活化的脊髓损伤也能产生脊髓痛觉过敏，导致中枢疼痛综合征。中枢神经性疼痛与反应结合蛋白(CREB)转录因子的磷酸化有关。
慢性疼痛始于外周，由神经损伤(神经性疼痛)或炎症引起，两种原因导致的疼痛都是阻碍有效治疗的主要临床问题。在人和哺乳动物模型中，神经损伤后的持续疼痛与初级感觉神经元的长期过度兴奋(LTH)有关，这些初级感觉神经元的轴突位于受损神经节上。由于对受损部位的疼痛感觉神经元细胞体和轴突电刺激敏化的不断增加，长期过度兴奋呈现出来。这些变化导致在静止或无伤害刺激时从感觉神经元中释放动作电位，引起中枢神经系统的高阶神经元持续激发、脊柱痛觉过敏和持续疼痛。
美国专利文献US6476007涉及中枢神经系统炎性痛觉过敏的机理，没有考虑外周神经系统的作用。瞄准中枢神经系统的疼痛通路具有许多重大弊端。首先，脊髓中的神经元回路非常复杂，据此预测缓解疼痛的药物可能具有相反的效果。其次，血脑屏障将中枢神经系统的神经元与身体其它部分隔离，这是通常阻止大量治疗药物达到靶点的巨大障碍。第三，穿透血脑屏障的药物进入整个中枢神经系统，导致严重的毒副作用。而外周神经系统就没有这些障碍，DRG的结构特性显示，可针对初级感觉神经元的特定种群进行治疗。第四，只有当来自外周的信号传至脑的高级中枢才能感知到疼痛，而DRG的神经元是这些信号的入口。
活性PKG在疼痛抑制中起决定作用(见WO2006/102267)。外周神经系统受到损伤后，一氧化氮合酶(NOS)活性增加，导致一氧化氮(NO)产量增多。NO活化可溶性鸟苷环化酶(sGC)，从而增加环磷酸鸟苷(cGMP)水平，导致在C-型和A-δ型痛觉神经元的轴突中PKG活化。活性PKG然后从损伤位点沿轴突反向传播至神经元细胞体，丝裂原活化蛋白激酶-erk(MAPKerk)。然后，活性MAPKerk转移入细胞核，调整疼痛相关基因的表达，这些基因调节LTH的出现。因此抑制PKG可缓解疼痛，减少涉及伤害感受蛋白质的信使RNA(mRNA)水平。
因此，本领域需要能够选择性抑制外周神经系统中PKG活性的化合物。对活性PKG的抑制不仅可阻止它经外周神经系统传播，还可阻断它在细胞内的活性。美国专利申请US2008/0176920述及抑制PKG活化形式的化合 物，以及它们在缓解疼痛，特别是慢性疼痛综合征中的应用。在该专利申请中提到的活性较高的化合物为化合物46。本申请的发明人按照美国专利申请US2008/0176920的方法制备了化合物46及其类似物JK-02A，并进行了药物活性研究。研究结果显示，该类化合物水溶性不够好、PKG抑制活性不够高。因此，本申请的发明人设计了一类水溶性更好、抑制活性更高的化合物。
Figure PCTCN2014090226-appb-000001
发明内容
鉴于美国专利申请US2008/0176920中设计的化合物46水溶性较差、PKG抑制活性不够高，本发明对化合物46进行结构改造，通过添加水溶性较高的低分子聚乙二醇片段，使其水溶性明显增加、镇痛效果显著提高。
因此，一方面，本发明提供了式I的化合物或其药学上可接受的盐，
Figure PCTCN2014090226-appb-000002
其中R1和R2相同或不同，各自独立地选自卤素、C1-C6烷氧基、C1-C6烷基、C2-C6烯基和C2-C6炔基；R3选自H、卤素、取代或未被取代的C1-C6烷基、C3-C6环烷基、C2-C6烯基、C2-C6炔基、芳基和杂芳基；且n为0-15的整数。
所述化合物具有更高的水溶性，且对PKG具有更高的抑制活性，从而提供了显著增加的镇痛效果。
另一方面，本发明提供了式I的化合物或其药学上可接受的盐在制备治疗疼痛的药物中的应用。
另一方面，本发明提供了包含式I的化合物或其药学上可接受的盐以及一种或多种药学上可接受的载体的药物组合物。
另一方面，本发明提供了治疗疼痛的方法，所述方法包括向需要治疗的个体给药治疗有效量的本发明的化合物或其药学上可接受的盐或者本发明的药物组合物。
再一方面，本发明提供了制备式I的化合物或其药学上可接受的盐的方法。
以上被描述的和其他的特征通过下面的详细描述更加清楚。
附图说明
图1A和图1B示出各测试化合物低剂量治疗的Von Frey纤毛试验平均值。
图2A和图2B示出各测试化合物高剂量治疗的Von Frey纤毛试验平均值。
图3示出本发明的化合物JK-02H的Von Frey纤毛试验的剂量依赖性。
图4A和图4B示出各测试化合物低剂量治疗的负重试验平均值。
图5A和图5B示出各测试化合物高剂量治疗的负重试验平均值。
图6示出本发明的化合物JK-02H的负重试验的剂量依赖性。
具体实施方式
本申请的发明人发现，将US2008/0176920所公开的化合物46及具有类似结构的化合物JK-02A的6位-OCH2CH2CH3替换为低分子聚乙二醇片段后，所得化合物的水溶性提高，同时显示对PKG具有更高的抑制活性，表明镇痛效果显著提高。本申请的发明人还合成了与上述化合物具有类似结构的化合物，研究结果表明这些化合物的水溶性较其母体化合物均有明显提高，这将对其药理作用的提高有正面影响。
因此，本发明提供了用于治疗疼痛的具有更高PKG抑制活性的通式I的化合物或其药学上可接受的盐，
Figure PCTCN2014090226-appb-000003
其中R1和R2相同或不同，各自独立地选自卤素(如F或Cl)、C1-C6烷氧基、C1-C6烷基、C2-C6烯基和C2-C6炔基；R3选自H、卤素、取代或未被取代的C1-C6烷基、C3-C6环烷基、C2-C6烯基、C2-C6炔基、芳基和杂芳基；且n为0-15的整数。
在本发明的优选实施方案中，R1和R2各自独立地选自卤素和C1-C6烷氧基；更优选地，R1为F，且R2为F或OCH3。
在本发明的优选实施方案中，R3选自H和取代或未被取代的C1-C6烷基；更优选地，R3选自H和取代或未被取代的C1-C3烷基；最优选地，R3为H、甲基或丙基。
在本发明的优选实施方案中，n为1-10的整数；更优选地，n为1-6的整数。
在本发明的优选实施方案中，R1为氟原子，R2为甲氧基，因而优选的化合物具有式II的结构
Figure PCTCN2014090226-appb-000004
其中R3和n如上文中所定义。
在本发明的其他优选实施方案中，R1和R2均为氟原子，因而优选的化合物具有式III的结构
Figure PCTCN2014090226-appb-000005
其中R3和n如上文中所定义。
具体地，本发明提供了如下优选的化合物：
Figure PCTCN2014090226-appb-000006
Figure PCTCN2014090226-appb-000007
这些优选化合物的水溶性较化合物46均有大幅度提高，具体分别为：JK-02H提高约5倍、JK-03M提高约4倍、JK-06H提高约10倍、JK-07M提高约8倍、JK-02P提高约3倍。
在弗氏完全佐剂(CFA)催生的大鼠痛觉过敏的疗效研究中，化合物JK-02H剂量为20mg/kg和5mg/kg时较空白溶媒均显示优异的抗痛觉过敏疗效；而化合物46仅在剂量为40mg/kg时才显示抗痛觉过敏疗效，在剂量为10mg/kg时几乎没有抗痛觉过敏疗效。
本领域技术人员会理解，本申请所公开的具有手性中心的化合物可以以旋光体和外消旋体的形式存在，并且一些化合物可以显示多种晶型。本发明包括所公开化合物的任何外消旋体、旋光体、多晶型或立体异构体、或其混合物。
具体地，本发明提供了如下优选的化合物：
Figure PCTCN2014090226-appb-000008
Figure PCTCN2014090226-appb-000009
在化合物具有足够碱性以形成酸加成盐的情况下，可以以盐的形式使用所述化合物。可接受的盐是与生理学上可接受的有机酸形成的酸加成盐，如甲苯磺酸盐、甲磺酸盐、乙酸盐、丙酸盐、羟基乙酸盐、丙酮酸盐、草酸盐、苹果酸盐、马来酸盐、富马酸盐、肉桂酸盐、扁桃酸盐、甲磺酸盐、乙磺酸盐、对甲苯磺酸盐、水杨酸盐、柠檬酸盐、丙二酸盐、酒石酸盐、琥珀酸盐、苯甲酸盐、抗坏血酸盐、α-酮戊二酸盐以及α-甘油磷酸盐等，或者是与生理学上可接受的无机酸形成的酸加成盐，如盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐、碳酸氢盐以及碳酸盐等。
本发明的另一方面提供了式I的化合物或其药学上可接受的盐在制备治疗疼痛的药物中的应用。
本发明的化合物可用于治疗各种疼痛，包括但不限于慢性疼痛、神经性疼痛、急性疼痛、癌痛、类风湿性关节炎疼痛、偏头痛、内脏痛等。本发明的化合物可用作在全身麻醉和监控麻醉护理时使用的止痛药物。常常联合使用不同性质的药物，以达到维持麻醉状态(例如记忆缺失、痛觉缺失、肌肉松弛和镇静)所需的平衡作用。联合用药包括吸入麻醉剂、安眠药、抗焦虑药、神经肌肉阻滞剂和阿片样物质。
本发明的化合物可以以不同的剂量和不同的频率来施用，这取决于如受试者的健康、年龄、体重和性别等情况。
本发明的另一方面提供了药物组合物，其包含式I的化合物或其药学上可接受的盐以及一种或多种药学上可接受的载体。
所述载体应被所给药的个体耐受且能将所述化合物递送至受侵害区域。
本申请描述的药物组合物的制剂可通过药学领域已知的任何方法来制备。通常此制备方法包括下述步骤：将活性成分与载体或一种或多种其它助剂组合，如有必要，可将产品包装成希望的单剂量或多剂量单元。除活性成分之外，药物组合物可进一步包含一种或多种其它的药学活性物质， 或该药物组合物可以与一种或多种其它的药学活性物质同时或相继使用。
本发明的药物组合物可以以无菌可注射的水性或油性悬浮液或溶液的形式制备、包装或销售。此悬浮液或溶液可根据已知技术配制，可包含除活性成分之外的其它成分，如分散剂、润湿剂或助悬剂等。此无菌可注射的制剂可使用无毒的稀释剂或溶液(如水或1,3-丁二醇)来制备。其它可接受的稀释剂和溶剂包括但不限于林格氏溶液、等渗氯化钠溶液、单甘油酯或二甘油酯等。
药物组合物的控制释放或持续释放制剂可使用常规技术来制备。活性成分的控制释放可由各种诱导因素如pH、温度、酶、水或其它生理条件或化合物来刺激。
包含本发明化合物的适宜药物组合物可以通过常规的方式给药，例如口服给药、局部给药、非胃肠道途径给药、口腔给药、鼻腔给药、阴道给药或直肠给药或通过吸入给药。为此，本发明的化合物可以通过现有技术中已知的方法被制备成各种形式，例如片剂、胶囊剂、水溶液剂或油溶液剂、混悬剂、乳剂、霜剂、软膏剂、凝胶剂、鼻腔喷雾剂、栓剂、很细的粉末剂或吸入用气雾剂，对于非胃肠道途径给药而言(包括静脉内注射、肌内注射或输液)，可以使用灭菌水或油溶液剂或混悬剂或灭菌乳剂。
本发明的另一方面提供了治疗疼痛的方法，所述方法包括向需要治疗的个体给药改善疼痛有效量的式I的化合物或其药学上可接受的盐或者包含改善疼痛有效量的式I的化合物或其药学上可接受的盐以及药学上可接受的载体的药物组合物。
使用本发明的化合物或其药学上可接受的盐的治疗方法可用于哺乳动物疼痛的预防和治疗，所述哺乳动物可以为人类。
本发明的另一方面提供了通式I的化合物的制备方法，所述方法包括以下步骤：
a)按照以下方案制备4-[(2-R1-3-R2-6-羟基)苯甲酰基]苯甲酸甲酯(中间体3)
Figure PCTCN2014090226-appb-000010
b)按照以下方案制备1-N-叔丁氧羰基-3,4-二氨基吡咯烷(中间体10)
Figure PCTCN2014090226-appb-000011
c)按照以下方案制备1H-吲唑-6-羧酸(中间体13)
Figure PCTCN2014090226-appb-000012
d)按照以下方案制备通式(I)的化合物
Figure PCTCN2014090226-appb-000013
其中X为卤素，优选Cl或Br；且R1、R2、R3和n如上文中所定义。
具体地，本发明提供了制备式I的化合物或其药学上可接受的盐的方法，所述方法包括：
(1)使化合物3
Figure PCTCN2014090226-appb-000014
与化合物14
Figure PCTCN2014090226-appb-000015
在碱的存在下进行亲核取代反应，得到化合物15，
Figure PCTCN2014090226-appb-000016
其中X为氯、溴或碘原子，且R1、R2、R3和n如上文中所定义；
优选地，所述亲核取代反应在有机溶剂中进行，所述有机溶剂选自乙腈、丙腈、二甲基甲酰胺(DMF)、二甲基乙酰胺、1,3-二甲基-2-咪唑啉酮(DMI)、二甲基亚砜(DMSO)、六甲基磷酰三胺(HMPA)、四氢呋喃、二氧六环、丙酮、甲苯、二甲苯、乙醚、丙醚、异丙醚、甲基叔丁基醚、正己烷、环己烷、甲基环己烷和正庚烷，及它们中任意一种或多种的任意比例的组合；
优选地，所述碱选自碳酸钾、碳酸钠、碳酸铯、碳酸钙、氢氧化钾、氢氧化钠、氢氧化锂、氢氧化钙、氢化钠、氢化钾、氢化钙、金属钠、金属钾、甲醇钠、乙醇钠、叔丁醇钾、丁基锂、苯基锂、二异丙氨基锂(LDA)、六甲基二硅胺基锂(LiHMDS)、二甲胺、二乙胺、三乙胺、二异丙基乙胺、哌啶、吡啶、1,8-二氮杂二环[5.4.0]十一碳-7-烯(DBU)、1,5-二氮杂二环[4.3.0]壬-5-烯(DBN)、四丁基氢氧化铵及其组合；
(2)使所述化合物15在碱的存在下进行水解反应，得到化合物16，
Figure PCTCN2014090226-appb-000017
其中R1、R2、R3和n如上文中所定义；
优选地，所述水解反应在有机溶剂中进行，所述有机溶剂选自水、甲醇、乙醇、正丙醇、异丙醇、正丁醇、异丁醇、叔丁醇、四氢呋喃、乙腈、丙腈、DMF、二甲基乙酰胺、DMI、DMSO、HMPA、四氢呋喃、二氧六环和丙酮，及它们中任意一种或多种的任意比例的组合；
优选地，所述碱选自氢氧化钾、氢氧化钠、氢氧化锂、氢氧化钙、氢氧化钡、氢氧化铜、三氯化铝、三氯化硼、三溴化铝、三溴化硼、氰化钠、 氰化钾、碳酸铯、碳酸铜、碘化锂、硼氢化钠、氢化钠、氢化钾、氢化钙及其组合；
(3)使所述化合物16与化合物10
Figure PCTCN2014090226-appb-000018
在缩合剂的存在下进行酰胺化反应，得到化合物17
Figure PCTCN2014090226-appb-000019
其中R1、R2、R3和n如上文中所定义；
优选地，所述酰胺化反应在有机溶剂中进行，所述有机溶剂选自甲苯、二甲苯、氯苯、乙腈、四氢呋喃、二氧六环、二氯甲烷、三氯甲烷、四氯化碳、乙醚、丙醚、异丙醚、甲基叔丁基醚、正己烷、环己烷、甲基环己烷和正庚烷，及它们中任意一种或多种的任意比例的组合；
优选地，所述缩合剂选自N-羟基-7-偶氮苯并三氮唑(HOAt)、1-羟基苯并三氮唑(HOBt)、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU)、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)、双(2-氧代-3-噁唑烷基)次磷酰氯(BOP-Cl)、1H-苯并三唑-1-氧基三(1-吡咯烷基)膦六氟磷酸盐(PyBOP)、1,3-二环己基碳二亚胺(DCC)、N,N'-羰基二咪唑(CDI)、1-乙基-(3-二甲基氨基丙基)碳二亚胺(EDC)、N,N'-二异丙基碳二亚胺(DIC)、4-二甲氨基吡啶(DMAP)及其组合；
(4)使所述化合物17与化合物13
Figure PCTCN2014090226-appb-000020
在缩合剂的存在下进行酰胺化反应，得到化合物18
Figure PCTCN2014090226-appb-000021
其中Boc代表叔丁氧羰基保护基，且R1、R2、R3和n如上文中所定义；
优选地，所述酰胺化反应在有机溶剂中进行，所述有机溶剂选自甲苯、二甲苯、氯苯、乙腈、四氢呋喃、二氧六环、二氯甲烷、三氯甲烷、四氯化碳、乙醚、丙醚、异丙醚、甲基叔丁基醚、正己烷、环己烷、甲基环己烷和正庚烷，及它们中任意一种或多种的任意比例的组合；
优选地，所述缩合剂选自HOAt、HOBt、HATU、HBTU、BOP-Cl、PyBOP、DCC、CDI、EDC、(DIC)、DMAP及其组合；
(5)使所述化合物18在脱保护试剂的存在下进行脱保护反应，得到式I的化合物，并任选地进一步形成其药学上可接受的盐，
优选地，所述脱保护反应在有机溶剂中进行，所述有机溶剂选自甲苯、二甲苯、氯苯、乙腈、四氢呋喃、二氧六环、二氯甲烷、三氯甲烷、四氯化碳、乙醚、丙醚、异丙醚、甲基叔丁基醚、正己烷、环己烷、甲基环己烷和正庚烷，及它们中任意一种或多种的任意比例的组合；
优选地，所述脱保护试剂选自三氟乙酸、盐酸、氢溴酸、氢碘酸、磷酸、硫酸、甲磺酸、对甲苯磺酸、乙酰氯、三氯化铝以及三氟化硼。
本发明还提供了制备式I的化合物的新的中间体，这些中间体为式15、式16、式17或式18的化合物：
Figure PCTCN2014090226-appb-000022
其中Boc、R1、R2、R3和n如上文中所定义。
定义
当在此使用时，术语“类似物”是指在结构上与另一化合物相似，但并不一定是其异构体的化合物。
当在此使用时，术语“有效量”是指足以产生所需效应的量。
当在此使用时，术语“抑制”是指化合物减少或阻止所描述功能的能 力。
当在此使用时，术语“烷基”是指直链或支链烷基基团。
当在此使用时，术语“C1-C6烷基”表示具有1个至6个碳原子的直链或支链烷基。通常C1-C6烷基包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、戊基、己基等。
当在此使用时，术语“烯基”是指包括至少一个双键的烯式不饱和直链或支链烃基。
当在此使用时，术语“C2-C6烯基”表示具有2个至6个碳原子和至少一个双键的烯式不饱和直链或支链烃基。通常C2-C6烯基包括但不限于：乙烯基、1-丙烯基、2-丙烯基、1,3-丁二烯基、1-丁烯基、己烯基、戊烯基等。
当在此使用时，术语“炔基”是指包括至少一个叁键的不饱和直链或支链烃基。
当在此使用时，术语“C2-C6炔基”表示具有2个至6个碳原子和至少一个叁键的不饱和直链或支链烃基。通常C2-C6炔基包括但不限于：乙炔基、1-丙炔基、2-丙炔基、1-丁炔基、2-丁炔基、1-戊炔基等。
当在此使用时，术语“C3-C6环烷基”表示环丙基、环丁基、环戊基、环己基等。
当在此使用时，术语“芳基”是指具有一个或两个芳环的单环或双环C5-C10碳环体系，包括但不限于：苯基、苄基、萘基、四氢萘基、茚满基、茚基等。
当在此使用时，术语“杂芳基”是指具有1个或2个芳环且环中除碳原子外还含有1至3个杂原子的单环或双环体系，其中所述杂原子选自氮、氧和硫。杂芳基的实例包括但不限于：呋喃基、噻吩基、吡啶基等。
当在此使用时，术语“杂环基”是指环中含有碳原子和1至3个杂原子的单环或双环体系，其中所述杂原子选自氮、氧和硫。
通常中间体没有完全进行结构确证，通常用质谱(MS)或核磁(NMR)分析评估其纯度。
当在此使用时，以下缩写具有如下含义：
CDCl3：氘代氯仿
DCM：二氯甲烷
DMF：N,N-二甲基甲酰胺
HATU：2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐
DIPEA：N,N-二异丙基乙胺
DMSO：二甲基亚砜
NMR：核磁共振
现在参考下述的实施例描述本发明。提供这些实施例仅为举例说明，绝不是限制本发明的范围。
实施例中所用的3,4-二氟苯甲醚、3-氨基-4-甲基苯甲酸、3-氟-4-甲氧基苯酚、3,4-二甲氧基苯酚、2-氟-4-甲氧基苯甲醛、间甲苯甲醚、2-丁炔酸购自百灵威科技有限公司，六甘醇、七甘醇单甲醚购自嘉兴博美生物技术有限公司，对甲酰基苯甲酸甲酯购自上海邦成化工有限公司，其余试剂均购自国药集团化学试剂北京有限公司。
实施例
实施例1：(3R,4R)-3-{4-[2,3-二氟-6-(5-羟基-3-氧杂戊烷-1-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-02H)的制备
中间体(1A)
4-[(2,3-二氟-6-甲氧基苯基)羟甲基]苯甲酸甲酯
Figure PCTCN2014090226-appb-000023
向冷却至-78℃的3,4-二氟苯甲醚(20.0g,0.139mol)的无水四氢呋喃(500mL)溶液中加入2.5M的正丁基锂(61.6mL,0.154mol)，继续搅拌2h，加入对甲酰基苯甲酸甲酯(25.2g,0.154mol)的无水四氢呋喃(500mL)溶液，反应混合物在10h内缓慢升至室温。加入蒸馏水(1.0L)淬灭反应，用乙酸乙酯(3×1.0L)提取，混合有机层用无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离得淡黄色固体状中间体(1A)29.4g，收率68.6％。
MASS(ESI+)m/z＝165(M+H)+。
1H NMR(400MHz,CDCl3):3.78(s,3H),3.88(s,3H),4.30(d,1H),6.36(d,1H),6.71(d,1H),7.23(d,1H),7.37(d,2H),7.94(d,2H)。
中间体(2A)
4-[(2,3-二氟-6-甲氧基)苯甲酰基]苯甲酸甲酯
Figure PCTCN2014090226-appb-000024
在室温氮气保护下向氯铬酸吡啶(PCC,26.2g,121.7mmol)的二氯甲烷(500mL)溶液中加入中间体(1A)(25g，81.1mmol)的二氯甲烷(250mL)溶液，反应混合物继续在室温下搅拌4h。过滤，滤液减压浓缩，残留物经硅胶柱分离得淡黄色固体状中间体(2A)20.8g，收率83.7％。
MASS(ESI+)m/z＝309(M+H)+。
1H NMR(400MHz,CDCl3):3.69(s,3H),3.94(s,3H),6.95(d,1H),7.35(d,1H),7.82(d,2H),8.09(d,2H)。
中间体(3A)
4-[(2,3-二氟-6-羟基)苯甲酰基]苯甲酸甲酯
Figure PCTCN2014090226-appb-000025
在-78℃下向中间体(2A)(15g,49.0mmol)的二氯甲烷(500mL)溶液中加入三溴化硼(24.5g,97.8mmol)，混合物搅拌2h，加入蒸馏水(250mL)，分液，水层用二氯甲烷(2×250mL)提取，混合有机层用饱和食盐水(500mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离得淡黄色固体状中间体(3A)13.6g，收率95.0％。
MASS(ESI+)m/z＝307(M+H)+。
1H NMR(400MHz,CDCl3):3.99(s,3H),6.89(d,1H),7.26(d,1H),7.74(d,2H),8.18(d,2H),11.29(s,1H)。
中间体((3R,4R)-4)
(3R,4R)-1-N-苄基-3,4-二羟基吡咯烷-2,5-二酮
Figure PCTCN2014090226-appb-000026
L-(+)-酒石酸(150.1g,1.0mol)的50％甲醇水溶液(200mL)中缓慢加入苄胺(107.2g,1.0mol)。反应混合物在50℃加热搅拌至澄清，减压浓缩。向残留物中加入二甲苯(3L)，反应混合物在油浴中回流分水8h。冷却后减压浓缩，用无水乙醇(2×150mL)带去痕量的二甲苯。向残留物中加入无水乙醇(700mL)，搅拌加热至回流，并回流保温30min。冷却至室温，过滤，滤饼用无水乙醇(3×100mL)洗涤，干燥得白色针状中间体((3R,4R)-4)153.9g，收率69.6％。合并滤液和洗液，浓缩至300mL，加入活性炭(30g)，回流搅拌30min，趁热过滤，滤饼用热乙醇(100mL)洗涤。合并滤液和洗液，静止冷却结晶，过滤，又得到中间体((3R,4R)-4)33.4g(15.1％)。两次共得到中间体((3R,4R)-4)187.3g，合计收率为84.7％。
MASS(ESI+)m/z＝222(M+H)+。
1H NMR(400MHz,DSO-d6):4.38(d,2H),4.53(d,1H),4.58(d,1H),6.29(d,2H),7.24(m,2H),7.27(m,1H),7.33(m,2H)。
中间体((3S,4S)-5)
(3S,4S)-1-N-苄基-3,4-二羟基吡咯烷
Figure PCTCN2014090226-appb-000027
在氮气保护下，向冷却至0℃后的LiAlH4(61.2g,1.6mol)的四氢呋喃(3.6L)溶液中慢慢加入中间体((3R,4R)-4)(132.7g,0.6mol)。反应混合物回流12h，冷却至室温，在冰水浴中滴加乙酸乙酯(144mL)。剧烈搅拌下依次滴入蒸馏水(61.2mL)、5％NaOH(61.2mL)和蒸馏水(183.6mL)。过滤，滤饼用热四氢呋喃(2×1.2L)洗涤。合并滤液和洗液，减压浓缩，残留物经硅胶柱分离纯化得浅黄色油状物，经乙酸乙酯重结晶后得白色固体状中间体((3S,4S)-5)82.3g，收率71.0％。
MASS(ESI+)m/z＝194(M+H)+。
1H NMR(400MHz,DSO-d6):2.30(dd,2H),2.74(dd,2H),3.46(d,1H),3.57(d,1H),3.84(m,2H),4.84(br s,2H),7.20-7.35(m,5H)。
中间体((3S,4S)-6)
(3S,4S)-3,4-二羟基吡咯烷
Figure PCTCN2014090226-appb-000028
将中间体((3S,4S)-5)(77.3g,0.4mol)溶于80％的乙醇水溶液(2.4L)中，加入10％Pd/C(7.0g)，室温通氢(0.07MPa)反应2d。过滤除去催化剂，滤液减压浓缩，残留物用无水乙醇(2×250mL)带除痕量水分得黄色油状中间体((3S,4S)-6)37.5g，收率90.9％。
MASS(ESI+)m/z＝104(M+H)+。
1H NMR(400MHz,DSO-d6):2.60(m,2H),3.02(m,2H),3.83(m,2H),4.81(br s,3H)。
中间体((3S,4S)-7)
(3S,4S)-1-N-叔丁氧羰基-3,4-二羟基吡咯烷
Figure PCTCN2014090226-appb-000029
剧烈搅拌下，向中间体((3S,4S)-6)(30.9g,0.30mol)和碳酸氢钠(218.9g,25.8mol)的50％二氧六环水溶液中滴加二碳酸二叔丁酯(98.2g,0.45mol)。反应混合物在室温下搅拌2h，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得白色固体状中间体((3S,4S)-7)51.6g，收率84.6％。
MASS(ESI+)m/z＝204(M+H)+。
1H NMR(400MHz,DSO-d6):1.39(s,9H),3.11(dd,2H),3.34(dd,2H),3.86(m,2H),5.06(d,2H)。
中间体((3S,4S)-8)
(3S,4S)-1-N-叔丁氧羰基-3,4-二甲基磺酰氧基吡咯烷
Figure PCTCN2014090226-appb-000030
向冷却至0℃后的中间体((3S,4S)-7)(50.8g,0.25mol)的二氯甲烷(1.6L)溶液中加入三乙胺(140mL,1.0mol)和甲基磺酰氯(58mL,0.75mol)。去除冰盐浴后，反应混合物自然升至室温，在室温继续搅拌6h。反应混合物依次 用饱和氯化铵和饱和食盐水洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得白色固体状中间体((3S,4S)-8)84.7g，收率94.3％。
MASS(ESI+)m/z＝360(M+H)+。
1H NMR(400MHz,CDCl3):1.47(s,9H),3.12(s,6H),3.71(m,2H),3.79(d,1H),3.82(d,1H),5.19(br s,2H)。
中间体((3R,4R)-9)
(3R,4R)-1-N-叔丁氧羰基-3,4-二叠氮基吡咯烷
Figure PCTCN2014090226-appb-000031
向中间体((3S,4S)-8)(71.9g,0.20mol)的DMF(1.8L)溶液中加入NaN3(143.0g,2.2mol)。反应混合物在90℃下加热24h，减压蒸干，残留物用乙酸乙酯(900mL)稀释，用蒸馏水(3×180mL)洗涤。水层用乙酸乙酯(180mL)提取，混合有机层用饱和食盐水(90mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得黄色油状中间体((3R,4R)-9)34.4g，收率67.9％。
MASS(ESI+)m/z＝254(M+H)+。
1H NMR(400MHz,CDCl3):1.45(s,9H),3.34(m,2H),3.65(m,2H),3.94(m,2H)。
中间体((3R,4R)-10)
(3R,4R)-1-N-叔丁氧羰基-3,4-二氨基吡咯烷
Figure PCTCN2014090226-appb-000032
将中间体((3R,4R)-9)(30.4g,0.12mol)溶于无水甲醇(500mL)中，加入10％Pd/C(12.8g，12mmol)，室温通氢(0.1MPa)反应18h。过滤除去催化剂，滤液减压浓缩得黄色油状中间体((3R,4R)-10)23.4g，收率96.9％。
MASS(ESI+)m/z＝202(M+H)+。
1H NMR(400MHz,CDCl3):1.48(s,9H),1.64-1.99(m,4H),3.14-3.37(m, 2H),3.38-3.52(m,2H),3.52-3.78(m,2H)。
中间体(11)
3-氨基-4-甲基苯甲酸甲酯
Figure PCTCN2014090226-appb-000033
将3-氨基-4-甲基苯甲酸(60.5g,0.40mol)溶于无水甲醇(1.5L)中，搅拌冷却至5℃。缓慢滴加氯化亚砜(103.6g,0.87mol)，滴完后回流搅拌6h。冷却至室温，反应液减压浓缩，残留物用冰水(1.2L)稀释，加5％的NaHCO3中和至pH7.5。水层用乙酸乙酯(3×600mL)提取，混合有机层用饱和食盐水(2×500mL)洗涤，经无水硫酸钠干燥，过滤，滤液减压浓缩得白色固体状中间体(11)62.7g，收率94.9％。
MP 114-116℃
1H NMR(400MHz,CDCl3):2.19(s,3H),3.73(br s,2H),3.87(s,3H),7.09(m,1H),7.34-7.37(m,2H)。
中间体(12)
1H-吲唑-6-羧酸甲酯
Figure PCTCN2014090226-appb-000034
将中间体(11)(57.8g,0.35mol)溶于冰醋酸(1.5L)中，缓慢滴加NaNO2(24.2g,0.35mol)的蒸馏水(350mL)溶液。反应液在室温搅拌5h，减压浓缩，向残留物中加入蒸馏水(500mL)，用乙酸乙酯(3×1.0L)提取。有机层用蒸馏水(1.5L)和食盐水(1.5L)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得浅黄色固体状中间体(12)49.5g，收率80.8％。
MASS(ESI+)m/z＝177(M+H)+。
1H NMR(400MHz,CDCl3):3.96(s,3H),7.80-7.85(m,2H),8.14(s,1H),8.27(s,1H)。
中间体(13)
1H-吲唑-6-羧酸
Figure PCTCN2014090226-appb-000035
将中间体(12)(44.0g,0.25mol)溶于四氢呋喃(500mL)中，加入2NLiOH水溶液(200mL,0.40mol)，反应液在50℃搅拌4h。冷却至室温，减压蒸除四氢呋喃，残留物加蒸馏水(200mL)稀释，用1N HCl酸化至pH 3.5，加乙酸乙酯(3×500mL)提取，混合有机层用食盐水(500mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得浅黄色固体状中间体(13)34.7g，收率85.6％。
MASS(ESI+)m/z＝163(M+H)+。
1H NMR(400MHz,CD3OD):7.79-7.87(m,2H),8.14(s,1H),8.29(s,1H)。
中间体(15-02H)
4-[2,3-二氟-6-(5-羟基-3-氧杂戊烷-1-氧基)]苯甲酰基苯甲酸甲酯
Figure PCTCN2014090226-appb-000036
将中间体(3A)(6.00g,20.52mmol)、2-(2-氯乙氧基)乙醇(7.67g,61.56mmol)、K2CO3(8.51g,61.56mmol)和KI(0.34g,2.05mmol)溶于DMF(125mL)中，氮气保护下回流过夜。冷却至室温，加入蒸馏水(100mL)，用乙酸乙酯(3×200mL)提取，混合有机层用饱和食盐水(300mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(15-02H)5.17g，收率66.2％。
MASS(ESI+)m/z＝381(M+H)+。
中间体(16-02H)
4-[2,3-二氟-6-(5-羟基-3-氧杂戊烷-1-氧基)]苯甲酰基苯甲酸
Figure PCTCN2014090226-appb-000037
将中间体(15-02H)(5.00g,13.15mmol)溶于四氢呋喃(100mL)中，加入2N LiOH水溶液(42mL,21.00mmol)，反应液在50℃搅拌4h。冷却至室温，减压蒸除四氢呋喃，残留物加蒸馏水(40mL)稀释，用1N HCl酸化至pH 3.5，加乙酸乙酯(3×100mL)提取，混合有机层用食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得浅黄色固体状中间体(16-02H)4.28g，收率88.9％。
MASS(ESI+)m/z＝367(M+H)+。
中间体(17-02H)
(3R,4R)-3-氨基-4-{4-[2,3-二氟-6-(5-羟基-3-氧杂戊烷-1-氧基)苯甲酰基]}苯 甲酰氨基吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000038
向冷却至0℃的中间体(16-02H)(4.00g,10.92mmol)、中间体((3R,4R)-10)(4.40g,21.84mmol)和DIPEA(2.82g,21.84mmol)的DMF(50mL)溶液中加入HATU(4.57g,12.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水(100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(17-02H)3.87g，收率64.5％。
MASS(ESI+)m/z＝550(M+H)+。
中间体(18-02H)
(3R,4R)-3-{4-[2,3-二氟-6-(5-羟基-3-氧杂戊烷-1-氧基)]苯甲酰基}苯甲酰氨 基-4-(1H-吲唑-6-甲酰氨基)吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000039
向冷却至0℃的中间体(17-02H)(3.50g,6.37mmol)、中间体(13)(1.14g, 7.01mmol)和DIPEA(2.47g,19.11mmol)的DMF(50mL)溶液中加入HATU(2.67g,7.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水(100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(18-02H)3.76g，收率85.1％。
MASS(ESI+)m/z＝694(M+H)+。
产品(JK-02H)
(3R,4R)-3-{4-[2,3-二氟-6-(5-羟基-3-氧杂戊烷-1-氧基)]苯甲酰基}苯甲酰氨 基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐
Figure PCTCN2014090226-appb-000040
向冷却至0℃的中间体(18-02H)(3.50g,5.05mmol)的乙酸乙酯(100mL)溶液中加入4mol/L的氯化氢乙酸乙酯溶液(100mL)，反应混合物在室温搅拌2h。过滤，滤饼用乙酸乙酯洗涤，真空减压干燥得浅黄色固体状产品(JK-02H)2.75g，收率86.4％。
MASS(ESI+)m/z＝594(M+H)+。
实施例2：(3R,4R)-3-{4-[2,3-二氟-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-03M)的制备
中间体(15-03M)
4-{[2,3-二氟-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲酸甲酯
Figure PCTCN2014090226-appb-000041
2,5,8-三氧杂癸烷基-10-对甲苯磺酸酯
冰盐浴冷却下向三甘醇单甲醚(49.2g,0.30mol)和吡啶(35.5g,0.45mol) 的混合溶液中滴加对甲苯磺酰氯(85.7g,0.45mol)的二氯甲烷(125mL)溶液，控制滴加速度，使反应溶液低于20℃，室温搅拌过夜。加入蒸馏水(100mL)，用6N的NaOH溶液将反应液pH调至4.0，分液，有机层用蒸馏水(2×50mL)洗涤。向有机层中加入蒸馏水(100mL)，用6N的HCl溶液将pH调至1.5，分液，有机层用蒸馏水(2×50mL)洗涤。有机层用无水硫酸钠干燥，过滤，减压蒸除溶剂得浅黄色油状物74.4g，收率78.0％。
1H NMR(400MHz,CDCl3):2.46(s,3H),3.38(s,3H),3.54(t,2H),3.62(m,6H),3.70(t,2H),4.17(t,2H),7.35(d,2H),7.81(d,2H)。
10-溴-2,5,8-三氧杂癸烷
冰盐浴冷却下向2,5,8-三氧杂癸烷基-10-对甲苯磺酸酯(39.8g,0.13mol)的DMF(40mL)溶液中缓慢加入溴化锂(16.3g,0.19mol)，控制加入速度，使反应溶液低于20℃，室温剧烈搅拌过夜。加入乙酸乙酯(125mL)，搅拌1h后过滤，滤液用蒸馏水(2×100mL)洗涤。有机层用无水硫酸钠干燥，过滤，滤液减压浓缩，残留物减压蒸馏得浅黄色油状物26.8g，收率90.8％。
1H NMR(400MHz,CDCl3):3.34(s,3H),3.42(t,2H),3.49-3.53(m,2H),3.59-3.66(m,6H),3.77(t,2H)。
4-{[2,3-二氟-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲酸甲酯
将中间体(3A)(6.00g,20.52mmol)、10-溴-2,5,8-三氧杂癸烷(13.98g,61.56mmol)、K2CO3(8.51g,61.56mmol)和KI(0.34g,2.05mmol)溶于DMF(125mL)中，氮气保护下回流过夜。冷却至室温，加入蒸馏水(100mL)，用乙酸乙酯(3×200mL)提取，混合有机层用饱和食盐水(300mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(15-03M)6.24g，收率69.4％。
MASS(ESI+)m/z＝439(M+H)+。
中间体(16-03M)
4-{[2,3-二氟-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲酸
Figure PCTCN2014090226-appb-000042
将中间体(15-03M)(6.00g,13.69mmol)溶于四氢呋喃(100mL)中，加入2N LiOH水溶液(44mL,22.00mmol)，反应液在50℃搅拌4h。冷却至室温，减压蒸除四氢呋喃，残留物加蒸馏水(40mL)稀释，用1N HCl酸化至pH 3.5，加乙酸乙酯(3×100mL)提取，混合有机层用食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得浅黄色固体状中间体(16-03M)5.32g，收率91.6％。
MASS(ESI+)m/z＝425(M+H)+。
中间体(17-03M)
(3R,4R)-3-氨基-4-{4-[2,3-二氟-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯 甲酰氨基吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000043
向冷却至0℃的中间体(16-03M)(4.63g,10.92mmol)、中间体((3R,4R)-10)(4.40g,21.84mmol)和DIPEA(2.82g,21.84mmol)的DMF(50mL)溶液中加入HATU(4.57g,12.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水(100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(17-03M)4.88g，收率73.5％。
MASS(ESI+)m/z＝608(M+H)+。
中间体(18-03M)
(3R,4R)-3-{4-[2,3-二氟-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲酰氨基 -4-(1H-吲唑-6-甲酰氨基)吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000044
向冷却至0℃的中间体(17-03M)(3.87g,6.37mmol)、中间体(13)(1.14g,7.01mmol)和DIPEA(2.47g,19.11mmol)的DMF(50mL)溶液中加入HATU(2.67g,7.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水(100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(18-03M)4.27g，收率89.2％。
MASS(ESI+)m/z＝752(M+H)+。
产品(JK-03M)
(3R,4R)-3-{4-[2,3-二氟-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲酰氨基 -4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐
Figure PCTCN2014090226-appb-000045
向冷却至0℃的中间体(18-03M)(4.00g,5.32mmol)的乙酸乙酯(100mL)溶液中加入4mol/L的氯化氢乙酸乙酯溶液(100mL)，反应混合物在室温搅拌2h。过滤，滤饼用乙酸乙酯洗涤，真空减压干燥得浅黄色固体状产品(JK-03M)3.07g，收率83.9％。
MASS(ESI+)m/z＝652(M+H)+。
实施例3：(3R,4R)-3-{4-[2,3-二氟-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-06H)的制备
中间体(15-06H)
4-{[2,3-二氟-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)]苯甲酰基}苯甲 酸甲酯
Figure PCTCN2014090226-appb-000046
17-羟基-3,6,9,12,15-五氧杂十七烷基-1-对甲苯磺酸酯
冰盐浴冷却下向六甘醇(127.0g,0.45mol)和吡啶(35.5g,0.45mol)的混合溶液中滴加对甲苯磺酰氯(85.7g,0.45mol)的二氯甲烷(125mL)溶液，控制滴加速度，使反应溶液低于20℃，室温搅拌过夜。加入蒸馏水(100mL)，用6N的NaOH溶液将反应液pH调至4.0，分液，有机层用蒸馏水(2×50mL)洗涤。向有机层中加入蒸馏水(100mL)，用6N的HCl溶液将pH调至1.5，分液，有机层用蒸馏水(2×50mL)洗涤。有机层用无水硫酸钠干燥，过滤，减压蒸除溶剂，残留物经硅胶柱分离得浅黄色油状物75.4g，收率38.4％。
1H NMR(400MHz,CDCl3):2.37(s,3H),2.90(s,1H),3.50-3.62(m,22H),4.08(t,2H),7.27(d,2H),7.72(d,2H)。
17-溴-3,6,9,12,15-五氧杂十七烷-1-醇
冰盐浴冷却下向17-羟基-3,6,9,12,15-五氧杂十七烷基-1-对甲苯磺酸酯(56.7g,0.13mol)的DMF(40mL)溶液中缓慢加入溴化锂(16.3g,0.19mol)，控制加入速度，使反应溶液低于20℃，室温剧烈搅拌过夜。加入乙酸乙酯(125mL)，搅拌1h后过滤，滤液用蒸馏水(2×100mL)洗涤。有机层用无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离得浅黄色油状物34.2g，收率76.2％。
1H NMR(400MHz,CDCl3):3.15(s,1H),3.39(t,2H),3.53-3.66(m,20H),3.73(t,2H)。
4-{[2,3-二氟-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)]苯甲酰基}苯甲 酸甲酯
将中间体(3A)(6.00g,20.52mmol)、17-溴-3,6,9,12,15-五氧杂十七烷-1-醇(21.25g,61.56mmol)、K2CO3(8.51g,61.56mmol)和KI(0.34g,2.05mmol)溶于DMF(125mL)中，氮气保护下回流过夜。冷却至室温，加入蒸馏水(100mL)，用乙酸乙酯(3×200mL)提取，混合有机层用饱和食盐水(300mL)洗涤， 无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(15-06H)7.16g，收率62.7％。
MASS(ESI+)m/z＝557(M+H)+。
中间体(16-06H)
4-{[2,3-二氟-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)]苯甲酰基}苯甲 酸
Figure PCTCN2014090226-appb-000047
将中间体(15-06H)(7.00g,12.58mmol)溶于四氢呋喃(100mL)中，加入2N LiOH水溶液(40mL,20.00mmol)，反应液在50℃搅拌4h。冷却至室温，减压蒸除四氢呋喃，残留物加蒸馏水(40mL)稀释，用1N HCl酸化至pH 3.5，加乙酸乙酯(3×100mL)提取，混合有机层用食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得浅黄色固体状中间体(16-06H)5.95g，收率87.2％。
MASS(ESI+)m/z＝543(M+H)+。
中间体(17-06H)
(3R,4R)-3-氨基-4-{4-[2,3-二氟-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)] 苯甲酰基}苯甲酰氨基吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000048
向冷却至0℃的中间体(16-06H)(5.92g,10.92mmol)、中间体((3R,4R)-10)(4.40g,21.84mmol)和DIPEA(2.82g,21.84mmol)的DMF(50mL)溶液中加入HATU(4.57g,12.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水(100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶 柱分离纯化得浅黄色固体状中间体(17-06H)4.86g，收率61.3％。
MASS(ESI+)m/z＝726(M+H)+。
中间体(18-06H)
(3R,4R)-3-{4-[2,3-二氟-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)]苯甲 酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000049
向冷却至0℃的中间体(17-06H)(4.62g,6.37mmol)、中间体(13)(1.14g,7.01mmol)和DIPEA(2.47g,19.11mmol)的DMF(50mL)溶液中加入HATU(2.67g,7.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水(100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(18-02H)4.53g，收率81.8％。
MASS(ESI+)m/z＝870(M+H)+。
产品(JK-06H)
(3R,4R)-3-{4-[2,3-二氟-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)]苯甲 酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐
Figure PCTCN2014090226-appb-000050
向冷却至0℃的中间体(18-06H)(4.50g,5.17mmol)的乙酸乙酯(100mL)溶液中加入4mol/L的氯化氢乙酸乙酯溶液(100mL)，反应混合物在室温搅拌2h。过滤，滤饼用乙酸乙酯洗涤，真空减压干燥得浅黄色固体状产品(JK-06H)3.32g，收率79.6％。
MASS(ESI+)m/z＝770(M+H)+。
实施例4：(3R,4R)-3-{4-[2,3-二氟-6-(2,5,8,11,14,17,20-七氧杂二十二烷-22-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-07M)的制备
中间体(15-07M)
4-{[2,3-二氟-6-(2,5,8,11,14,17,20-七氧杂二十二烷-22-氧基)]苯甲酰基}苯甲 酸甲酯
Figure PCTCN2014090226-appb-000051
22-氯-2,5,8,11,14,17,20-七氧杂二十二烷
在氮气保护下向七甘醇单甲醚(24.5g,72.0mmol)和吡啶(5.70g,72.0mmol)的CHCl3(60mL)溶液中缓慢滴入氯化亚砜(10.95g,92.0mmol)的CHCl3(15mL)溶液，反应液回流搅拌3h。加入蒸馏水(300mL)，分液，有机层用无水硫酸钠干燥，减压浓缩得黄色油状物23.6g，收率91.3％。产品无须纯化，直接用于下步反应。
4-{[2,3-二氟-6-(2,5,8,11,14,17,20-七氧杂二十二烷-22-氧基)]苯甲酰基}苯甲 酸甲酯
将中间体(3A)(6.00g,20.52mmol)、22-氯-2,5,8,11,14,17,20-七氧杂二十二烷(22.09g,61.56mmol)、K2CO3(8.51g,61.56mmol)和KI(0.34g,2.05mmol)溶于DMF(125mL)中，氮气保护下回流过夜。冷却至室温，加入蒸馏水(100mL)，用乙酸乙酯(3×200mL)提取，混合有机层用饱和食盐水(300mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(15-07M)7.45g，收率59.1％。
MASS(ESI+)m/z＝615(M+H)+。
中间体(16-07M)
4-{[2,3-二氟-6-(2,5,8,11,14,17,20-七氧杂二十二烷-22-氧基)]苯甲酰基}苯甲 酸
Figure PCTCN2014090226-appb-000052
将中间体(15-07M)(7.00g,11.39mmol)溶于四氢呋喃(100mL)中，加入2N LiOH水溶液(36mL,18.00mmol)，反应液在50℃搅拌4h。冷却至室温，减压蒸除四氢呋喃，残留物加蒸馏水(40mL)稀释，用1N HCl酸化至pH 3.5，加乙酸乙酯(3×100mL)提取，混合有机层用食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得浅黄色固体状中间体(16-07M)6.14g，收率89.8％。
MASS(ESI+)m/z＝601(M+H)+。
中间体(17-07M)
(3R,4R)-3-氨基-4-{4-[2,3-二氟-6-(2,5,8,11,14,17,20-七氧杂二十二烷-22-氧 基)]苯甲酰基}苯甲酰氨基吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000053
向冷却至0℃的中间体(16-07M)(6.56g,10.92mmol)、中间体((3R,4R)-10)(4.40g,21.84mmol)和DIPEA(2.82g,21.84mmol)的DMF(50mL)溶液中加入HATU(4.57g,12.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水(100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(17-07M)5.93g，收率69.3％。
MASS(ESI+)m/z＝784(M+H)+。
中间体(18-07M)
(3R,4R)-3-{4-[2,3-二氟-6-(2,5,8,11,14,17,20-七氧杂二十二烷-22-氧基)]苯甲 酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000054
向冷却至0℃的中间体(17-07M)(4.99g,6.37mmol)、中间体(13)(1.14g,7.01mmol)和DIPEA(2.47g,19.11mmol)的DMF(50mL)溶液中加入HATU(2.67g,7.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水(100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(18-07M)5.17g，收率87.4％。
MASS(ESI+)m/z＝928(M+H)+。
产品(JK-07M)
(3R,4R)-3-{4-[2,3-二氟-6-(2,5,8,11,14,17,20-七氧杂二十二烷-22-氧基)]苯甲 酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐
Figure PCTCN2014090226-appb-000055
向冷却至0℃的中间体(18-07M)(4.69g,5.05mmol)的乙酸乙酯(100mL)溶液中加入4mol/L的氯化氢乙酸乙酯溶液(100mL)，反应混合物在室温搅拌2h。过滤，滤饼用乙酸乙酯洗涤，真空减压干燥得浅黄色固体状产品(JK-07M)3.61g，收率82.7％。
MASS(ESI+)m/z＝828(M+H)+。
实施例5：(3R,4R)-3-{4-[2,3-二氟-6-(4,7-二氧杂壬烷-9-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-02P)的制备
中间体(15-02P)
4-{[2,3-二氟-6-(3,6-二氧杂壬烷-1-氧基)]苯甲酰基}苯甲酸甲酯
Figure PCTCN2014090226-appb-000056
3,6-二氧杂壬烷-1-醇
在冰水浴冷却下向二甘醇(424.5g,4.0mol)的四氢呋喃(2.5L)溶液中加入NaH(60％,25.0g,0.63mol)，随后滴入1-溴丙烷(24.6g,0.20mol)，混合物回流12h。减压蒸除溶剂，向残余物中加入蒸馏水(2.5L)，用乙酸乙酯(3×2.0L)，混合有机层用无水硫酸钠干燥，减压蒸除溶剂得无色油状物23.2g，收率78.3％。产品无须纯化，直接用于下步反应。
3,6-二氧杂壬烷基-1-对甲苯磺酸酯
冰盐浴冷却下向3,6-二氧杂壬烷-1-醇(44.5g,0.30mol)和吡啶(35.5g,0.45mol)的混合溶液中滴加对甲苯磺酰氯(85.7g,0.45mol)的二氯甲烷(125mL)溶液，控制滴加速度，使反应溶液低于20℃，室温搅拌过夜。加入蒸馏水(100mL)，用6N的NaOH溶液将反应液pH调至4.0，分液，有机层用蒸馏水(2×50mL)洗涤。向有机层中加入蒸馏水(100mL)，用6N的HCl溶液将pH调至1.5，分液，有机层用蒸馏水(2×50mL)洗涤。有机层用无水硫酸钠干燥，过滤，减压蒸除溶剂得浅黄色油状物61.5g，收率67.8％。
1H NMR(400MHz,CDCl3):0.93(s,3H),1.59(m,2H),2.45(s,3H),3.45(m,2H),3.51(t,2H),3.57(m,2H),3.67-3.73(m,2H),4.16(t,2H),7.34(d,2H),7.80(d,2H)。
1-溴-3,6-二氧杂壬烷
冰盐浴冷却下向3,6-二氧杂壬烷基-1-对甲苯磺酸酯(39.3g,0.13mol)的DMF(40mL)溶液中缓慢加入溴化锂(16.3g,0.19mol)，控制加入速度，使反应溶液低于20℃，室温剧烈搅拌过夜。加入乙酸乙酯(125mL)，搅拌1h后过滤，滤液用蒸馏水(2×100mL)洗涤。有机层用无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离得无色油状物24.3g，收率88.5％。
1H NMR(400MHz,CDCl3):0.92(t,3H),1.61(m,2H),3.43(m,2H),3.47(m,2H),3.59(m,2H),3.67(m,2H),3.82(t,2H)。
4-{[2,3-二氟-6-(3,6-二氧杂壬烷-1-氧基)]苯甲酰基}苯甲酸甲酯
将中间体(3A)(6.00g,20.52mmol)、1-溴-3,6-二氧杂壬烷(13.00g,61.56 mmol)、K2CO3(8.51g,61.56mmol)和KI(0.34g,2.05mmol)溶于DMF(125mL)中，氮气保护下回流过夜。冷却至室温，加入蒸馏水(100mL)，用乙酸乙酯(3×200mL)提取，混合有机层用饱和食盐水(300mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(15-02P)5.92g，收率68.3％。
MASS(ESI+)m/z＝423(M+H)+。
中间体(16-02P)
4-{[2,3-二氟-6-(3,6-二氧杂壬烷-1-氧基)]苯甲酰基}苯甲酸
Figure PCTCN2014090226-appb-000057
将中间体(15-02P)(5.00g,11.84mmol)溶于四氢呋喃(100mL)中，加入2N LiOH水溶液(38mL,19.00mmol)，反应液在50℃搅拌4h。冷却至室温，减压蒸除四氢呋喃，残留物加蒸馏水(40mL)稀释，用1N HCl酸化至pH 3.5，加乙酸乙酯(3×100mL)提取，混合有机层用食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得浅黄色固体状中间体(16-02P)4.49g，收率92.8％。
MASS(ESI+)m/z＝409(M+H)+。
中间体(17-02P)
(3R,4R)-3-氨基-4-{4-[2,3-二氟-6-(3,6-二氧杂壬烷-1-氧基)]苯甲酰基}苯甲酰 氨基吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000058
向冷却至0℃的中间体(16-02P)(4.46g,10.92mmol)、中间体((3R,4R)-10)(4.40g,21.84mmol)和DIPEA(2.82g,21.84mmol)的DMF(50mL)溶液中加入HATU(4.57g,12.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水 (100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(17-02P)4.76g，收率73.7％。
MASS(ESI+)m/z＝592(M+H)+。
中间体(18-02P)
(3R,4R)-3-{4-[2,3-二氟-6-(3,6-二氧杂壬烷-1-氧基)]苯甲酰基}苯甲酰氨基 -4-(1H-吲唑-6-甲酰氨基)吡咯烷-1-甲酸叔丁酯
Figure PCTCN2014090226-appb-000059
向冷却至0℃的中间体(17-02P)(3.77g,6.37mmol)、中间体(13)(1.14g,7.01mmol)和DIPEA(2.47g,19.11mmol)的DMF(50mL)溶液中加入HATU(2.67g,7.01mmol)。反应混合物在室温下搅拌过夜，加入蒸馏水(100mL)，用乙酸乙酯(3×100mL)提取，混合有机层用饱和食盐水(150mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物经硅胶柱分离纯化得浅黄色固体状中间体(18-02H)4.13g，收率88.1％。
MASS(ESI+)m/z＝736(M+H)+。
产品(JK-02P)
(3R,4R)-3-{4-[2,3-二氟-6-(3,6-二氧杂壬烷-1-氧基)]苯甲酰基}苯甲酰氨基 -4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐
Figure PCTCN2014090226-appb-000060
向冷却至0℃的中间体(18-02P)(3.50g,4.76mmol)的乙酸乙酯(100mL)溶液中加入4mol/L的氯化氢乙酸乙酯溶液(100mL)，反应混合物在室温搅拌2h。过滤，滤饼用乙酸乙酯洗涤，真空减压干燥得浅黄色固体状产品(JK-02H)2.86g，收率89.4％。
MASS(ESI+)m/z＝636(M+H)+。
实施例6：(3R,4R)-3-{4-[2-氟-3-甲氧基-6-(5-羟基-3-氧杂戊烷-1-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-M02H)的制备
中间体(1B)
4-[(2-氟-3-甲氧基-6-苄氧基苯基)羟甲基]苯甲酸甲酯
Figure PCTCN2014090226-appb-000061
1-甲氧基-2-氟-4-苄氧基苯
将3-氟-4-甲氧基苯酚(7.5g,52.8mmol)和碳酸铯(34.5g,105.8mmol)溶于乙腈(400mL)中，搅拌下缓慢滴入溴化苄(18.1g,105.8mmol)。反应混合物回流搅拌5h，减压浓缩。残余物溶于乙醚(2.0L)中，加入蒸馏水(3×300mL)洗涤，有机相用无水硫酸钠干燥，减压浓缩，残留物经硅胶柱分离得淡黄色油状物10.8g，收率88.1％。
MASS(ESI+)m/z＝233(M+H)+。
1H NMR(400MHz,CDCl3):3.76(s,3H),5.01(s,2H),6.83(d,1H),6.91(d,1H),7.03(s,1H),7.31(m,1H),7.37(m,2H),7.42(m,2H)。
4-[(2-氟-3-甲氧基-6-苄氧基苯基)羟甲基]苯甲酸甲酯
采用实施例1的方法，用1-甲氧基-2-氟-4-苄氧基苯代替3,4-二氟苯甲醚作为原料合成标题化合物。
MASS(ESI+)m/z＝397(M+H)+。
1H NMR(400MHz,CDCl3):3.76(s,3H),3.88(s,3H),4.29(d,1H),5.01(s,2H),6.36(d,1H),6.83(d,1H),6.91(d,1H),7.31(m,1H),7.37(m,2H),7.42(m,2H),7.47(d,2H),7.95(d,2H)。
中间体(2B)
4-[(2-氟-3-甲氧基-6-苄氧基)苯甲酰基]苯甲酸甲酯
Figure PCTCN2014090226-appb-000062
采用实施例1的方法，用中间体(1B)代替中间体(1A)作为原料合成标题化合物。
MASS(ESI+)m/z＝395(M+H)+。
1H NMR(400MHz,CDCl3):3.76(s,3H),3.94(s,3H),5.01(s,2H),6.93(d,1H),7.27(d,1H),7.31(m,1H),7.37(m,2H),7.42(m,2H),7.82(d,2H),8.09(d,2H)。
中间体(3B)
4-[(2-氟-3-甲氧基-6-羟基)苯甲酰基]苯甲酸甲酯
Figure PCTCN2014090226-appb-000063
将中间体(2B)(15.0g,38.0mmol)和20％Pd(OH)2/C(3.0g)溶于THF-MeOH(1:1,250mL)中，用氢气置换空气三次，通氢反应4h。过滤，滤液减压浓缩，残留物经硅胶柱分离得淡黄色固体状中间体(3B)10.6g，收率91.7％。
MASS(ESI+)m/z＝305(M+H)+。
1H NMR(400MHz,CDCl3):3.78(s,3H),3.98(s,3H),6.89(d,1H),7.25(d,1H),7.74(d,2H),8.17(d,2H),11.30(s,1H)。
产品(JK-M02H)
(3R,4R)-3-{4-[2-氟-3-甲氧基-6-(5-羟基-3-氧杂戊烷-1-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐
Figure PCTCN2014090226-appb-000064
采用实施例1的方法，用中间体(3B)代替中间体(3A)作为原料合成标题化合物。
MASS(ESI+)m/z＝606(M+H)+。
实施例7：(3R,4R)-3-{4-[2-氟-3-甲氧基-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-M03M)的制备
Figure PCTCN2014090226-appb-000065
采用实施例2的方法，用中间体(3B)代替中间体(3A)作为原料合成标题化合物。
MASS(ESI+)m/z＝664(M+H)+。
实施例8：(3R,4R)-3-{4-[2-氟-3-甲氧基-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-M06H)的制备
Figure PCTCN2014090226-appb-000066
采用实施例3的方法，用中间体(3B)代替中间体(3A)作为原料合成标题化合物。
MASS(ESI+)m/z＝782(M+H)+。
实施例9：(3R,4R)-3-{4-[2-氟-3-甲氧基-6-(2,5,8,11,14,17,20-七氧杂二十二烷-22-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-M07M)的制备
Figure PCTCN2014090226-appb-000067
采用实施例4的方法，用中间体(3B)代替中间体(3A)作为原料合成标题 化合物。
MASS(ESI+)m/z＝840(M+H)+。
实施例10：(3R,4R)-3-{4-[2-氟-3-甲氧基-6-(4,7-二氧杂壬烷-9-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-M02P)的制备
Figure PCTCN2014090226-appb-000068
采用实施例5的方法，用中间体(3B)代替中间体(3A)作为原料合成标题化合物。
MASS(ESI+)m/z＝648(M+H)+。
实施例11：(3R,4R)-3-{4-[2,3-二甲氧基-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-MM06H)的制备
中间体(3C)
4-[(2,3-二甲氧基-6-羟基)苯甲酰基]苯甲酸甲酯
Figure PCTCN2014090226-appb-000069
采用实施例6的方法，用3,4-二甲氧基苯酚代替3-氟-4-甲氧基苯酚作为原料合成标题化合物。
MASS(ESI+)m/z＝317(M+H)+。
1H NMR(400MHz,CDCl3):3.79(s,6H),3.98(s,3H),6.36(d,1H),6.72(d,1H),7.74(d,2H),8.17(d,2H),11.29(s,1H)。
产品(JK-MM06H)
(3R,4R)-3-{4-[2,3-二甲氧基-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1-氧基)] 苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐
Figure PCTCN2014090226-appb-000070
采用实施例3的方法，用中间体(3C)代替中间体(3A)作为原料合成标题化合物。
MASS(ESI+)m/z＝794(M+H)+。
实施例12：(3R,4R)-3-{4-[2,3-二甲氧基-6-(5-羟基-3-氧杂戊烷-1-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-MM02H)的制备
Figure PCTCN2014090226-appb-000071
采用实施例1的方法，用中间体(3C)代替中间体(3A)作为原料合成标题化合物。
MASS(ESI+)m/z＝602(M+H)+。
实施例13：(3R,4R)-3-{4-[2-氟-3-乙烯基-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-E03M)的制备
中间体(3D)
4-[(2-氟-3-乙烯基-6-羟基)苯甲酰基]苯甲酸甲酯
Figure PCTCN2014090226-appb-000072
2-氟-4-甲氧基苯乙烯
向甲基三苯基溴化膦(35.7g,0.10mol)的四氢呋喃(850mL)溶液中滴入正丁基锂(2.5M的四氢呋喃溶液,40mL)，滴完后继续搅拌15min，得到黄色溶液。分批加入2-氟-4-甲氧基苯甲醛(15.4g,0.10mol)，溶液颜色逐渐变 浅。加完后继续搅拌2h，加入饱和氯化铵溶液，反应混合物用二氯甲烷提取，有机相用无水硫酸钠干燥，过滤，减压浓缩，残留物经硅胶柱分离得淡黄色油状物11.8g，收率77.5％。
MASS(ESI+)m/z＝153(M+H)+。
1H NMR(400MHz,CDCl3):3.79(s,3H),5.24(dd,1H),5.58(dd,1H),6.84(dd,1H),6.98(dd,1H),7.09(d,1H),7.47(d,1H)。
4-[(2-氟-3-乙烯基-6-羟基)苯甲酰基]苯甲酸甲酯
采用实施例1的方法，用2-氟-4-甲氧基苯乙烯代替3,4-二氟苯甲醚作为原料合成标题化合物。
MASS(ESI+)m/z＝301(M+H)+。
1H NMR(400MHz,CDCl3):3.98(s,3H),5.24(dd,1H),5.58(dd,1H),6.78(d,1H),6.98(dd,1H),7.40(d,1H),7.73(d,2H),8.15(d,2H),11.29(s,1H)。
产品(JK-E03M)
(3R,4R)-3-{4-[2-氟-3-乙烯基-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲 酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-E03M)
Figure PCTCN2014090226-appb-000073
采用实施例2的方法，用中间体(3D)代替中间体(3A)作为原料合成标题化合物。
MASS(ESI+)m/z＝660(M+H)+。
实施例14：(3R,4R)-3-{4-[2-氟-3-丙炔基-6-(2,5,8-三氧杂癸烷-10-氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐(JK-PM06H)的制备
中间体(3E)
4-{[2-甲基-3-(1-丙炔基)-6-羟基]苯甲酰基}苯甲酸甲酯
Figure PCTCN2014090226-appb-000074
2-甲基-4-甲氧基碘化苯
将间甲苯甲醚(24.4g,0.20mol)溶于乙腈(800mL)中，加入N-碘代丁二酰亚胺(67.5g,0.30mol)。将反应液升温至82℃，保温搅拌2h。减压蒸除溶剂，加入乙醚(800mL)，依次用亚硫酸氢钠水溶液和蒸馏水洗涤，有机相用无水硫酸钠干燥，过滤，减压浓缩，残留物经硅胶柱分离得淡黄色油状物43.2g，收率87.1％。
MASS(ESI+)m/z＝249(M+H)+。
1H NMR(400MHz,CDCl3):2.39(s,3H),3.77(s,3H),6.47(dd,1H),6.81(d,1H),7.65(d,1H)。
1-(2-甲基-4-甲氧基)苯基-1-丙炔
将2-甲基-4-甲氧基碘化苯(24.8g,0.10mol)、2-丁炔酸(11.7g,0.14mol)、碘化亚铜(0.38g,2.0mmol)、三苯基膦(1.05g,4.0mmol)和碳酸钾(41.5g,0.30mol)溶于二甲基亚砜(1.0L)中，充氮气保护。将反应液升温至100℃，保温搅拌24h。冷却至室温，用蒸馏水(3.0L)稀释，加乙酸乙酯提取(3×1.0L)，有机相用无水硫酸钠干燥，过滤，减压浓缩，残留物经硅胶柱分离得淡黄色油状物13.1g，收率81.8％。
MASS(ESI+)m/z＝161(M+H)+。
1H NMR(400MHz,CDCl3):2.05(s,3H),2.34(s,3H),3.82(s,3H),6.84(dd,1H),6.98(d,1H),7.35(d,1H)。
4-{[2-甲基-3-(1-丙炔基)-6-羟基]苯甲酰基}苯甲酸甲酯
采用实施例1的方法，用1-(2-甲基-4-甲氧基)苯基-1-丙炔代替3,4-二氟苯甲醚作为原料合成标题化合物。
MASS(ESI+)m/z＝309(M+H)+。
1H NMR(400MHz,CDCl3):2.05(s,3H),2.34(s,3H),3.98(s,3H),6.78(d,1H),7.40(d,1H),7.73(d,2H),8.15(d,2H),11.30(s,1H)。
产品(JK-PM06H)
(3R,4R)-3-{4-[2-甲基-3-(1-丙炔基)-6-(17-羟基-3,6,9,12,15-五氧杂十七烷-1- 氧基)]苯甲酰基}苯甲酰氨基-4-(1H-吲唑-6-甲酰氨基)吡咯烷盐酸盐
Figure PCTCN2014090226-appb-000075
采用实施例3的方法，用中间体(3E)代替中间体(3A)作为原料合成标题化合物。
MASS(ESI+)m/z＝786(M+H)+。
实施例15：化合物的水溶解度实验
在室温条件下，在10mL具玻璃磨口塞的量筒中加入约0.1g的待测样品(固体物质必须研磨成粉末)，逐渐增加水的体积，在每次加入0.5mL水后，搅拌混合10min，用肉眼检查是否存在有样品不溶。如果加水10mL后，样品或样品中某些部分仍不溶时，试验在100mL量筒中继续进行。各化合物在室温下的溶解度如下表1所示：
表1实验化合物的溶解度
Figure PCTCN2014090226-appb-000076
从表1数据可以看出，增加低分子聚乙二醇片段后的化合物水溶性较原化合物有较大幅度提高，有些化合物提高了近10倍。
实施例16：化合物JK-02H、化合物46以及JK-02A对弗氏完全佐剂(CFA)催生的大鼠痛觉过敏的疗效研究
1.研究设计
化合物46、JK-02A的制备参照美国专利申请US2008/0176920，化合物JK-02H的制备参照实施例1。对同批动物进行单剂量给药后再洗脱的重复疗效研究。动物14天的生存研究包括两个阶段：在注射CFA后第7天进行单次低剂量治疗评估，第14天进行单次高剂量治疗评估。
1.1动物
1.1.1种属：SD大鼠
1.1.2提供者：北京维通利华实验动物技术有限公司
1.1.3总数量：40只，8只用于最大耐受量(MTD)试验，32只用于疗效研究
1.1.4性别：雌性
1.1.5鼠龄：5-7周，7天适应期后为6-8周
1.1.6体重：150-170g，研究开始时为180-200g
1.2分组和治疗
在注射CFA后第7天，根据大鼠体重、Von Frey纤毛试验基准值和负重试验基准值，使用计算机产生的随机程序将大鼠进行随机分组。在CFA注射后第7天和第14天，对动物分别进行相应的单剂量治疗(空白溶媒和三种试验药品)，评估试验药品的疗效。在第7天进行单次低剂量给药后和第14天进行单次高剂量给药后，对所有试验动物分别进行低剂量和高剂量痛觉过敏疗效评估。研究分组情况、每组动物数量和治疗模式见表2。
表2分组和治疗
Figure PCTCN2014090226-appb-000077
1.3CFA注射
1.3.1试剂：4mg/kg CFA
1.3.2途径：左后足底注射
1.3.3体积：50μL/只
1.3.4频率：在第0天上午9:00单次注射
1.4配药过程
1.4.1空白溶媒：10％的羟丙基β环糊精
1.4.1.1途径：静脉注射
1.4.1.2体积：4mL/kg
1.4.1.3频率：分别在第7天和第14天上午9:00进行单次注射
1.4.1.4配制方法：将1.0g羟丙基β环糊精溶于5mL生理盐水中，涡旋振荡并超声，直至获得均匀溶液，加生理盐水定容至10mL。最后，将溶液过0.22μm滤膜。所有过程都在超净工作台内完成。
1.4.2试验药品：化合物46
1.4.2.1途径：静脉注射
1.4.2.2体积：4mL/kg
1.4.2.3频率：分别在第7天和第14天上午9:00进行单次注射
1.4.2.4配制方法：将25.0mg化合物46溶于5mL含20％羟丙基β环糊精的生理盐水中，涡旋振荡并超声，直至获得均匀溶液，加生理盐水定容至10mL，得到浓度为2.5mg/mL(10mg/kg)的溶液。最后，将溶液过0.22μm滤膜。所有过程都在超净工作台内完成。
1.4.3试验药品：化合物JK-02A
1.4.3.1途径：静脉注射
1.4.3.2体积：4mL/kg
1.4.3.3频率：分别在第7天和第14天上午9:00进行单次注射
1.4.2.4配制方法：将25.0mg化合物46溶于5mL含20％羟丙基β环糊精的生理盐水中，涡旋振荡并超声，直至获得均匀溶 液，加生理盐水定容至10mL，得到浓度为2.5mg/mL(10mg/kg)的溶液。最后，将溶液过0.22μm滤膜。所有过程都在超净工作台内完成。
1.4.4试验药品：化合物JK-02H
1.4.4.1途径：静脉注射
1.4.4.2体积：4mL/kg
1.4.4.3频率：分别在第7天和第14天上午9:00进行单次注射
1.4.4.4配制方法：将12.5mg化合物JK-02H溶于5mL含20％羟丙基β环糊精的生理盐水中，涡旋振荡并超声，直至获得均匀溶液，加生理盐水定容至10mL。得到浓度为1.25mg/mL(5mg/kg)的溶液。最后，将溶液过0.22μm滤膜。所有过程都在超净工作台内完成。
表3给药方法
Figure PCTCN2014090226-appb-000078
2.测量参数和试验方法
在CFA注射后第7天和第14天测量机械性痛觉过敏和负重，随后给予对照或试验药品。在第7天和第14天对所有研究动物分别在三个时间点和四个时间点进行Von Frey纤毛和负重试验。第14天之所以增加一个时间点，是因为观察到高剂量受试药在当天第3个时间点仍然具有较强的镇痛作用。
2.1Von Frey纤毛试验
在研究前和研究中通过测量所有试验动物组的Von Frey纤毛(Bioseb,France)阀值来测量左足的机械性痛觉过敏。纤毛垂直于足底表面不断增加拉力。取重复2-3次刺激(以g为单位)的平均值来评估足撤回的阀值。
在第7天进行三次Von Frey纤毛试验，包括治疗前、给药后15min和2h共三个时间点。在一周的洗脱期过后，即在CFA注射后的第14天，在单次高剂量给药后再进行四次Von Frey纤毛试验，包括治疗前、给药后15min、2h和5h共四个时间点。
2.2负重试验
大鼠的体重不均衡地分配在注射CFA足和对侧足上，由重量平衡变化仪(Bio-medical,USA)来测量。通过嵌入地板的测力板记录后足承载的重负荷，以此来测量箱体内动物的负重情况。在10秒内测量注射CFA足和对侧足的平均负重(以g为单位)。本实验测试的时间间隔与Von Frey纤毛试验的间隔一致。
2.3统计分析
所有结果均以平均值±标准偏差表示。本实验用one-way ANOVA进行各组间的数据分析，随后再进行多重比较，p＜0.05认为具有显著性差异。
3.研究结果
动物个体的Von Frey纤毛试验基准和承重试验基准列在附录I中。
3.1Von Frey纤毛试验
低剂量治疗的Von Frey纤毛试验组平均值见表4和图1。
高剂量治疗的Von Frey纤毛试验组平均值见表5和图2。
动物个体的Von Frey纤毛试验值汇总在附录II中。JK-02H的Von Frey纤毛试验的剂量依赖性见图3。
表4低剂量的Von Frey纤毛试验(平均值±标准偏差)
Figure PCTCN2014090226-appb-000079
注释：“*”与溶媒组比较p＜0.05
“**”与溶媒组比较p＜0.01
表5高剂量组的Von Frey纤毛试验(平均值±标准偏差)
Figure PCTCN2014090226-appb-000080
注释：“*”与溶媒组比较p＜0.05
“**”与溶媒组比较p＜0.01
3.2负重试验
低剂量治疗的负重试验组平均值见表6和图4。
高剂量治疗的负重试验组平均值见表7和图5。
动物个体的负重试验值汇总在附录III中。
JK-02H的负重试验的剂量依赖性见图6。
表6低剂量的负重试验数据(平均值±标准偏差)
Figure PCTCN2014090226-appb-000081
注释：“*”与溶媒组比较p＜0.05
“**”与溶媒组比较p＜0.01
表7高剂量的负重试验数据(平均值±标准偏差)
Figure PCTCN2014090226-appb-000082
Figure PCTCN2014090226-appb-000083
注释：“*”与溶媒组比较p＜0.05
“**”与溶媒组比较p＜0.01
4.结论
单次注射化合物46、化合物JK-02A和化合物JK-02H后，分别以Von Frey纤毛和负重试验评估其治疗大鼠CFA催生的痛觉过敏的疗效。结果表明，化合物JK-02H在高剂量(20mg/kg)和低剂量(5mg/kg)时与溶媒组比较，均显示出明显的抗痛觉过敏疗效；而化合物46和化合物JK-02A仅在高剂量(40mg/kg)时具有抗痛觉过敏疗效，在低剂量(10mg/kg)时药效则不明显。另外，化合物JK-02H在高剂量延长测试时间至5h，仍有非常明显的疗效，提示化合物JK-02H可能有较长的半衰期。
附录I动物个体的Von Frey纤毛试验基准和承重试验基准
Figure PCTCN2014090226-appb-000084
Figure PCTCN2014090226-appb-000085
附录II动物个体的Von Frey纤毛试验值
Figure PCTCN2014090226-appb-000086
Figure PCTCN2014090226-appb-000087
附录III动物个体的承重试验值
Figure PCTCN2014090226-appb-000088
Figure PCTCN2014090226-appb-000089

Claims (22)

  1. 式I的化合物或其药学上可接受的盐，
    Figure PCTCN2014090226-appb-100001
    其中R1和R2相同或不同，各自独立地选自卤素、C1-C6烷氧基、C1-C6烷基、C2-C6烯基和C2-C6炔基；R3选自H、卤素、取代或未被取代的C1-C6烷基、C3-C6环烷基、C2-C6烯基、C2-C6炔基、芳基和杂芳基；且n为0-15的整数。
  2. 根据权利要求1所述的化合物或其药学上可接受的盐，其中R1和R2各自独立地选自卤素和C1-C6烷氧基。
  3. 根据权利要求1所述的化合物或其药学上可接受的盐，其中R1为F，且R2为F或OCH3。
  4. 根据权利要求1所述的化合物或其药学上可接受的盐，所述化合物具有式II的结构：
    Figure PCTCN2014090226-appb-100002
  5. 根据权利要求1所述的化合物或其药学上可接受的盐，所述化合物具有式III的结构：
    Figure PCTCN2014090226-appb-100003
  6. 根据权利要求1所述的化合物或其药学上可接受的盐，其中R3选自 H和取代或未被取代的C1-C6烷基。
  7. 根据权利要求1所述的化合物或其药学上可接受的盐，其中R3选自H和取代或未被取代的C1-C3烷基。
  8. 根据权利要求1所述的化合物或其药学上可接受的盐，其中R3为H、甲基或丙基。
  9. 根据权利要求1所述的化合物或其药学上可接受的盐，其中n为1-10的整数。
  10. 根据权利要求1所述的化合物或其药学上可接受的盐，其中n为1-6的整数。
  11. 根据权利要求1所述的化合物或其药学上可接受的盐，其中所述化合物选自
    Figure PCTCN2014090226-appb-100004
    Figure PCTCN2014090226-appb-100005
  12. 根据权利要求1所述的化合物或其药学上可接受的盐，其中所述化合物选自：
    Figure PCTCN2014090226-appb-100006
  13. 权利要求1-12中任一项所述的化合物或其药学上可接受的盐，其中所述药学上可接受的盐选自盐酸盐、硫酸盐、酒石酸盐和柠檬酸盐。
  14. 权利要求1-13中任一项所述的化合物或其药学上可接受的盐在制备治疗疼痛的药物中的应用。
  15. 药物组合物，所述药物组合物包含权利要求1-13中任一项所述的化合物或其药学上可接受的盐以及一种或多种药学上可接受的载体。
  16. 治疗疼痛的方法，所述方法包括向需要治疗的个体给药治疗有效 量的权利要求1-13中任一项所述的化合物或其药学上可接受的盐或者权利要求15所述的药物组合物。
  17. 制备权利要求1-13中任一项所述的化合物或其药学上可接受的盐的方法，所述方法包括：
    (1)使化合物3
    Figure PCTCN2014090226-appb-100007
    与化合物14
    Figure PCTCN2014090226-appb-100008
    在碱的存在下进行亲核取代反应，得到化合物15
    Figure PCTCN2014090226-appb-100009
    其中X为氯、溴或碘原子，且R1、R2、R3和n如权利要求1-12中任一项所定义；
    (2)使所述化合物15在碱的存在下进行水解反应，得到化合物16
    Figure PCTCN2014090226-appb-100010
    其中R1、R2、R3和n如权利要求1-12中任一项所定义；
    (3)使所述化合物16与化合物10
    Figure PCTCN2014090226-appb-100011
    在缩合剂的存在下进行酰胺化反应，得到化合物17
    Figure PCTCN2014090226-appb-100012
    其中R1、R2、R3和n如权利要求1-12中任一项所定义；
    (4)使所述化合物17与化合物13
    Figure PCTCN2014090226-appb-100013
    在缩合剂的存在下进行酰胺化反应，得到化合物18
    Figure PCTCN2014090226-appb-100014
    其中Boc代表叔丁氧羰基保护基，且R1、R2、R3和n如权利要求1-12中任一项所定义；
    (5)使所述化合物18在脱保护试剂的存在下进行脱保护反应，得到权利要求1-12中任一项的化合物，并任选地进一步形成权利要求1-13中任一项的药学上可接受的盐。
  18. 根据权利要求17所述的方法，其中在所述步骤(1)中，所述碱选自碳酸钾、碳酸钠、碳酸铯、碳酸钙、氢氧化钾、氢氧化钠、氢氧化锂、氢氧化钙、氢化钠、氢化钾、氢化钙、金属钠、金属钾、甲醇钠、乙醇钠、叔丁醇钾、丁基锂、苯基锂、二异丙氨基锂、六甲基二硅胺基锂、二甲胺、二乙胺、三乙胺、二异丙基乙胺、哌啶、吡啶、1,8-二氮杂二环[5.4.0]十一碳-7-烯、1,5-二氮杂二环[4.3.0]壬-5-烯、四丁基氢氧化铵及其组合。
  19. 根据权利要求17所述的方法，其中在所述步骤(2)中，所述碱选自氢氧化钾、氢氧化钠、氢氧化锂、氢氧化钙、氢氧化钡、氢氧化铜、三氯化铝、三氯化硼、三溴化铝、三溴化硼、氰化钠、氰化钾、碳酸铯、碳酸铜、碘化锂、硼氢化钠、氢化钠、氢化钾、氢化钙及其组合。
  20. 根据权利要求17所述的方法，其中在所述步骤(3)和所述步骤(4)中，所述缩合剂各自独立地选自N-羟基-7-偶氮苯并三氮唑、1-羟基苯并三氮唑、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、双(2-氧代-3-噁唑烷基)次磷酰氯、1H-苯并三唑-1-氧基三(1-吡咯烷基)膦六氟磷酸盐、1,3-二环己基碳二亚胺、N,N'-羰基二咪唑、1-乙基-(3-二甲基氨基丙基)碳二亚胺、N,N'-二异丙基碳二亚胺、4-二甲氨基吡啶及其组合。
  21. 根据权利要求17所述的方法，其中在所述步骤(5)中，所述脱保护试剂选自三氟乙酸、盐酸、氢溴酸、氢碘酸、磷酸、硫酸、甲磺酸、对甲苯磺酸、乙酰氯、三氯化铝以及三氟化硼。
  22. 通式15、16、17或18的化合物，
    Figure PCTCN2014090226-appb-100015
    其中R1、R2、R3和n如权利要求1-12中任一项所定义。
PCT/CN2014/090226 2013-11-04 2014-11-04 具有更高蛋白激酶g抑制活性的化合物及其制备方法 Ceased WO2015062555A1 (zh)

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