WO2013120438A1 - 治疗或缓解疼痛的物质 - Google Patents

治疗或缓解疼痛的物质 Download PDF

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WO2013120438A1
WO2013120438A1 PCT/CN2013/071515 CN2013071515W WO2013120438A1 WO 2013120438 A1 WO2013120438 A1 WO 2013120438A1 CN 2013071515 W CN2013071515 W CN 2013071515W WO 2013120438 A1 WO2013120438 A1 WO 2013120438A1
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pain
receptor
subunit
group
gaba
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French (fr)
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张旭
李帅
鲍岚
叶阳
姚胜
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Shanghai Institutes for Biological Sciences SIBS of CAS
Shanghai Institute of Materia Medica of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
Shanghai Institute of Materia Medica of CAS
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Priority to US14/378,812 priority Critical patent/US20150374705A1/en
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Priority to JP2014556911A priority patent/JP6061956B2/ja
Priority to EP13749318.5A priority patent/EP3025716B1/en
Publication of WO2013120438A1 publication Critical patent/WO2013120438A1/zh
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    • 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
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/9426—GABA, i.e. gamma-amino-butyrate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2842—Pain, e.g. neuropathic pain, psychogenic pain

Definitions

  • the present invention is in the field of biotechnology; more specifically, the present invention relates to a substance for treating or relieving pain. Background technique
  • Pain is a sensory expression that is different from touch, pressure, heat or cold. Patients often describe pain in terms such as sharp pain, dull pain, soreness, stinging, cutting pain, or burning pain.
  • the pain caused by the injury nerve and manifested as hyperalgesia is generally called “neurogenic” pain; the pain caused by stimulating the nociceptor is called “nociceptive” pain.
  • pain can be divided into two categories: acute pain and chronic pain.
  • Acute pain is caused by damage to the skin, body structure or internal organs and/or harmful stimuli caused by the disease, or by abnormal function of the muscle or viscera that does not cause actual tissue damage; and chronic pain can be defined as a disease that continues beyond the usual course of acute disease. Or a reasonable time for the injury to be cured, or associated with a chronic pathological process that causes persistent pain, or pain that recurs at intervals of months or years, and if there is still pain after the cure has been reached, it is considered chronic pain.
  • chronic pain can be chronic non-remission or relapse, and the definition of definition between acute and chronic pain is not only a semantic difference, but an important clinical correlation, such as acute pain. With good control, it will develop into chronic pain.
  • Acute and chronic pain differ in the etiology, pathology, and diagnosis and treatment.
  • chronic pain is caused by chronic pathological processes in the body's structure or internal organs, or by extended or sometimes permanent skill disorders in the peripheral or central nervous system or both.
  • chronic pain can sometimes be attributed to psychological mechanisms or / and environmental factors.
  • Acute pain generally refers to non-neurological pain, including common conditions such as arthritis pain, musculoskeletal pain, post-operative pain, and fibromyalgia. Most of these pains are thought to be caused by soft tissue and bone damage, such as arthritis pain, musculoskeletal pain, and post-operative pain, which cause inflammation in the normally functioning nervous system, and pain is only an inflammatory process. result.
  • Chronic pain includes neuropathic pain, inflammatory pain, and cancer pain. These pains can be associated with hyperalgesia and/or abnormal pain, where hyperalgesia refers to increased sensitivity to typical noxious stimuli and abnormal sensation refers to increased sensitivity to typical non-noxious stimuli.
  • Somatic-derived pain is caused by peripheral sensory nerve injury or infection, including but not limited to peripheral nerve injury, herpes virus infection, diabetes, burning, avulsion of blood vessels or plexus, neuralgia, amputation and nodular veins Pain caused by tube inflammation, neurogenic pain is also caused by chronic environmental poisoning, human immunodeficiency virus infection, hypothyroidism, uremia or vitamin deficiency caused by nerve damage, clinical manifestations including but not limited to inflammatory pain, bone and joint pain , trigeminal neuralgia, cancer pain, diabetic neuropathy, leg hyperactivity syndrome, postherpetic neuralgia, burning pain, avulsion of the brachial plexus, occipital neuralgia, gout, phantom limbs, burns and other forms of neuralgia, Neurological and spontaneous pain syndrome.
  • peripheral sensory nerve injury or infection including but not limited to peripheral nerve injury, herpes virus infection, diabetes, burning, avulsion of blood vessels or plexus, neuralgi
  • Neuropathic pain is generally considered to be chronic pain caused by damage or lesions in the peripheral or central nervous system. Lesions associated with neuropathic pain include long-term peripheral or central neuronal sensitization, central sensitization associated with impaired nervous system inhibition and/or excitatory function, and abnormal interactions between parasympathetic and sympathetic nervous systems. Many clinical symptoms are associated with neurogenic pain or form the basis of neuropathic pain, such as diabetes, postoperative pain from severing, lower back pain, cancer, chemical damage or toxins, other serious surgeries, and compression from traumatic damage. Peripheral nerve damage, nutritional deficiencies, infections (such as herpes zoster, HIV).
  • Analgesics are drugs that relieve pain by increasing the pain threshold of the patient without losing consciousness. Analgesics can have an anti-allergic sensitivity in individuals with hyperalgesia.
  • prescription analgesics approved by the US Food and Drug Administration (FDA) fall into two categories: opioids and anti-inflammatory drugs.
  • opioids belong to different classifications.
  • Opioid analgesics act by mimicking the body's endogenous opioids, endorphins and enkephalins, which are produced by the body to help alleviate pain, through opioids acting throughout the central and peripheral nervous systems. Receptors to block pain.
  • Anti-inflammatory drugs include non-selective non-anti-inflammatory analgesics and specific
  • COX-2 inhibitors attempt to reduce inflammation caused by chemical transmitters such as prostaglandins caused by damage to the body.
  • opioid analgesics Of all the opioid analgesics, morphine remains the most widely used analgesic. However, in addition to its therapeutic properties, it has several disadvantages, including respiratory depression, decreased gastrointestinal motility (causing constipation), nausea and vomiting. Tolerance and physical dependence also limit the clinical use of opioids.
  • Anti-inflammatory analgesics including aspirin and other salicylic acid compounds, can prevent the enlargement of the inflammatory process and temporarily reduce the pain, however, the above drugs are not effective for neuropathic pain. And many anti-inflammatory drugs, especially non-anti-inflammatory drugs, can cause gastrointestinal side effects, especially when taken orally. This side effects include causing gastrointestinal ulcers and erosions, which often become as serious as the need for hospitalization. It can even be fatal.
  • Gabapentin is currently the first-line clinical drug for the treatment of neuropathic pain and has been widely used. Gabapentin has significant side effects, including lethargy, dizziness, walking instability, and fatigue, which are common in the early stages of medication. Children occasionally become irritable and will disappear after stopping the drug.
  • analgesics which are very safe and resistant, and are not addictive.
  • the ideal analgesic will reduce or eliminate the patient's feeling of pain, produce analgesia in the occurrence of various types of pain, and the effect is satisfactory regardless of oral or other modes of administration, with minimal or no side effects, and does not produce Drug tolerance and drug dependence.
  • GABA Gamma-aminobutyric acid
  • GABA A receptor which is a ligand gate
  • GABA B receptor which is a member of the G protein-coupled receptor superfamily.
  • the GABA A receptor subunits in mammals are found to have subunits such as ⁇ 1-6, ⁇ 1-4, ⁇ 1-3, ⁇ , ⁇ , ⁇ , and pl-2, of which ⁇ subunit, ⁇ subunit, and ⁇ subunit.
  • the base pair is essential for the formation of a complete functional GABA A receptor, and the alpha subunit is critical for the binding of benzodiazepine to the GABA A receptor.
  • the GABA A receptor containing ⁇ 1, ⁇ 2, ⁇ 3 or ⁇ 5 subunits which is mainly capable of binding to benzodiazepine, has been reported to have a role in mediating and relaxing muscle relaxation of GABA A receptors containing ⁇ subunits. And contains (2 and (x3 receptors mainly mediate anticonvulsant effects.
  • GABA A receptors The central nervous system suppresses pain, and in 2008, an agonist of the GABA A receptor comprising the ⁇ 2 and ⁇ 3 subunits was reported to significantly inhibit neuropathic pain and inflammatory pain at the spinal level. Whether the inhibitor or inverse agonist of GABA A receptor can inhibit pain is unclear. It has been reported in the literature that GABA can cause an excitatory current in the rat or human peripheral nervous system, while the GABA A receptor agonist Muscimol can inhibit formalin-induced pain at low doses, while high doses promote Fuer. Marlin-induced pain. The role of GABA A receptors in pain is unclear, and the widespread expression of GABA A receptors in the central nervous system limits the use of ligands in the treatment of pain.
  • the ⁇ 5-containing GABA A receptor accounts for less than 5% of the GABA A receptor in the mammalian brain, and has a very low expression level in the cerebral cortex, but GABA in the hippocampus of the brain. The proportion of A receptors is greater than 20%, and other brain regions are hardly expressed.
  • a5-GABA A receptors may be associated with cognition (Collinson N et al, Enhanced learning and memory and altered GABAergic synaptic transmission in mice lacking the alpha 5 subunit of the GABAA receptor J Neurosci. 2002 Jul . l ;22(13):5572-80.)
  • A5I and MRK016 are compounds developed by Merck for the treatment of cognitive-related diseases, and A5I was found in clinical phase II trials. Nephrotoxicity due to too low solubility of the compound; and MRK016 has problems in safety. In Phase I clinical trials, the drug was found to cause side effects such as top-heavy, headache, and nausea and vomiting.
  • the role of (x5-GABA A receptor in the peripheral nervous system has not been reported.
  • the peripheral nervous system is composed of the somatic nervous system and the autonomic nervous system.
  • the somatic nervous system is divided into the sensory nervous system (; afferent nerve) and the motor nervous system.
  • Two types of (efferent nerves) are distributed to various parts of the body. Neurons that transmit nerve impulses from the periphery to the nerve center are called primary sensory neurons.
  • In the sensory nervous system only the thoracic nerve is rooted in the ribs. Walking on the lower edge, forming intercostal nerves, innervating the skin and muscles of the chest wall and abdominal wall.
  • the rest of the sensory nerves are joined by several adjacent nerves, forming nerve plexus, such as cervical plexus, brachial plexus, lumbar plexus and plexus.
  • nerve plexus such as cervical plexus, brachial plexus, lumbar plexus and plexus.
  • a number of nerves are separated from each nerve plexus and distributed to the skin, muscles, etc. of the neck, upper chest, upper limbs, lower limbs, and perineum.
  • the primary sensory neurons of the dorsal root ganglia are divided into different sizes according to the diameter of the cells. Functional groups, usually divided into small cells, medium cells, and large cells.
  • small and medium cells are cells that transmit nociceptive (pain), some Small cell free nerve endings to noxious stimuli sensitive or persistent, called nociceptors has been found comprising a (main x5-GABA A receptors are expressed in small neurons, increased expression of nerve and cut (Xiao model HS et al, Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain.” Proc Natl Acad Sci US A. 2002 Jun 1 1 ;99(12):8360-5.), but whether it is Closely related to injury or pain, there is no in-depth research in this field.
  • the object of the present invention is to provide a substance and a method for treating or relieving pain, and also to propose a new pain and analgesia. Drug screening method.
  • the inverse agonist of the a5-GABA A receptor is a ligand that selectively binds to the benzodiazepine binding site of the receptor.
  • the a5-GABA A receptor ligand binding to a (capacity x5-GABA A receptor is greater than binding capacity to contain ⁇ or ⁇ 2 or ⁇ 3 subunit of the GABA A receptor.
  • the a5-GABA A receptor inverse agonist ligand efficiency of GABA-A receptors A5 efficiency of greater than or inverse agonism comprising ⁇ ⁇ 2 or ⁇ 3 subunit receptor GAB ⁇ ⁇ .
  • the inverse agonist of the a5-GABA A receptor is a compound having a structure represented by the formula (I) and ( ⁇ ) structure or a drug thereof
  • R' a represents halogen; or d_ 6 alkyl, C 3 _ 7 cycloalkyl, C 4 _ 7 cycloalkenyl, C 6 -8 bicycloalkyl, C 6 _ 10 aryl, C 3 _ 7 Heterocycloalkyl, defined or containing 6 atoms, wherein 1, 2 or 3 are nitrogen or contain 5 atoms; wherein 1, 2 or 3 are independently selected from oxygen, nitrogen or sulfur and wherein oxygen or sulfur is not More than one aromatic ring heteroaryl, or bis(d- 6 )alkylamino group, any of these groups may be optionally substituted with one or more substituents selected from the group consisting of: halogen, R 3 , OR 3 , OC(0)R 3 , NR 4 R 5 , NR 4 R 5 (C 1 -6 )alkyl, NR 4 R 5 C(0), NR 4 R 5 C(0) (Cw) alkyl, CN , cyano (Cw) alkyl
  • R lb represents d- 6 alkyl, C 3 _ 7 cycloalkyl, C 4 _ 7 cycloalkenyl, aryl, C 3 -7 heterocycloalkyl, heterocyclic aryl, two
  • R 3 represents C 2 -6 alkyl, ⁇ 6 6 alkenyl, C 2 -6 block, ⁇ 6 6 cycloalkyl, C 3 -6 cycloalkyl (d- 6 ) alkyl, cyano (d- 6)
  • An alkyl group, a hydroxy (d- 6 ) alkyl group and R 3 is optionally 1, 2 or 3 fluoro.
  • R 4 and R 5 are each independently hydrogen, d 6 alkyl, ⁇ 6 6 alkenyl, C 2 -6 block, -6 cycloalkyl or CF 3 , or R 4 and R 5 are co-linked thereto
  • the nitrogen atoms together form a 4-7 membered heteroaliphatic ring containing the nitrogen atom and one additional heteroatom selected from the group consisting of 0, N and S, the ring being capable of being interrupted by one or more R 3 groups Optional replacement.
  • R 6 is C 6 _ 1Q aryl, C 6 _ 1Q aryl (C1-6) alkyl, heteroaryl or heteroaryl (d- 6 )alkyl. wherein heteroaryl is as defined above, and R 6 is optional substituted by one, two or three substituents independently selected from the following group: a halogen atom and d_ 4 alkyl, C 2 _ 4 alkenyl, C 2 _ 4 block basis, C1-4 alkoxy, C 2 _ a 4 -alkenyloxy group and a C 2 - 4 -oxy group, wherein each group is unsubstituted or substituted with one, two or three halogen atoms.
  • X' is NR 4 R 5 ; or X' is a 5-membered heteroaryl ring containing 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur, and at most one of the heteroatoms is oxygen or sulfur, Or a 6-membered heteroaryl ring containing 1, 2 or 3 nitrogen atoms, optionally fused to a benzene or pyridine ring and optionally substituted by RW and/or R y and/or R z substituted, wherein R w is halogen, R 3 , OR 3 , OC(0) R 3 , C(0)OR 3 , NR 4 R 5 , NR 4 C(0)R 5 , OH , three (C 1 -6 alkane Methylsilyl 6 methoxy.4 alkyl, CN or R 6 , R Y is halogen, R 3 , OR 3 , OC(0) R 3 , NR 4 R 5 , NR 4 C(0) R 5 , NR 4
  • Z' represents a 5-membered heteroaryl ring containing 1, 2 or 3 hetero atoms independently selected from the group consisting of oxygen, nitrogen and sulfur, and at most one hetero atom is oxygen or sulfur, and provided that one atom is oxygen or sulfur At least one nitrogen atom is also present, or a 6-membered heteroaryl ring other than pyrazine containing 2 or 3 nitrogen atoms, and any one of the rings may be one or more selected from the group consisting of optionally substituted with: halo, R 3, OR 3, OC (0) R 3, NR 4 R 5, NR 4 R 5 (. C 1 6) alkyl, NR 4 R 5 C (0 ), CN, cyano (d. 6 ) fluorenyl or R 6 .
  • the inverse agonist of the a5-GABA A receptor further comprises: an isomer, a precursor of a compound having the structure represented by the formula (I and II).
  • the inverse agonist of the a5-GABA A receptor is a compound having the structure represented by formula (III or V) or a pharmaceutical thereof (III), (V),
  • R LE represents bromine, thienyl, tert-butyl, phenyl or furyl
  • R LD represents aryl, d. 6 fluorenyl, C 3 . 7 cyclodecyl 1 is a nitrogen atom containing 1, 2 or 3 a 5-membered heterocyclic ring, wherein at most one of the heteroatoms is oxygen or sulfur, or a 6-membered heteroaryl ring containing 1, 2 or 3 nitrogen atoms, and any of the 5 or 6-membered heteroaromatic rings
  • the one may be optionally substituted by one or more substituents selected from the group consisting of: d. 6 fluorenyl, amino, pyridyl, CF 3 , aryl (d.
  • the a5-GABA A receptor Inverse agonists also include: isomers, precursors of compounds having the structure shown by formula (III or V).
  • the inverse agonist of the a5-GABA A receptor is the following compound or a pharmaceutically acceptable salt thereof, which is:
  • the inverse agonist of the a5-GABA A receptor is more preferably the following compound or a drug thereof:
  • the inverse agonist of the a5-GABA A receptor is a chemically acceptable salt having the structure represented by the formula (VI)) -
  • halogen d 7 embankment group, C 2 7 alkynyl group, a cycloalkyl group, C1-7 alkoxy embankment, OCF 3, -NHR, -NHC ( 0) R or -NHS02R;..
  • R is hydrogen, d. 7 alkyl, d.
  • R 2 is halogen, hydrogen, -C ( 0) 0(C 1-4 )alkyl, C(0)NHCH 2 CCH, C(0)NHCH 2 CH 2 , S0 2 CH 2 CH 3 , C 1-7 alkyl, d. 7 substituted by halogen .
  • embankment, cyano, C 3 6 cycloalkyl; n is 0, 1, 2 or 3.
  • the inverse agonist of the a5-GABA A receptor is the following compound or a pharmaceutically acceptable compound thereof
  • the inverse agonist of the a5-GABA A receptor is more preferably the following compound or a pharmaceutically acceptable compound thereof
  • the a5-GABA A receptor binding receptor inverse agonist is the expression of peripheral nerve receptors but a5-GABA A with the hub can not effectively expressed receptor receptor a5-GABA A Combine.
  • the pain is chronic pain associated with peripheral nerves.
  • the chronic pain is neuropathic pain, inflammatory pain, and cancer pain.
  • the pain condition includes (but is not limited to): headache, facial pain, neck pain, shoulder pain, back pain, chest pain, abdominal pain, back pain, back pain, lower limb pain, musculoskeletal pain, Physical pain, vascular pain, replacement page (Article 26) Gout, arthritis pain, pain associated with somatic-like mental disorders, visceral pain, pain caused by infectious diseases (such as AIDS and post-herpetic neuralgia), bone pain, sickle cell anemia, autoimmune diseases, Multiple sclerosis or inflammation-related pain, acute and chronic inflammatory pain, cancer pain, neuropathic pain, injury or surgery-induced pain, cancer pain, nociceptive pain, diabetes, peripheral neuropathy, post-pustular neuralgia, trigeminal Neuralgia, lumbar or cervical radiculopathy, glossopharyngeal neuralgia, autonomic reflex pain, reflex sympathetic dystrophy, nerve root avulsion, cancer, chemical damage, toxins, nutritional deficiencies, viral or bacterial infections, regression Osteoarth
  • a method of screening for a medicament for preventing, ameliorating or treating pain comprising:
  • step (2) administering the candidate substance to the system of step (1), and observing the binding of the candidate substance to (x5-GABA A receptor; if the candidate substance is capable of binding to (x5-GABA A , the candidate substance is prevention, improvement or A potential drug for treating pain.
  • the binding to the a5-GABA A receptor is: selective binding to a benzodiazepine binding site.
  • the candidate substance is not effective to bind to the centrally expressed (x5-GABA A receptor.
  • the candidate substance is a small amount that passes through or does not pass through the animal body.
  • the substance of the blood-brain barrier, in the tissue distribution of animals, the distribution of candidate compounds in brain tissue is less than 50% of the distribution in plasma tissue.
  • the a5-GABA A consists of an ⁇ 5 subunit of the GABA A receptor, a ⁇ subunit (such as ⁇ 2, ⁇ 3), and a ⁇ subunit (such as ⁇ 1, ⁇ 3).
  • the step (2) includes observing the inhibition of the candidate substance (for example, inhibiting 20% or more, more preferably inhibiting 40% or more, and more preferably inhibiting 50% or more).
  • GABA passes through the GABA A receptor.
  • the condition of the induced current; if the candidate substance is capable of inhibiting the current induced by GABA through the GABA A receptor, the candidate substance is a potential drug for preventing, ameliorating or treating pain.
  • the step (1) further comprises the step of: providing a control group selected from the group consisting of: a system comprising (eg, expressing) a GABA A receptor comprising an ⁇ subunit, comprising (eg, expressing a system comprising a GABA A receptor of the ⁇ 2 subunit and/or a system comprising (eg, expressing) a GABA A receptor comprising an ⁇ 3 subunit, the control GABA A receptor having intact GABA A receptor function; (2) wherein the method comprises: detecting a binding substance of the candidate substance to the a5-GABA A receptor in a system comprising (eg, expressing) (x5-GABA A receptor, and comparing with the control group, if the candidate substance
  • Ki (nM) to (x5-GABA A receptor) is statistically smaller (eg, 20% smaller, More preferably, it is 40% or more, more preferably, more preferably
  • the candidate substance is incapable of effectively inhibiting the central binding of the ligand of the a5-GABA A receptor (as can be identified by isotopic labeling methods). Giving the experimental animal a dose of candidate substance and Known (a specific ligand for the x5-GABA A receptor, which can be labeled (eg, isotopically labeled), and then tested whether the candidate inhibits the distribution of the known a5-GABA A receptor-specific ligand at the central If the candidate substance inhibits the known distribution of the specific ligand of the x5-GABA A receptor in the central portion by less than 60%, it indicates that the candidate is a potential drug for preventing, ameliorating or treating pain.
  • the candidate substance does not effectively cross the blood-brain barrier, and the distribution ratio of the candidate substance in the brain and plasma is less than 50%, indicating that the candidate is a potential drug for preventing, ameliorating or treating pain.
  • the step (1) further comprises the step of: providing a control group selected from the group consisting of: a system comprising (eg, expressing) a GABA A receptor comprising an ⁇ subunit, comprising (eg, expressing a system comprising a GABA A receptor of the ⁇ 2 subunit and/or a system comprising (eg, expressing) a GABA A receptor comprising an ⁇ 3 subunit, the control GABA A receptor having intact GABA A receptor function;
  • the method comprises: detecting, by an electrophysiological method, the inhibition of the GABA A -induced current by the candidate substance, if the test substance can be significantly inhibited (eg, inhibiting 20% or more, and more preferably suppressing 40% or more) , more preferably inhibits more than 50% of the a5-GABA A receptor-mediated current, but at the same time does not significantly inhibit the inclusion of cd or (2 or (x3 GABA A receptor-induced current, indicating that the candidate substance is preventive, A potential drug to improve
  • the candidate substance includes, but is not limited to, a small molecule compound (e.g., gamma-aminobutyric acid)
  • the system is selected from the group consisting of: a cell system (or cell culture system) (e.g., dorsal root ganglion cells), a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
  • a cell system or cell culture system
  • a subcellular system e.g., a cell system, or cell culture system
  • a solution system e.g., a tissue system, an organ system, or an animal system.
  • the method further comprises: performing further cell experiments and/or animal tests on the obtained potential substances to further select and determine substances that are truly useful for preventing, ameliorating or treating pain.
  • a method of preventing, ameliorating or treating pain comprising administering to a patient in need thereof a safe and effective amount of an inverse agonist of the a5-GABA A receptor or a pharmaceutically acceptable salt thereof.
  • the inverse agonist of the a5-GABA A receptor or a pharmaceutically acceptable salt thereof can be administered orally, subcutaneously, transdermally, intranasally or by an automatic injection device.
  • GABA A receptor ⁇ 5 subunit protein is elevated in the dorsal root ganglia under partial neuronal injury (Sredifice Nerve Injury, SNI).
  • GABA A receptor ⁇ 5 subunit protein is elevated in dorsal root ganglia under conditions of complete Freund's adjuvant-induced (CFA) inflammatory pain model.
  • CFA complete Freund's adjuvant-induced
  • the expression of the (alpha5 subunit protein) of the GABA A receptor was determined by immunohistochemistry.
  • B The protein level of the ⁇ 5 subunit of the GABA A receptor was determined by immunoblotting.
  • Figure 3 The GABA A receptor ct5 subunit protein in the dorsal root ganglia can be transported to the sciatic nerve.
  • GABA A receptor x5 subunit protein in the sciatic nerve, dorsal root ganglion cell, dorsal root nerve, and spinal dorsal horn.
  • Figure 4 In the SNI-induced pain model, MRK016 was administered. Or MRK016-M3 effectively inhibits neuropathic pain.
  • Rats with 10 days of SNI establishment were used as experimental animals, and MRK016-M3 was injected into the paws of rats to inhibit the effectiveness of neuropathic pain.
  • MRK016-M3 can not significantly increase the threshold of pain under the condition of intrathecal injection of siRNA specific for GABA A receptor ⁇ 5 subunit mRNA. In the case of injection of control siRNA, MRK016-M3 can significantly increase the threshold of pain. Figure 5. In a CFA-induced inflammatory pain model, administration of MRK016 or MRK016-M3 inhibited the pain response in rats.
  • MRK016 (2 mg/kg body weight) was intraperitoneally injected at 7 days, and the compound was found to be effective in inhibiting mechanical pain.
  • MRK016 (2 mg/kg body weight) was intraperitoneally injected for 3 days, and the compound was found to be effective in suppressing heat pain.
  • intraperitoneal injection of A5I can also effectively treat heat pain.
  • Compound C5 has no effect on pentylenetetrazol-induced epilepsy.
  • different doses of compound C5 were first administered subcutaneously at doses of 1 mg/kg, 3 mg/kg, 10 mg/kg and 15 mg/kg, respectively, after 0.5 hours. Injection of pentylenetetrazol 100 mg/kg, respectively, the test results showed that different doses of compound C5 had no effect on the pentylenetetrazol-induced seizures in the reaction order and latency.
  • the inventors have intensively studied for the first time to disclose a new method for treating pain, treating pain with an inverse agonist against the peripheral nerve a5-GABA A receptor.
  • the present invention discloses that the ct5 subunit protein of the GABA A receptor is mainly transported to the peripheral plexus or sciatic nerve, and the expression of the gene protein is elevated under the conditions of the SNI model and the CFA-induced inflammatory pain model.
  • the present inventors hypothesized that those inverse agonists of GABA A receptors that pass through or do not cross the blood-brain barrier can bind to peripheral nerves (x5-GABA A receptors, inhibit neuropathic pain and inflammatory pain).
  • peripheral nerves x5-GABA A receptors, inhibit neuropathic pain and inflammatory pain.
  • the ct5 subunit of the GABA A receptor is mainly expressed in the nervous system, such compounds are theoretically an analgesic drug with no side effects or less side effects.
  • gamma-aminobutyric acid A receptor is used interchangeably with “GABA A receptor”.
  • (a) ligand for a GABA A receptor comprising a ct5 subunit is defined as a compound capable of binding to an a5-GABA A receptor that modulates a pain response.
  • inverse agonist of a GABA A receptor comprising a ct5 subunit refers to a compound capable of binding to the a5-GABA A receptor, and the compound is capable of inhibiting GABA to a5-GABA A receptor The role.
  • treatment also includes prophylactic administration, once the condition is established, to alleviate or eliminate the condition.
  • patient is defined as any warm-blooded animal, such as, but not limited to, a mouse, a guinea pig, a dog, a horse, or a human, preferably a human.
  • acute pain is defined as pain caused by damage to the skin, body structure or internal organs and/or harmful irritation of the disease, or pain caused by abnormal function of the muscle or viscera that does not cause actual tissue damage. .
  • chronic pain is defined as a reasonable period of time beyond the usual course of the acute disease or the healing of the injury, or associated with a chronic pathological process that causes persistent pain, or pain that recurs at intervals of months or years, if It is considered to be chronic pain after the cure has been reached or after the usual treatment process.
  • the length of time required for pain depends on the nature of the pain and the course of the pain-related treatment. If the pain exceeds the usual course of treatment, the pain is chronic.
  • Chronic pain includes, but is not limited to, headache, facial pain, neck pain, shoulder pain, chest pain, abdominal pain, back pain, low back pain, lower limb pain, musculoskeletal pain, pain associated with somatic-like mental disorders, visceral pain, painful diabetes sexual neuropathy, vascular pain, gout, arthritic pain, cancer pain, autonomic reflex pain, pain caused by infectious diseases (such as AIDS and herpes zoster), pain caused by autoimmune diseases (rheumatoid) Pain, pain caused by acute and chronic inflammation, post-operative pain and pain after burns.
  • infectious diseases such as AIDS and herpes zoster
  • autoimmune diseases rheumatoid Pain
  • the medicament disclosed in the present invention is effective for treating chronic pain as defined above, and the medicament disclosed in the present invention can be used for treating pain sensitivity accompanying other disorders, including hyperalgesia, allodynia, pain enhancement, and pain memory enhancement, and the invention will improve Treatment of its pain.
  • headache can be divided into primary headaches and secondary headaches.
  • Primary headaches include tense headaches, migraine headaches, and cluster headaches, while secondary headaches are caused by other diseases. Pain-sensitive tissue of the head and face can cause various headaches when it is damaged or stimulated. These pain-sensitive tissues include the scalp, face, mouth and throat. Because they are mainly the muscles or blood vessels of the head, they are rich in nerves. Fiber, which is sensitive to pain, can cause headaches when these tissues are damaged.
  • facial pain includes, but is not limited to, trigeminal neuralgia, atypical facial pain, facial paralysis, and hemifacial spasm.
  • trigeminal neuralgia is a unique chronic painful condition, also known as painful convulsion, which refers to the appearance of transient, paroxysmal, and recurrent electric shock-like severe pain in the trigeminal distribution. Or accompanied by the same side tendon. Trigeminal neuralgia is divided into two types: primary and secondary. Primary trigeminal neuralgia refers to no clinical signs of the nervous system. No organic lesions are found. Secondary trigeminal neuralgia refers to clinical There are signs of the nervous system, and organic diseases such as tumors and inflammation are found.
  • typical facial pain refers to pain caused by a variety of causes. It is characterized by persistent burning pain, no intermittentness, and no special action or triggering. The pain is mostly bilateral, and the pain often exceeds the distribution of the trigeminal nerve and even the skin of the neck. The cause can be caused by sinusitis, malignant tumors, infection of the jaw and skull base, etc., or damage to the trigeminal nerve.
  • neck pain, back pain, shoulder pain refers to pain caused by acute and chronic muscle strain and degenerative changes and trauma of bones and joints.
  • Common diseases that cause pain in the neck, shoulders and upper limbs include neck and shoulder muscle fasciitis, ligament inflammation, cervical spondylosis, frozen shoulder, thoracic outlet syndrome, external humeral epicondylitis, or pain caused by autoimmune diseases.
  • rheumatoid arthritis, ankylosing spondylitis and rheumatoid arthritis other diseases that may cause neck pain, back pain, shoulder pain, neck and shoulder tumors, neuritis, arteriovenous diseases and Infection and pain involved in chest and abdominal organ diseases.
  • chest, abdomen and back pain refers to pain caused by diseases of the visceral and thoracic and abdominal tissues of the chest and abdomen, including but not limited to intercostal neuralgia, intercostal chondritis, angina pectoris, abdominal pain (acute abdominal visceral pain). And lumbar back myofascial syndrome.
  • Waist, lower limb pain refers to lower back, lumbosacral, sacral, hip, hip, and lower extremity pain. Waist and lower extremity pain is often not an independent disease, but a common feature of multiple diseases, diverse clinical manifestations, complex causes, degenerative and damage, including but not limited to lumbar disc herniation, acute lumbar sprain, sciatica , Osteoporosis, Third Lumbar Vertebrae Transverse Syndrome, Piriformis Syndrome, Knee Osteoarthritis, Tail Pain and Foot Root Pain.
  • muscle and bone pain includes, but is not limited to, myofascial pain, pain caused by trauma, and chronic Regional pain syndrome.
  • painful diabetes refers to pain caused by a neurological injury associated with diabetes, which is caused, at least in part, by decreased blood flow and hyperglycemia. Some diabetic patients do not develop neuropathy, and other patients develop the disease early. Diabetic neuropathic pain can be divided into single neuropathy and systemic polyneuropathy involving one or more lesion sites, which may be diffusion and symmetry. Often, it mainly involves the way of feeling (Merrit's Textbook of Neurology, 9th edition). The manifestations of diabetic neuropathy can include plant neurological dysfunction, leading to dysregulation including the heart, smooth muscles, and glands, causing hypotension, diarrhea, constipation, and sexual incompetence.
  • Diabetic neuropathy often develops in stages, early in the nerve ending area, autonomic or sensory neuropathy occurs in the foot, cranial neuropathy occurs around the face and around the eyes, intermittent pain and tingling, in the subsequent stages, Pain is stronger and more frequent. Finally, when pain loss occurs in a certain area, it occurs as a painless neuropathy. Since there is no pain as an indication of injury, the risk of serious tissue damage is greatly increased.
  • visceral pain includes, but is not limited to, irritable bowel syndrome (IBS), with or without chronic fatigue syndrome (CFS), inflammatory bowel disease (IBD), and interstitial cystitis. .
  • IBS irritable bowel syndrome
  • CFS chronic fatigue syndrome
  • IBD inflammatory bowel disease
  • interstitial cystitis interstitial cystitis
  • vascular pain is pain that results from one or more of the following factors.
  • the organization's injections are not appropriate.
  • occlusive arteriosclerosis occlusive thromboangiitis
  • acute arterial occlusion embolism
  • congenital arteriovenous stenosis vasospasm disease
  • Rayaud disease hand and foot cyanosis
  • acute venous closure thrombophlebitis
  • varicose veins And lymphedema examples include, but are not limited to, occlusive arteriosclerosis, occlusive thromboangiitis, acute arterial occlusion, embolism, congenital arteriovenous stenosis, vasospasm disease, Rayaud disease, hand and foot cyanosis, acute venous closure, thrombophlebitis, varicose veins And lymphedema.
  • cancer pain refers to the pain that occurs when a malignant tumor develops. Cancer pain is currently thought to have three mechanisms, namely: pain directly caused by cancer development, pain caused by cancer treatment, and cancer patients. Complicated with painful diseases.
  • autonomic reflex pain refers to pain caused by "reflex sympathetic atrophy.”
  • Reflex sympathetic atrophy sign refers to the body's acute and chronic pain, severe spontaneous pain, allergies to touch and pain, accompanied by edema and blood disorder, followed by dystrophic and atrophic symptoms of skin and musculoskeletal.
  • postoperative pain refers to a complex physiological response of the body to the disease itself and to tissue damage caused by surgery, which manifests itself as an unpleasant experience of psychology and behavior.
  • arthritic pain includes, but is not limited to, osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, psoriasis joint disease, gout, pseudogout, infectious arthritis, tendonitis, bursa Pain caused by diseases such as inflammation, bone damage and joint soft tissue inflammation.
  • post-herpetic neuralgia means that the herpes zoster rashes after healing in the original rash area The intense pain that persists under the skin for a long time.
  • nociceptive pain is pain caused by a process of tissue damage that induces noxious receptors, or by excitability caused by nociceptors.
  • the pain caused by the excitability of the nociceptors may be caused by the persistent noxious stimuli of the nociceptors or their sensitization or both, or they may be caused by these factors, and by their persistence, various reflex mechanisms and other factors And extended.
  • Pain refers to the neural mechanism by which harmful stimuli are detected. Pain involves two steps: peripheral nerve conduction damage stimulation and transmission of these signals to the central nervous system.
  • alkyl refers to a saturated, straight or branched aliphatic hydrocarbon group.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl.
  • the "alkyl group” is preferably a d- 8 alkyl group; more preferably a d- 6 alkyl group; more preferably a d- 4 alkyl group.
  • alkenyl denotes a straight or branched chain hydrocarbon radical containing at least one carbon to carbon double bond and at least 2 carbon atoms, preferably 2 to 6 carbon atoms.
  • the "alkenyl group” is preferably a C 2 -8 alkenyl group; more preferably a C 2 -6 alkenyl group; more preferably a C 2 _ 4 alkenyl group.
  • block group means having at least one carbon-carbon triple bond and at least 2 carbon atoms (preferably 2-8 carbon atoms; more preferably 2-6 carbon atoms; more preferably 2- Linear and branched hydrocarbon groups of 4 carbon atoms.
  • halogen used herein? , Cl, Br or I, especially F, C1 or Br.
  • alkoxy refers to an oxygen-containing alkyl group such as methoxy, ethoxy, n-propoxy, isopropoxy and the like. Preferably it contains 2-8 carbon atoms; more preferably 2-6 carbon atoms; more preferably 2-4 carbon atoms.
  • cycloalkyl refers to a cycloalkyl group having from 3 to 7 carbon atoms, such as cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl.
  • cycloalkenyl as used herein is defined as “cycloalkyl” and contains at least one unsaturated carbon-carbon double bond.
  • aryl refers to an aromatic system which may be a single ring or a polyaromatic ring which is originally fused or linked together such that at least a portion of the fused or linked ring forms a conjugated aromatic system.
  • Aryl groups include (but are not limited to): phenyl, naphthyl
  • heterocycle refers to a stable 4-7 membered monocyclic or stable polycyclic heterocycle which may be saturated, partially unsaturated or unsaturated, and which consists of a carbon atom and is selected from the group consisting of The 1-4 heteroatoms make up: N, 0 and S atoms. The N and S atoms can be oxidized.
  • the heterocyclic ring may also include any polycyclic ring in which any of the above heterocyclic rings may be fused to the aromatic ring.
  • heteroaryl refers to a heterocyclic aryl group.
  • isomer as used herein includes: conformational isomers, optical isomers (such as enantiomers and diastereomers), geometric isomers (such as cis and trans isomers).
  • optical isomers such as enantiomers and diastereomers
  • geometric isomers such as cis and trans isomers.
  • Inverse agonist of ⁇ -aminobutyric acid ruthenium receptor containing ⁇ 5 subunit and use thereof
  • Studies on the inverse agonist of the x5-GABA A receptor have been carried out for more than a decade, and new compounds have been synthesized one after another. Previous studies have suggested that it may treat learning and memory (cognitive) related diseases.
  • the inventors of the present inventors have found that an inverse agonist of the x5-GABA A receptor can effectively inhibit pain.
  • the inverse agonist of the x5-GABA A receptor is capable of binding to the (x5-GABA A receptor).
  • the compound which can bind to (x5-GABA a receptor benzodiazepine binding site the compound may be (x5-GABA a receptor inverse agonists. preferably some not effectively enter the blood brain barrier A compound that binds to the (X5-GABA A receptor in the central nervous system.
  • the ⁇ 5 subunit protein of the GABA A receptor is expressed in the dorsal root ganglia and is mainly transported to the periphery; whereas the ct5 subunit protein of the GABA A receptor is elevated in the CFA-induced inflammatory pain model and the SNI model.
  • the expression of the ct5 subunit protein of the GABA A receptor in the dorsal root ganglion can significantly inhibit inflammatory pain and neuropathic pain.
  • an inverse agonist of the (x5-GABA A receptor) can significantly inhibit inflammatory pain and neuropathic pain.
  • the ability of the inverse agonist of a5-GABA A to bind to the a5-GABA A receptor is greater than the ability to bind to a GABA A receptor comprising only the alpha or alpha 2 or alpha 3 subunit.
  • the present invention provides a (x5-GABA A receptor) as shown in the structural formula (I and ⁇ )
  • each group has the same definition as before.
  • the present invention provides a structure represented by (IV) of the ct5-GABA A receptor represented by the structural formula ( ⁇ ).
  • each group has the same definition as before.
  • Inverse agonists of 5-GABAA receptors also include those mentioned in the following patents: WO 201 1/107812 A2, WO 2010/1 12475 Al, WO 2010/104843 A2, WO 2010/002451 A1, WO 2010/127978 Al, WO 2010/127968 Al, WO 2010/097368 Al, WO 2010/125042 Al, WO 2010/028769 Al, WO 2010/094669 Al, WO 2010/127976 Al, WO 2010/127975 Al, WO 2009/ 000662 Al , WO 2009/071464 Al , WO 2009/071476 Al , WO 2009/071477 Al , WO 2008/154438 Al , WO 2008/154442 Al , WO 2008/154447 Al , WO 2007074089 Al , WO 2007/071598 Al , WO 2007/074078 A2, WO 2010/127974 Al, WO 2007/137954 Al, WO 2007/042420 Al, WO 2007/054444 A2,
  • Inverse agonists of the GABA A receptor comprising an alpha 5 subunit include, but are not limited to, the compounds described in the above patents.
  • the drugs for the brain barrier are:
  • M K016 can be in vivo Metabolism is MRK016-M1, MRK016-M2. MRK016-M3 and other structures.
  • MRK016-M3 and replacement page (Article 26) MRK016-M2 has an obvious reverse agonistic effect of the x5-GABA A receptor, but it does not effectively bind to the x5-GABA A receptor. This suggests that x5-GABA cannot effectively enter the blood-brain barrier. Inverse agonists of the A receptor are effective in the treatment of inflammatory pain and neuropathic pain, and are highly safe drugs.
  • the inventors have also discovered that It can effectively inhibit inflammatory pain and neuropathic pain. They can hardly cross the blood-brain barrier and do not directly affect the central function. They are drugs that have small central side effects and can effectively treat pain. These compounds are preferred compounds in the present invention.
  • the present invention also includes isomers, racemates, pharmaceutically acceptable salts, hydrates or precursors of the various compounds enumerated above.
  • the "pharmaceutically acceptable salt” means a salt formed by reacting the above compound with an inorganic acid, an organic acid, an alkali metal or an alkaline earth metal or the like.
  • These salts include, but are not limited to: (1) salts with the following inorganic acids: such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts with the following organic acids, such as acetic acid , lactic acid, citric acid, succinic acid, fumaric acid, gluconic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, tartaric acid, maleic acid, or refined ammonia acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, ni
  • salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, ammonium or water-soluble amine salts such as N-methylglucamine salts, and lower alkanolammonium salts.
  • other pharmaceutically acceptable amine salts such as methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine
  • a salt an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trishydroxyethylamine salt, and an amine salt formed from morpholine, piperazine, and lysine, respectively, or other conventional "prodrugs" " form.
  • the compound has one or more asymmetric centers. Thus, these compounds may exist as racemic mixtures, as individual enantiomers, as individual diastereomers, as mixtures of diastere
  • precursor of a compound means a compound which is converted into a compound of the formula I, or a compound of the formula I, by a metabolic or chemical reaction of a precursor of the compound in a patient after administration by an appropriate method. Salt or solution.
  • Precursors of the compounds include, but are not limited to, carboxylates, carbonates, phosphates, nitrates, sulfates, sulfone esters, sulfoxide esters, amino compounds, carbamates, azo compounds, phosphoramides of the compounds. , glucoside, ether, acetal, etc.
  • MRK-016 a GABAA receptor alpha5 subtype-selective inverse agonist. J Pharmacol Exp Ther. 2009 Nov;331(2):470-84 .
  • MRK016, MRK016-M3, and a5IA are all inverse agonists that bind to benzodiazepine binding site selectivity a5, MRK016 (MRK-016) and its major metabolites (Ml, M2, M3)
  • the binding affinity (Affinity) and potency (Efficacy) of each subtype of GABA A receptor are shown in Table 1. Screening method
  • an inverse agonist of the a5-GABA A receptor can be screened for binding to (x5-GABA A receptor) based on this characteristic by selectively binding to the (x5-GABA A receptor to exert a pain-inhibiting effect).
  • the substance of the body After that, a drug that is really useful for suppressing pain can be found from the substance.
  • the present invention provides methods of screening prevention, amelioration or treatment of pain, said method comprising: (1) providing a system a5-GABA A receptor, the a5-GABA A receptor for GABA A subject having a complete (2)
  • the candidate substance is administered to the system of step (1) to observe the binding of the candidate substance to the (x5-GABA A receptor; if the candidate substance is capable of binding to the (x5-GABA A receptor receptor,
  • the candidate substance is a potential drug for preventing, ameliorating or treating pain.
  • the system comprising the ct5 subunit of the GABA A receptor may be, for example, a cell (such as a dorsal root ganglion cell) system, and the cell may be endogenous.
  • ct5 cells expressing GABA a subunit receptor may also be a subcellular system, solution system. , tissue system, organ system or animal system (such as animal models, preferably animal models of non-human mammals, such as rats, rabbits, sheep, monkeys, etc.) and the like.
  • a control group in the screening, in order to make it easier to observe the difference in binding of the ct5 subunit comprising the GABA A receptor to the candidate substance, a control group may be provided, and the control group may not be added.
  • candidate substances may include, but are not limited to, small molecule compounds (inverse agonists of the GABA A receptor), polypeptides, ligands.
  • the candidate substance is a substance that does not cross the blood-brain barrier of the animal.
  • the method further comprises: performing further cell experiments and/or animal tests on the obtained potential substances to further select and determine substances that are truly useful for preventing, ameliorating or treating pain.
  • the present invention also includes a pain-suppressing substance obtained by the screening method.
  • Detection of whether a compound binds to a receptor or a partial subunit of a receptor is a technique known in the art. It has been done to test whether a compound is an inverse agonist or antagonist against the a5-GABA A receptor.
  • GABA A is used in the international application patents WO 92/22652 and WO 94/13799.
  • the combination of ⁇ 5, ⁇ 3 and ⁇ 2 of the receptor detects whether a compound binds to the receptor; in the process of drug screening, Goeders et al. (Goeders NE and Kuhar MJ, Benzodiazepine binding in vivo with [ 3 H] are usually used. Ro 15-1788. Life Sci, 1985 Jul, 37(4): 345-355).
  • Detection of a ligand that binds to the a5-GABA A receptor is an antagonist, agonist or inverse agonist.
  • MRK016 can effectively inhibit the binding of ( 3 H)Ro-15-1788 in the central, while MRK016-M3 does not significantly inhibit the binding of (1H)RO-15-1788 in the central. It can also be determined by detecting the drug in different tissues, such as detecting the distribution of drugs in the brain and plasma to determine whether the drug can effectively enter the blood-brain barrier.
  • the present invention provides for the safe and effective treatment of acute and chronic pain by providing a method of treating pain.
  • a method of treating pain according to the present invention comprises administering to a patient in need of such treatment an effective amount of an inverse agonist of the X5-GABA A receptor.
  • the invention is particularly applicable to elderly patients suffering from pain, liver function and people with poor renal function.
  • Chronic pain is a common condition for most elderly people, and pain is often thought to be a concomitant symptom of organ aging and disease in the elderly.
  • Pain in the elderly mainly includes joints from the joints of the bones and joints, back and neck pain, headaches and other chronic diseases. The most common manifestations are joint pain, stiffness and limited mobility.
  • Degenerative osteoarthrosis includes articular cartilage degeneration and bone hyperplasia (spur or osteophyte). In people over 50 years of age, the incidence is second only to heart disease, and the incidence rate can reach 60 in people over 65 years old. % ⁇ 90%.
  • Osteoarthritis is the most common cause of pain. More than 80% of the elderly over the age of 60 have osteoarthritis, such as joint pain, ringing during movement, walking obstacles and morning stiffness. In severe cases, there may be joint swelling. However, elderly patients are often accompanied by other chronic diseases such as hypertension, diabetes and heart disease. In order to treat the corresponding diseases, it is necessary to take a variety of different drugs at the same time, and the elderly are less tolerant to drugs and prone to adverse reactions.
  • the present invention provides a method of treating pain, and more particularly a method of treating acute pain and chronic pain.
  • the method is to treat pain with a ligand for the a5-GABA A receptor, and these drugs can be administered orally, subcutaneously, transdermally, intranasally or by an automatic injection device.
  • the ligand for treating pain is directed against an inverse agonist of the x5-GABA A receptor, which specifically targets (x5-GABA A receptor, cannot effectively cross the blood-brain barrier, but at the same time It may also be an antagonist or inverse agonist of a GABA A receptor comprising alpha or alpha 2 or alpha 3.
  • the ligand may also be an antagonist of the GABA A receptor.
  • the method of treating pain in the present invention can be prepared and administered in various administration forms such as oral and parenteral.
  • the compounds of the present invention may be administered by injection, i.e., intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally or intraperitoneally; the compounds of the present invention may also be administered by inhalation, for example Intranasal inhalation; further, the compounds of the invention may be administered transdermally.
  • the compound of the present invention can be used to prepare a pharmaceutical composition.
  • the pharmaceutically acceptable carrier can be a solid or a liquid.
  • the solid preparation includes a powder, a tablet, a pill, a capsule, a sachet, a suppository and a dispersible granule.
  • the solid carrier can be one or more. They can be used as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.
  • the carrier is a finely divided solid, and the compound of the invention is present in the mixture with the finely divided active component.
  • the compound is mixed with the desired binding carrier in an appropriate ratio and compressed into the desired shape and size.
  • Powders and tablets preferably contain from 5 to 70% active compound, suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, carboxymethylcellulose, carboxymethyl Sodium cellulose, low melting wax and cocoa butter.
  • suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, carboxymethylcellulose, carboxymethyl Sodium cellulose, low melting wax and cocoa butter.
  • cachets or lozenges, tablets, powders, capsules, pills, cachets, and lozenges may be solid dosage forms suitable for oral administration.
  • Formulations in solution form include solutions, suspensions and emulsions such as water or aqueous propylene glycol solutions.
  • aqueous solution suitable for oral administration can be prepared by dissolving the active ingredient in water and adding a suitable coloring agent, flavoring agent, emulsifier and thickening agent as needed.
  • Aqueous suspensions suitable for oral administration can be prepared by dissolving the finely divided active component in aqueous viscous materials such as natural or synthetic gums, resins, methylcellulose, sodium hydroxymethylcellulose, and other well known suspensions. Agent.
  • solid form preparations which are intended to be converted shortly before use to liquid formulations for oral administration.
  • liquid forms include solutions, suspensions and emulsions, which may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, synthetic or natural sweeteners, dispersing agents, thickening agents and supplements. Solvents, etc.
  • the pharmaceutical preparation is preferably in a unit dosage form, in which the preparation is subdivided into unit doses containing appropriate amounts of the active ingredient, which may be a packaged preparation containing a quantity of preparation such as a packaged tablet , capsules and powders in vials or capsules.
  • the unit dosage form can also be a capsule, tablet, cachet or lozenge itself, or any suitable amount of any of these powders may be present in the package.
  • the amount of the active ingredient in a unit dose preparation may vary depending on the particular application and the effectiveness of the active ingredient, and may be adjusted from 0.01 mg to about 0.1 go.
  • the drug may be in the form of a capsule of from 0.1 to about 3 mg per day. Administration is three times and, if desired, the composition may also contain other compatible therapeutic agents.
  • the compounds used in the present invention are administered at a starting dose of from 0.001 mg to 10 mg/kg body weight per day.
  • these dosages may vary depending on the needs of the patient, the severity of the condition being treated, and the compound employed.
  • treatment begins with a smaller dose that is less than the optimal dose of the compound, after which a small increase in the dose is achieved.
  • the total daily dose can be subdivided into fractionated doses per day if needed.
  • compositions of this invention may also be used in combination with other therapeutic agents or adjuvants, including but not limited to morphine, gabapentin, and the like.
  • the present invention provides a medicament for treating pain which is not only effective but has no obvious side effects, and another object of the present invention is to provide a special patient population such as an elderly person having liver or kidney function.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in the "Molecular Cloning Experimental Guide” (Science Press, 2002) compiled by conventional conditions such as J. Sambrook et al., or according to the manufacturer. Recommended conditions. Percentages and parts are by weight unless otherwise stated.
  • mice Male Sprague-Dawley rats (200-250 g) were placed in cages in groups of 6 and all animals were placed on a 12-hour light/cycle (ie 12 hours of illumination and 12 hours of darkness per day), with any food and water. All trials were performed without the observer's knowledge of the medication.
  • Partial nerve injury model (SNI): intraperitoneal injection of 10% chloral hydrate, resulting in anesthesia in rats, incision of the skin on the upper limbs of the hind limbs, longitudinal separation of muscles, exposure of the main sciatic nerve and its branches under the sacral nerve, sputum total
  • the muscles and skin are sutured layer by layer, and then antibiotics are taken by intraperitoneal injection for antibacterial prevention.
  • the specific method can be referred to (Decosterd I and Woo If CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. 2000 Aug; Pain 87(2): 149-58.).
  • CFA Complete Finnish Adjuvant (CFA)-induced Inflammatory Pain Model: After 10% chloral hydrate was intraperitoneally injected (0.3 ml/100 g body weight), CFA 200 ⁇ l was injected subcutaneously into both hind toes and feet.
  • CFA liquid preparation method CFA liquid (physiological saline of Mycobacterium tuberculosis: oil suspension) sold by American Sigma Company.
  • Sciatic nerve and dorsal root nerve ligation model Normal rats, injected with 10% chloral hydrate, causing anesthesia in rats, incision of the skin on the upper limbs of the hind limbs, longitudinal separation of muscles, exposure of the sciatic nerve trunk with ligation of the gut; Nerve ligation, routine alcohol disinfection after shaving on the buttocks, longitudinal 2 cm incision at the lumbar 3 and lumbar vertebrae, blunt From the muscle tissue on both sides of the spine, the expander expands the muscles and skin, so that the surgical field is exposed sufficiently large, the waist 3 vertebrae are destroyed with a surgical forceps, and the dorsal nerves of the waist 5 and the waist 6 are exposed, and the gut is ligated with the gut. After 24 hours, the rats were perfused and then taken, and the dorsal root ganglia, dorsal root nerve, spinal dorsal horn and sciatic nerve were taken.
  • Pentylenetetrazol-induced epilepsy test Rats were first injected with C5 or 70% PGE400, and half an hour later, rats were injected subcutaneously with 100 mg/kg of pentylenetetrazol, and pentylenetetrazol was dissolved in saline, and then the rats were observed. Convulsion reaction. The convulsion score was performed using the Fatholahi grading method. Grade 0, no response; Grade 1, rhythmic mouth and facial twitching; Grade 2, body wandering migratory sputum; Grade 3, whole body myoclonus, buttocks up; Grade 4, body Flip to one side; level 5, tilt up position, body tonic attack. Observe for 30 minutes after each administration. The maximum number of seizures in rats and the latency of pentylenetetrazol injection to induce the maximum response level were counted.
  • the rat sacral cancer pain model reflects the metastatic cancer pain model in clinical patients.
  • 10% chloral hydrate was injected, the rats were anesthetized, the skin was cut in the upper limbs of the hind limbs, the muscles were longitudinally separated, the tibia was exposed, and the 3x10 3 MRMT of the homologous rat was injected into the medullary cavity of the tibia.
  • the rats showed significant pain-sensitive responses.
  • the rats in the operation model were perfused with the fixative at different time points, and fixed for 1.5 hours.
  • the tissue soaked for 10 hours in 10% sucrose for 24 hours or more was cut into 14 ⁇ slices in a cryostat, and placed in -20. °C refrigerator to save.
  • Sections and antibodies (anti-015-0 8 4 receptor (8 & & 03 ⁇ 42) goat-derived, effective dilution ratio 1: 100 ⁇ 200) were incubated overnight, then washed three times with 0.01 M PBS, then the secondary antibody was incubated for 40 minutes. Then wash three times with 0.01 M PBS, protected from light. Finally, observe under the microscope and take a film.
  • Immunofluorescence intensity The immunofluorescence intensity was quantitatively analyzed by Image-pro.
  • the dorsal root ganglia of the material used were RIPAC50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, And sodium ortho vanadate, sodium fluoride, EDTA, leupeptin and other inhibitors. Cracking, boiling at 100 °C for 5 minutes to denature the protein, waiting for the protein sample to cool After pipetting 10-20 ⁇ , the sample was added to the prepared pores.
  • the SDS gel containing the target strip is cut, and the membrane transfer operation is performed, that is, the negatively charged protein is transferred to the PVDF membrane with the positive electrode by electroporation, and in the blocking solution containing 5% skim milk powder. Block for 1 hour to reduce non-specific binding of the antibody. After the end of the blocking, the PVDF membrane was infiltrated in a blocking solution containing primary antibody at 4 ° C overnight or at room temperature for 1-2 hours.
  • the PVDF membrane was washed three times with TBST eluate for a total of 1 hour, and then The secondary antibody with horseradish peroxidase was incubated for 1 hour at room temperature. After incubation of the secondary antibody, it was washed with TBST eluent for 1 hour. Finally, the PVDFA membrane and the horseradish peroxidase substrate ECL-PLUS were reacted for 5 minutes in the dark to generate a fluorescent signal, and then X-ray film was used. Obtain a fluorescent signal on the PVDF membrane. The film is fixed by the developer and fixer. (4) Pain detection method
  • the rats were injected with CFA solution for 24 hours on the plantar, and the heat-induced sensitivity test was used to determine the contractile response of rats to thermal radiation stimulation.
  • the rats were placed in a plexiglass box with a transparent glass plate, and the paws of the rats were stimulated with a thermal radiometer (BME-410C; CAMS).
  • the thermal pain threshold is the length of time that the light source shines on the rat's foot and reaches the rat's reflex response.
  • the dose is 3 mg/kg as an injection.
  • the siRNA was configured as an injectable solution according to the methodology described in EX 500.
  • the rats were then anesthetized and the siRNA solution was injected from the site of the waist 5 or waist 6 into the spinal cord.
  • the siRNA sequence for the a5-GABA A receptor mRNA is: 5 'GGUGCGAACAGACAUCUAUTT 3 ⁇
  • the control siRNA sequence is: 5 'UUCUCCGAACGUGUCACGUTT 3'.
  • Example 1 Increased expression of the ⁇ 5 subunit of GABA A receptor under SNI model conditions
  • Immunohistochemistry experiments showed that the SNI model can induce an increase in the expression of the ⁇ 5 subunit protein of the GABA A receptor.
  • the expression of the ct5 subunit protein of the GABA A receptor was increased 14 days after the SNI model preparation, and the control was the dorsal root ganglion section of the normal rat (normal), as shown in Fig. 1A.
  • the results showed that the expression of the ct5 subunit of GABA A receptor was increased in the dorsal root ganglia. More interestingly, the fluorescence signal intensity of the ct5 subunit protein of GABA A receptor was significantly increased in small cells.
  • GABA A receptor ct5 subunit protein was found to be elevated in small cell expression in dorsal root ganglia. Statistics show that the expression level of GABA A receptor ct5 subunit is increased in the dorsal root ganglia. Western blotting showed that the protein amount of the GABA A receptor ct5 subunit was significantly increased in the inflammatory pain model (Fig. 2B), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a positive control.
  • GABA A receptor ct5 subunit protein was found to be elevated in small cell expression in dorsal root ganglia.
  • Statistics show that the expression level of GABA A receptor ct5 subunit is increased in the dorsal root ganglia.
  • Western blotting showed that the protein amount of the GABA A receptor ct5 subunit was significantly increased in the inflammatory pain model (Fig. 2B), and glyceraldehyde-3-phosphate dehydrogenas
  • the ⁇ 5 subunit protein of the GABA A receptor is expressed in the dorsal root ganglia and transported to the sciatic nerve in the model of sciatic nerve and dorsal root nerve ligation, and the amount of GABA A receptor ct5 subunit protein is observed.
  • Figure 3 shows.
  • FIG. 3 shows that the GABA A receptor ct5 subunit protein can be transported to the sciatic nerve with little transport to the dorsal root nerve.
  • CGRP calcium phosphate phosphate
  • CGRP calcium phosphate
  • GABA A receptor subunit ct5 sciatic nerve was significantly higher than the amount of dorsal root ganglia, spinal cord and less in the amount of protein in the GABA A receptor subunits ct5, glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) was used as a positive control.
  • GAPDH glyceraldehyde-3-phosphate dehydrogenase
  • the SNI model mimics neuropathic pain associated with nerve damage, which can be directly caused by trauma and crush, or can be caused indirectly by a range of diseases such as infections, cancer, metabolic diseases, toxins, Nutritional deficiencies, immune dysfunction, and musculoskeletal changes.
  • Neuropathic pain is one of the most difficult pains in the current clinical practice, which seriously affects the quality of life of patients.
  • Gabapentin is currently the first line of treatment for neuropathic pain, but gabapentin can cause side effects such as drowsiness, dizziness, unstable walking and fatigue.
  • the present inventors have found that the compound MRK016 and its metabolite MRK016-M3 can effectively inhibit pain.
  • subcutaneous injection of MRK016 was effective in inhibiting neuropathic pain, and the results showed that pain was significantly inhibited 1 hour after the drug was administered, as shown in Fig. 4A.
  • MRK016-M3 The inventors examined whether MRK016-M3 can inhibit neuropathic pain. Through dose-dependent testing, MRK016-M3 was also found to be effective in inhibiting neuropathic pain, as shown in Figure 4B. In this experiment, the inventors used rats established with the SNI model for 10 days to 20 days as experimental animals, and found that doses of 10 mg/kg, 3 mg/kg, and 1 mg/kg were administered 1 hour after oral administration. Both can significantly inhibit the mechanical pain threshold.
  • the present inventors simultaneously examined the effect of gabapentin in the treatment of neuropathic pain.
  • the SNI model of the 1 day rat as an experimental animal found that gabapentin can effectively treat neuropathic pain, as shown in Fig. 4C; but MRK016-M3
  • the dose is much smaller than that of gabapentin, and the effect of treating pain is similar.
  • MRK016-M3 acts through the peripheral nerve
  • the inventors established the SNI model as a laboratory animal for 10 days as a laboratory animal, and administered MRK016-M3 (1 mg/ml) 100 ⁇ l to the paw of the rat.
  • MRK016-M3 1 mg/ml
  • neuropathic pain can also be significantly suppressed, as shown in Fig. 4D.
  • the role of the a5-GABA A receptor in the SNI model was examined by gene knockout experiments.
  • the inventors targeted the GABA A receptor ⁇ 5 subunit by intrathecal injection.
  • the siRNA of the base mRNA was statistically found to significantly knock out the expression of the GABA A receptor ⁇ 5 subunit protein in the dorsal root ganglia, as shown in Fig. 4 ⁇ .
  • GABA A receptor ⁇ 5 subunit protein was significantly decreased in the dorsal root ganglia after injection of specific siRNA.
  • the control siRNA did not significantly change the GABA A receptor ct5 subunit protein.
  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a positive control.
  • GABA A receptor ct5 subunit was found to significantly increase the threshold for pain, and non-specific siRNA had no significant effect on pain, as shown in Figure 4F.
  • the inventors injected siRNA against the GABA A receptor ⁇ 5 subunit into the rat spinal cord 7 days after the SNI model was established, and then in the SNI model for 9 days, 1 day, 13 days, 15 days, and 17 days.
  • the pain threshold of the rats was separately measured.
  • the pain threshold was measured 4 days after the establishment of the SNI model, followed by injection of control siRNA and siRNA against the GABA A receptor ⁇ 5 subunit. The effect of MRK016-M3 on the pain threshold of rats was examined after 4 days.
  • MRK016-M3C was orally administered in rats injected with control siRNA, 3 mg/kg) To significantly inhibit neuropathic pain; in rats injected with specific siRNA against the GABA A receptor ct5 subunit,
  • MRK016-M3 oral, 3 mg/kg had no significant effect, as shown in Figure 4G.
  • X5-GABA A receptors are biologically active substances that are potential substances for the prevention and treatment of pain.
  • Inflammatory pain is generally caused by inflammation of the tissue or organ due to inflammation of the lesion, and there is acute pain and chronic pain.
  • the tissue injury pathological pain model is a model similar to clinical inflammatory pain, in which the CFA-induced inflammatory pain model reflects various indicators of clinical chronic inflammatory pain.
  • the inflammatory response begins at the injection site 2-3 hours after CFA injection, peaking at 6-24 hours, and pain sensitivity and edema can last for 1-2 weeks (Ladarola et al" Differential activation of spinal cord dynorphin and enkephalin neurons During hyperalgesia: evidence using cDNA hybridization.” Brain Res. 1988, vol. 455, No. 2, pp: 205-12), the model has significant inflammatory pain characteristics and a long duration.
  • the present inventors have found that the compound MRK016 and its metabolite MRK016-M3 can effectively inhibit inflammatory pain.
  • Intraperitoneal injection of MRK016 can effectively inhibit inflammatory pain.
  • the present inventors injected MRK016 (2 mg/kg body weight) intraperitoneally and found that the compound can effectively inhibit mechanical pain (Fig. 5C); In the inflammatory pain model, it is sensitive to heat pain for 3 days.
  • intraperitoneal injection of MRK016 (2 mg / kg body weight)
  • the compound was found to be effective in inhibiting heat pain (Fig. 5D); the experimental results showed that MRK016 can significantly inhibit inflammatory pain 1 hour after the fight.
  • Oral administration of MRK016-M3 can also effectively inhibit inflammatory pain.
  • the present inventors detected mechanical pain in the model of inflammatory pain for 3 days to detect thermal pain and inflammatory pain for 7 days, and the results showed that MRK016-M3 was administered 1 hour after the fight. Significant inhibition of inflammatory pain, as shown in Figures 5E and F.
  • the experiment also proved that: the inverse agonist acting on the peripheral nerve (x5-GABA A receptor can also treat inflammatory pain.
  • the experiment shows: can bind a5-GABA A receptor but not cross the blood brain barrier Specific inverse agonists can effectively inhibit inflammatory pain, providing a new screening method for screening clinical drugs for potential analgesia.
  • Example 6 a5-GABA A receptor inverse agonist A5I treatment of neuropathic pain and Inflammatory pain
  • ⁇ 5 ⁇ ( ⁇ 5 ⁇ ) is an inverse agonist specifically developed for the a5-GABA A receptor by Merck. In clinical trials, it was found that the drug caused certain toxicity problems due to water solubility problems.
  • the inventors found that intraperitoneal injection of A5I (3 mg/kg body weight) can also effectively treat inflammatory pain, as shown in Figures 6C and D.
  • the inflammatory pain model was established for 3 days to detect the effect of A5I on thermal pain.
  • the pain threshold was measured at 0.5, 1.0, 2.0, and 3.0 hours after the drug was administered, and the A5I was detected on the machine 7 days after the establishment of the inflammatory pain model.
  • the effect of the pain threshold was tested for mechanical pain thresholds at 0.5, 1.0, 2.0, and 24 hours after drug administration.
  • Different subunits of the GABA A receptor are expressed in cell lines, including the human renal epithelial cell line 293 cell line or the L(tk-) cell line.
  • the cells were cultured in a medium, and the cells were used as a cell model for screening drugs for inhibiting pain.
  • An et subunit, beta subunit and gamma subunit are essential for the formation of a complete functional GABA A receptor.
  • the inventors established the following cell model:
  • the composition contains the a5-GABA A receptor;
  • a GABA A receptor comprising an ⁇ -purine subunit
  • the ⁇ 3 subunit (see GenBank accession number 000000808.3), the ⁇ 3 subunit and the ⁇ 2 subunit were simultaneously expressed in the 293 cell line to constitute a GABA A receptor containing the ⁇ 3 subunit.
  • the cell membrane is then extracted and the protein concentration is detected, and the competitive binding of the test substance to the radiolabeled compound (for example, 1 nM [1H]Ro-0151788) is detected, and the Ki value of the substance to be tested is calculated.
  • the above method can be referred to the literature (BallardTM et al., R04938581, a novel cognitive enhancer acting at GABAA alpha5 subunit-containing receptors. Psychopharmacology (Berl). 2009 Jan; 202(1-3): 207-23).
  • the candidate is a potential drug for preventing, ameliorating or treating pain.
  • the candidate is a potential drug for preventing, ameliorating or treating pain.
  • Those skilled in the art are aware of a number of methods that can be used to assess the ability of a5-GABA A receptor-containing ligands to bind to (x5-GABA A receptors, including but It is not limited to the above method.
  • the inventors detected the reverse activation efficiency of the substance to be tested by an electrophysiological method. This method can refer to the literature
  • test substance can significantly inhibit GABA-induced current through the a5-GABA A receptor, but at the same time can not significantly inhibit GABA-induced cd-containing or (x2 or (x3 GABA A receptor-induced current, it indicates that the candidate is preventive) , amelioration or treatment of pain in a medicament.
  • field of the art can be used to evaluate a number of methods understood ligand receptor a5-GABA a inverse agonists of GABA-a receptors efficiency of A5, including but not limited to the above-described method.
  • the blood-brain barrier is detected by detecting the substance to be tested effectively by the blood-brain barrier model (BBBM).
  • BBBM blood-brain barrier model
  • brain microvascular endothelial cells Brain microvessl endothelial cells
  • astrocytes were co-cultured, and then the test substance was tested for effective passage through the cell membrane.
  • HTS high throughput
  • MRK016, MRK016-M3, and a5IA were tested as candidate substances.
  • the results showed that in Table 2, MRK016, MRK016-M3 and ⁇ 5 ⁇ have similar affinity to each subtype of GABA A receptor, but to a5-GABA A receptor.
  • the inverse agonistic efficiency (Efficacy) is much higher than the reverse agonistic efficiency of the GABA A receptor containing ⁇ or ⁇ 2 or ⁇ 3, so that they are useful for inhibiting pain in mammals.
  • MRK016-M3 cannot be worn significantly
  • the blood-brain barrier is theoretically an effective analgesic drug with no side effects.
  • the binding factor Ki (nM) of the candidate substance in the test group to the ct5 subunit of ⁇ -aminobutyric acid A is smaller than the other subunits (cd subunit, (x2 subunit or (x3 subunit)), this candidate is indicated.
  • the test substance can significantly inhibit GABA-induced currents induced by the ABA5 subunit GABA A receptor, it does not significantly inhibit GABA-induced cd-containing or (x2 or (The current induced by the GABA A receptor of x3 indicates that the candidate is a drug for prevention, amelioration or treatment of pain, or both.
  • the above indicates that the candidate is a drug for preventing, ameliorating or treating pain.
  • the animals are administered intraperitoneally (or by oral administration) with a subcutaneous injection of the candidate substance, and the distribution of the candidate substance in the animal is determined at an appropriate time after the treatment.
  • the binding efficiency of the test compound to the central a5-GABA A receptor can be detected by the following two methods.
  • the first method detects the distribution of the drug to be tested in the animal. In this process, whether competitive or inhibition e.g. isotope labeled substance (3 H) Ro- 15- 1788 in hippocampus tissue binding, it can not exceed 60% of the suppression efficiency by detecting a substance to be detected.
  • the experimental animal is given a dose of the candidate substance and (1H)Ro-15-1788 in the experimental animal, and then it is detected whether the candidate substance inhibits the known (x5-GABA A receptor specific ligand in the central distribution, if the candidate substance inhibits The distribution of (1H)Ro- 15- 1788 in the central center is less than 60%, indicating that the candidate is a potential drug for preventing, improving or treating pain.
  • the second method detects the tissue distribution of the test compound in the animal.
  • the test substance is injected (or orally) into the animal, and then the brain tissue and plasma tissue are extracted after 0.25-2.0 hours of injection, and the concentration of the drug is detected by chromatography-mass spectrometry (L-MS), if the compound
  • L-MS chromatography-mass spectrometry
  • reaction solution was evaporated, then washed with water (20 mL) rinse, and then extracted with ethyl acetate (20 mL x 3), the organic layer was dried over MgS0 4, evaporated to a low pressure to give 2 g of product (4-t-butyl-3- (hydroxyalkyl Methyl) -1 hydrogen-pyrazole-5-ol) is a white solid.
  • Example 9 C2, C3, C4 compounds can inhibit neuropathic pain and inflammatory pain
  • the inventors examined the therapeutic analgesic effects of C2, C3, C4 (Fig. 7A) compounds in the SNI model and in the inflammatory pain model.
  • Figure 7A shows the chemical structural formula of compounds C2, C3, and C4.
  • Figure 7B shows that intraperitoneal injection of C2, C3, and C4 compounds is effective in treating neuropathic pain.
  • C2, C3, and C4 compounds 3 mg/kg body weight
  • the pain threshold was measured 1 hour after the drug was administered. The results showed that the compound can effectively inhibit neurogenicity. pain.
  • Example 8 A method for obtaining ethyl 5-hydroxymethyl-isoxazole-3-carboxylate can be referred to in Example 8.
  • Ethyl 5-hydroxymethyl-isoxazole-3-carboxylate (Reference Example 8, 1.5 g; 8.76 mmol) and 1 M sodium hydroxide (18 mL, 18 mmol) were stirred at room temperature for 1.5 hr. Brine (40 mL) was added and the pH was adjusted to 2 with 6N hydrochloric acid. Extract with ethyl acetate (6 x 60 mL). It was then dried over MgSO.sub.4, evaporated.
  • Step 1 Replace the page (Article 26) (a) ⁇ ,-( ⁇ chlorophenylindol-4-yl)-5-(hydroxymethyl)isoxazol-3-carbohydrazide N'-(l-chloro ohydrazide
  • N,-(l-chlorophenylindol-4-yl)-5-(hydroxymethyl)isoxazole-3-carboxylic acid ethyl ester (900 mg, 2.82 mmol) and ethylamine quinadine (154 mg, 0.13 mmol, 0.4 eq.) was refluxed in xylene (60 mL) for 3 h.
  • DCM CH 2 C1 2
  • Example 11 C5, C6, C7, C8 compounds can inhibit neuropathic pain and inflammatory pain
  • the compounds C5, C6, C7 and C8 synthesized in the above examples were dissolved in 70% PGE400 at a dose of 3 mg/kg body weight, respectively, as an injection.
  • the inventors examined the analgesic effects of C5, C6, C7 and C8 compounds in the SNI model and in the inflammatory pain model.
  • Figure 8A shows the chemical structural formulas of the compounds C5, C6, C7 and C8.
  • Figure 8B shows that intraperitoneal injection of C5, C6, C7 and C8 compounds is effective in the treatment of neuropathic pain.
  • C5, C6, C7, and C8 compounds 3 mg/kg body weight
  • C5 has the best analgesic effect in behavioral testing.
  • Figure 8E subcutaneous C5 was administered at a dose of 1 mg/kg body weight and 3 mg/kg body weight, respectively, and then 0.5 hours, 1 hour, 2 hours, 3 hours, 6 hours, and 12 hours after drug administration.
  • the pain threshold of the rats was examined, and it was confirmed that the compound can effectively inhibit neuropathic pain.
  • morphine was used as a positive control and injected subcutaneously at a dose of 10 mg/kg.
  • the inventors also examined whether C5 affects the central nervous system response, and we examined whether C5 potentially induces epileptic response.
  • the inventors examined the distribution of this type of compound in rat tissues.
  • the test substance is first injected subcutaneously into the animal, and then brain tissue and plasma tissue are extracted 1 hour after the injection, and the concentration of the drug is detected by chromatography-mass spectrometry (L-MS), if the compound is in brain tissue and plasma tissue.
  • L-MS chromatography-mass spectrometry
  • the ratio of brain/plasma ratio is less than 0.1, indicating that the candidate is a potential drug with less side effects and can treat pain.

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Abstract

一种含α亚基的γ-氨基丁酸A受体的反向激动剂用于制备预防、改善或治疗疼痛的药物中的用途。

Description

治疗或缓解疼痛的物质
技术领域
本发明属于生物技术领域； 更具体地， 本发明涉及治疗或缓解疼痛的物质。 背景技术
疼痛是一种不同于触、 压、 热或冷的感觉表达。 患者经常用如锐痛、 钝痛、 酸痛、 刺痛、 切割性疼痛或灼烧痛的此类术语描述疼痛。 一般将由伤害神经引起、 表现为痛觉 过敏的疼痛称为 "神经源性" 痛； 而将通过刺激伤害感受体引起的疼痛称为 "伤害性" 痛。
从临床上来讲， 疼痛又可分为两类： 急性痛和慢性痛。 急性痛由皮肤、 身体结构或 内脏的损伤和 /或疾病产生的有害刺激引起，或由不产生实际组织损害的肌肉或内脏的异 常功能引起； 而慢性痛可以定义为持续超出急性疾病通常的病程或损伤治愈的合理时 间， 或与引起持续疼痛的慢性病理过程有关， 或疼痛以一定间隔复发数月或数年， 如果 在应该已经达到治愈后仍存在疼痛， 则认为是慢性痛。 对于本发明的目的， 慢性痛可以 是慢性非缓解的或是复发的， 急性痛和慢性痛之间定义的差异不仅仅是语义上的差异， 而是具有重要的临床相关性， 例如急性痛不能得到很好的控制， 就会发展成为慢性痛。
急性和慢性痛在病因学机制、 病理学和诊断治疗方面不同。 与急性痛的暂时性相对 比， 慢性痛由身体结构或内脏中的慢性病理过程引起， 或由外周或中枢神经系统或这两 者延长的、 有时是永久性的技能障碍引起。 此外， 慢性痛有时可以归因于心理学机制或 /和环境因素。
急性痛一般是指非神经病性疼痛， 包括常见病症， 如关节炎疼痛、 肌肉骨骼疼痛、 手术后疼痛和纤维肌痛。 这些疼痛中的大多数被认为是由软组织和骨损伤引起的， 如关 节炎疼痛、肌肉骨骼疼痛和手术后疼痛，是在正常发挥功能的神经系统中导致炎症反应， 而疼痛仅仅是炎症过程的结果。
慢性痛包括神经源性痛、 炎性痛和癌症痛。 这些疼痛可以与痛觉过敏和 /或异常痛 觉有关， 其中痛觉过敏是指对典型有害刺激的敏感性增高而异常痛觉是指对典型非有害 刺激的敏感性增高。
躯体源性的疼痛是由外周感觉神经损伤或感染引起，它包括但不限于由外周神经损 伤、 疱疹病毒感染、 糖尿病、 灼痛、 血管或神经丛撕脱、 神经痛、 截肢和结节性脉管炎 引起的疼痛， 神经源性痛还由慢性环境中毒、 人免疫缺陷病毒感染、 甲状腺功能减退、 尿毒症或维生素缺乏造成的神经损伤引起， 其临床表现包括但不限于炎症疼痛、 骨关节 疼痛、 三叉神经痛、 癌症疼痛、 糖尿病神经病、 腿多动综合症、 疱疹后神经痛、 灼痛、 臂血管神经丛撕脱、 枕部神经痛、 痛风、 幻肢、 烧伤及其它形式的神经痛、 神经性和自 发性疼痛综合征。 神经源性痛一般被认为是由外周或中枢神经系统被损害或病变引起的慢性痛。与神 经源性痛有关的病变包括长期外周或中枢神经元致敏、与神经系统抑制和 /或兴奋功能受 到损害有关的中枢敏化以及副交感神经和交感神经神经系统之间的异常相互作用。 许多 临床症状与神经源性痛有关或构成神经源性痛的基础，例如糖尿病、切断术的术后疼痛、 背下部疼痛、 癌症、 化学损伤或毒素、 其他严重手术、 由创伤性损害压迫引起的外周神 经损害、 营养缺乏、 感染 (如带状疱疹、 HIV)等。
使疼痛受到注意的刺激水平称为 "疼痛阈值" 。 镇痛剂为通过使患者提高痛阈而不 丧失意识来缓解疼痛的药物。在患痛觉过敏的个体中，镇痛药可具有抗痛觉敏感的作用。 目前， 美国食品和药品管理局 (FDA)所批准的处方镇痛药仅分为两类药物： 阿片类和抗 炎药。 麻醉药属于不同的分类。 阿片类镇痛药通过模拟机体的内源性阿片类物质即内啡 肽和脑啡肽 (其由机体产生以有助于减轻疼痛)而起作用， 通过作用于整个中枢和外周神 经系统的阿片类受体来阻断疼痛。 抗炎药包括非选择性非 类抗炎镇痛药和特异的
COX-2抑制剂， 试图降低由机体损伤引起的前列腺素等化学递质所产生的炎症。
在所有的阿片类镇痛剂中， 吗啡仍然是最广泛使用的镇痛剂。 但是除了它的治疗特 性外， 它有一些缺点， 包括呼吸抑制、 胃肠运动降低 (引起便秘)、 恶心和呕吐。 耐受性 和物理依赖性也限制了阿片类化合物的临床使用。 抗炎类镇痛剂包括阿司匹林和其他水 杨酸化合物， 能够阻止炎症过程的扩大， 并暂时性地减轻疼痛， 然而上述药物对于神经 源性痛效果并不显著。 并且很多抗炎药物， 特别是非 类抗炎药会引起胃肠道副作用， 尤其在口服时， 这种副作用包括引起胃肠道溃疡和糜烂， 这些通常无症状的副作用会变 得严重到需要住院， 甚至可以致命。 加巴喷丁 （Gabapentin) 是当前治疗神经源性痛的 一线临床药物， 得到广泛的应用。 加巴喷丁具有明显的副作用， 包括嗜睡、 眩晕、 行走 不稳或和疲劳感， 这些副作用常见于用药早期。儿童偶尔会急躁易怒， 停药以后会消失。
根据这些现实， 需要更有效的镇痛剂， 它具有很好的安全性和耐药性， 并且无成瘾 性。 理想的镇痛剂将减轻或消除患者对疼痛的感觉， 在各类疼痛发生时产生痛觉缺失， 无论口服还是其他给药方式其作用均令人满意， 产生最小副作用或不产生副作用， 并且 不产生药物耐受和药物依赖的倾向。
γ-氨基丁酸 (GABA)是哺乳动物中枢神经系统中重要的抑制性神经递质， 有两类 GABA受体存在于自然界中， 一类是 GABAA受体， 该类受体为配体门控离子通道超家 族的成员， 另一类是 GABAB受体， 该类受体是为 G蛋白偶连受体超家族的成员。 哺乳 动物中的 GABAA受体亚基被发现的有 α1-6、 β1-4、 γ1-3、 δ、 ε、 θ和 pl-2等亚基， 其 中 α亚基、 β亚基和 γ亚基对形成一个完整的功能型 GABAA受体是必不可少的， 而 α 亚基对苯二氮卓与 GABAA受体的结合是至关重要的。 主要能够与苯二氮卓结合的是含 有 α1、 α2、 α3或 α5亚基的 GABAA受体， 已有文献已经报道过包含 αΐ亚基的 GABAA 受体主要介导镇定和肌肉松弛的作用， 而包含 ( 2和 (x3的受体主要介导抗惊厥的作用。 关于 GABAA受体在疼痛中的作用研究比较广泛， 特别是 GABAA受体的激动剂可以在 中枢神经系统抑制疼痛，在 2008年报道包含 α2和 α3亚基的 GABAA受体的激动剂在脊 髓水平明显抑制神经源性痛和炎性痛。 GABAA受体的抑制剂或者反向激动剂是否能够 抑制疼痛尚不清楚。 已有文献报道， GABA在大鼠或人体外周神经系统可以引起一个兴 奋性的电流，而 GABAA受体的激动剂 Muscimol在低剂量可以抑制福尔马林诱导的疼痛， 而高剂量促进福尔马林诱导的疼痛。 GABAA受体在疼痛中的作用尚不清楚， 而 GABAA 受体在中枢神经系统表达的广泛性限制了一应配体在治疗疼痛方面的应用。
含 α5的 GABAA受体 （a5-GABAA受体） 在哺乳动物大脑的 GABAA受体中所占的 比例小于 5%， 在大脑皮层中表达水平非常低， 但在大脑海马组织中的 GABAA受体中所 占比例大于 20%， 其他大脑区域几乎不表达。 以前的工作已经提示 a5-GABAA受体很可 會 ^与认知有关 (Collinson N et al, Enhanced learning and memory and altered GABAergic synaptic transmission in mice lacking the alpha 5 subunit of the GABAA receptor J Neurosci. 2002 Jul. l ;22(13):5572-80.)
考虑到 (X5-GABAA受体的在大脑海马组织中特异性分布和功能研究， 包括默沙东在 内的许多制药公司从事于 (X5-GABAA受体配体的研究， 陆续有大量的化合物合成出来， 特别是针对 (x5-GABAA受体的化合物，期望能够治疗与认知有关的疾病。 A5I和 MRK016 是默沙东开发出来用于治疗认知相关疾病的化合物， A5I在临床 II期实验中发现由于化 合物的溶解度太低造成肾毒性； 而 MRK016在安全性上存在问题， 在 I期临床实验中， 发现该药物可以导致头重脚轻、 头痛和恶心呕吐等副作用。
关于 (x5-GABAA受体在外周神经系统的作用， 尚未有相关报道。 外周神经系统由躯 体神经系统和自主神经系统组成。 躯体神经系统分感觉神经系统 (；传入神经)和运动神经 系统 (传出神经)两大类， 分布到身体的各部分。 把神经冲动从外周传到神经中枢的神经 元叫初级感觉神经元， 在感觉神经系统中， 只有胸神经是一根根地在肋骨下缘行走， 形 成肋间神经， 支配胸壁和腹壁的皮肤和肌肉。 其余感觉神经都是相邻的几根神经结合起 来， 形成神经丛， 如颈丛、 臂丛、 腰丛和骶丛等， 从各个神经丛再分出许多神经， 分别 分布到颈部、 上胸、 上肢、 下肢和会阴部的皮肤和肌肉等处。 背根神经节的初级感觉神 经元按照细胞直径的大小分为不同的功能群， 通常分为小细胞、 中等细胞和大细胞。 通 常认为中小细胞是传递伤害性感受 （疼痛） 的细胞， 某些中小细胞的游离神经末梢对伤 害性或持续性刺激敏感， 称之为伤害性感受器。 已发现包含 (x5-GABAA受体主要表达在 小神经元， 并且在神经切断模型中表达升高 (Xiao HS et al, Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain." Proc Natl Acad Sci U S A. 2002 Jun 1 1 ;99(12):8360-5.)，但其是否与伤害或疼痛密切 相关本领域还没有深入的研究。 发明内容
本发明的目的在于提供治疗或缓解疼痛的物质和方法，同时也提出一种新的疼镇痛 药物筛选方法。
在本发明的第一方面， 提供 (x5-GABAA受体的反向激动剂或其药学上可接受的盐的 用途， 用于制备预防、 改善 (缓解)或治疗疼痛的药物。
在一个优选例中， 所述的 a5-GABAA受体的反向激动剂是选择性结合于该受体的苯 二氮卓结合位点的配体。
在另一优选例中， 所述的 a5-GABAA受体的配体结合于 (x5-GABAA受体的能力大于 结合于包含 αΐ或 α2或 α3亚基的 GABAA受体的能力。
在另一优选例中， 所述的 a5-GABAA受体的配体反向激动 a5-GABAA受体的效率大 于反向激动包含 αΐ或 α2或 α3亚基的 GAB ΑΑ受体的效率。
在另一优选例中， 所述的 a5-GABAA受体的反向激动剂是具有式 (I) 和 （Π ) 结构所 示的结构的化合物或其药
Figure imgf000005_0001
( II) ，
其中， R'a表示卤素； 或 d_6烷基、 C3_7环烷基、 C4_7环烯基、 C6_8二环烷基、 C6_10 芳基、 C3_7杂环烷基、 定义为或者含有 6个原子， 其中 1， 2或 3个为氮， 或者含有 5个 原子； 其中 1、 2或 3个独立选自氧、 氮或硫而且其中氧或硫不超过一个的芳环的杂芳 基， 或二 (d_6) 烷基氨基， 这些基团中的任意一个可以用一个或多个选自以下的取代基 任选取代： 卤素、 R3、 OR3、 OC(0)R3、 NR4R5、 NR4R5(C1 -6)烷基、 NR4R5C(0)、 NR4R5C(0) (Cw)烷基、 CN、 氰基 (Cw)烷基或 R6。
Rlb表示 d_6烷基、 C3_7环烷基、 C4_7环烯基、 芳基、 C3_7杂环烷基、 杂环芳基、 二
(Cl— 6) 院基氨基。
R3表示 C2_6烷基、 ^_6链烯基、 C2_6块基、 ^_6环烷基、 C3_6环烷基 （d_6)烷基、 氰 基 (d_6)烷基、 羟基 （d_6)烷基且 R3任选为 1、 2或 3氟代的。
R4和 R5每个独立为氢、 d_6烷基、 ^_6链烯基、 C2_6块基、 _6环烷基或 CF3， 或 R4和 R5与它们共同连接的氮原子一起形成 4-7元杂脂肪环， 该脂肪环含有所述氮原予 和一个任选自 0、 N和 S的其它杂原子， 所述环可以被一个或多个 R3基团任选取代。
R6为 C6_1Q芳基、 C6_1Q芳基 (C1-6)烷基、 杂芳基或杂芳基 (d_6)烷基. 其中杂芳基如 上定义， 而且 R6任选被一个、 两个或三个独立选自以下的取代基取代： 卤原子和 d_4 烷基、 C2_4链烯基、 C2_4块基、 C1-4烷氧基、 C2_4链烯氧基和 C2_4块氧基基团，其中每个 基团是未取代的或被一个、 两个或三个卤原子取代。
X'为 NR4R5; 或 X'为含有 1、 2、 3或 4个独立选自氧、 氮和硫的杂原子的 5元杂芳 环， 杂原子中最多有一个为氧或硫， 或为含有 1、 2或 3个氮原子的 6元杂芳环， 所述 5 或 6元杂芳环任选稠合于苯环或吡啶环上并且该杂芳环任选被 RW和 /或 Ry和 /或 Rz取代， 其中 Rw为卤素、 R3、 OR3、 OC(0) R3、 C(0)OR3、 NR4R5、 NR4C(0)R5、 OH、 三 (C1 -6烷 基)甲硅垸基 6垸氧基 .4烷基、 CN或 R6， RY为卤素、 R3、 OR3、 OC(0) R3、 NR4R5、 NR4C(0) R5、 、 NR4 R5(C1 -6)垸基或 CN而 RZ为 R3、 OR3 , OC(0) 3 , 前提是当 X'为吡 啶衍生物时， 该吡啶环任选为 N-氧化物形式， 并且前提是 X'为四唑衍生物时， 它被 d.4 烷基基团保护；或 X'为由一个、两个或三个独立选自以下的基团任选取代的苯基： 卤素、 氰基、 Ci.6垸基、 C2.6链烯基、 C2.6炔基和 C3.6环垸基。
Z'表示含有 1、 2或 3个独立选自氧、氮和硫的杂原子的 5元杂芳环， 最多有 1个杂 原子为氧或硫， 并且前提是当 1个原子为氧或硫时至少也存在 1个氮原子， 或为含有 2 或 3个氮原子的除吡嗪之外的 6元杂芳环， 所述环中的任何 1个可以由 1个或多个选自 以下的取代基任选取代：卤素、 R3、 OR3、 OC(0)R3、NR4R5、NR4R5(C 1.6)烷基、NR4R5C(0)、
Figure imgf000006_0001
CN、 氰基 (d.6)垸基或 R6。
在另一优选例中， 所述的 a5-GABAA受体的反向激动剂还包括： 具有式 (I和 II)所示 结构的化合物的异构体、 前体。
在另一优选例中， 所述的 a5-GABAA受体的反向激动剂是具有式 (III或 V)所示的结 构的化合物或其药学
Figure imgf000006_0002
( III ) ， (V) ,
其中， RLE表示溴、 噻吩基、 叔丁基、 苯基或呋喃基； RLD表示芳基， d.6垸基、 C3.7 环垸基 1为含有 1、 2或 3氮原子的 5元杂环， 杂原子中最多有一个为氧或硫， 或为含 有 1、 2或 3个氮原子的 6元杂芳环， 所述 5或 6元杂芳环所述环中的任何 1个可以由 1 个或多个选自以下的取代基任选取代： d.6垸基、 氨基、 吡啶基、 CF3、 芳基 (d.6)垸基、 吡啶基 (C )烷基、 卤素、 氰基、 氰基 (C^)垸基、 羟甲基、 羟基或其酮基互变异构体。 Z1表示羟基、 卤素、 羟基或氨基取代的 - 垸基、 ^ 4链烯基、 C2-C4链炔基、 d-C4 在另一优选例中， 所述的 a5-GABAA受体的反向激动剂还包括： 具有式 （III或 V) 所示结构的化合物的异构体、 前体。
在另一优选例中， 所述的 a5-GABAA受体的反向激动剂为下列化合物或其药学上可 接受的盐， 该化合物为：
Figure imgf000006_0003
替换页 （细则第 26条) 在另一优选例中， 所述的 a5-GABAA受体的反向激动剂， 更优选为下列化合物或其 药 该化合物为：
Figure imgf000007_0001
。
在另一优选例中， 所述的 a5-GABAA受体的反向激动剂是具有式 (VI))所示的结构的 化 接受的盐-
Figure imgf000007_0002
其中， 是卤素、 d.7垸基、 C2.7链炔基、 环烷基、 C1-7 垸氧基、 OCF3、 -NHR、 -NHC(0)R或 -NHS02R; R是氢、 d.7烷基、被卤素取代的 d.7垸基、杂芳基、 -(CH^O-Cw 垸基、 -NH-C1-7 烷基； R2 是卤素、 氢、 -C(0)0( C1-4)烷基、 C(0)NHCH2CCH、 C(0)NHCH2CH2、 S02CH2CH3 、 C1-7烷基、被卤素取代的 d.7垸基、氰基、 C3.6环烷基; n 是 0、 1、 2或 3。
在另一优选例中， 所述的 a5-GABAA受体的反向激动剂为下列化合物或其药学上可 接
Figure imgf000007_0003
另一优选例中， 所述的 a5-GABAA受体的反向激动剂更优选为下列化合物或其药学 上
Figure imgf000007_0004
在另一优选例中，所述的 a5-GABAA受体受体的反向激动剂是结合外周神经表达的 a5-GABAA受体但不能有效与中枢表达的 a5-GABAA受体受体相结合。
在另一优选例中， 所述的疼痛是外周神经相关的慢性痛。
在另一优选例中， 所述的慢性痛是神经源性痛、 炎性痛和癌症痛。
在另一优选例中， 所述的疼痛病包括 (但不限于)： 头痛、 面部痛、 颈痛、 肩痛、 背 痛、 胸痛、 腹痛、 背部痛、 腰痛、 下肢痛、 肌肉与骨骼疼痛、 躯体疼痛、 血管疼痛、 替换页 （细则第 26条） 痛风、 关节炎疼痛、 与躯体病样精神障碍有关的疼痛、 内脏疼痛、 感染性疾病 (如 AIDS 和带状疱疹后神经痛)导致的疼痛、 多骨疼痛、 镰刀细胞贫血、 自身免疫性疾病、多发性 硬化或炎症有关的疼痛、 急慢性炎症疼痛、 癌性疼痛、 神经源性痛、 损伤或手术引起的 疼痛、 癌性痛、 伤害感受性疼痛、 糖尿病、 外周神经病、 疤疹后神经痛， 三叉神经痛、 腰部或子宫颈神经根病、舌咽神经痛、 自主神经反射性疼痛、反射性交感神经营养不良、 神经根撕脱、 癌症、 化学损伤、 毒素、 营养缺乏、 病毒或细菌感染、 退行性骨关节病， 或它们的联合形式。
在本发明的另一方面， 提供一种筛选预防、 改善或治疗疼痛的药物的方法， 所述方 法包括：
(1) 提供一包含 a5-GABAA受体的体系，所述的 a5-GABAA受体具有完整的 GABAA 受体功能；
(2) 将候选物质给予步骤 (1)的体系， 观察候选物质与（x5-GABAA受体的结合情况； 若候选物质能够与（x5-GABAA结合，则该候选物质是预防、改善或治疗疼痛的潜在药物。
在一个优选例中， 所述的与 a5-GABAA受体结合为： 选择性结合于苯二氮卓结合位 点。
在另一优选例中， 所述的候选物质不能有效与中枢表达的（x5-GABAA受体相结合。 在另一优选例中， 所述的候选物质是少量穿过或不穿过动物体血脑屏障的物质， 在 动物体内组织分布中， 候选化合物在大脑组织分布低于在血浆组织分布的 50%。
在另一优选例中，所述的 a5-GABAA由 GABAA受体的 α5亚基、 β亚基 (如 β 2， β3)、 γ亚基 (如 γ1、 γ3)组成。
在另一优选例中， 所述的步骤 (2)中， 包括观察候选物质抑制 (如抑制 20%以上， 更 佳地抑制 40%以上， 更佳地抑制 50%以上） GABA通过 GABAA受体诱导的电流的情况； 若候选物质能够抑制 GABA通过 GABAA受体诱导的电流， 则该候选物质是预防、 改善 或治疗疼痛的潜在药物。
在另一优选例中， 步骤 (1)中， 还包括设置对照组的步骤， 所述的对照组选自： 包 含 （如表达） 含 αΐ亚基的 GABAA受体的体系、 包含 （如表达） 含 α2亚基的 GABAA受 体的体系和 /或包含 （如表达） 含 α3亚基的 GABAA受体的体系， 对照组 GABAA受体具 有完整的 GABAA受体功能； 且，步骤 (2)中，所述方法包括：在包含 （如表达） (x5-GABAA 受体的体系中， 检测候选物质与 a5-GABAA受体的结合情况， 并与对照组比较， 若候选 物质与（x5-GABAA受体的结合抑制常数 Ki (nM) (为表征拮抗剂与受体结合能力的物理 常数， 越小表明结合能力越强） 在统计学上小于 （如小 20%以上， 更佳地小 40%以上， 更佳地小 60%以上， 更佳地小 80%以上） 其与含 α1、 α2或 α3亚基的 GABAA受体的结 合抑制常数 Ki， 就表明该候选物是预防、 改善或治疗疼痛的潜在药物。
在另一优选例中，所述的候选物质不能有效抑制 a5-GABAA受体的配体在中枢的结 合 (如可通过同位素标记方法鉴定这种结合）。 在给予实验动物一定剂量的候选物质和已 知的（x5-GABAA受体的特异配体， 该特异配体可以被标记 (如同位素标记)， 然后检测该 候选物质是否抑制已知的 a5-GABAA受体特异配体在中枢的分布， 如果候选物质抑制已 知的（x5-GABAA受体的特异配体在中枢的分布小于 60%， 就表明该候选物是预防、 改善 或治疗疼痛的潜在药物。
在另一优选例中， 所述的候选物质不能有效穿过血脑屏障， 候选物质在大脑和血浆 中的分布比例小于 50%， 就表明该候选物是预防、 改善或治疗疼痛的潜在药物。
在另一优选例中， 步骤 (1)中， 还包括设置对照组的步骤， 所述的对照组选自： 包 含 （如表达） 含 αΐ亚基的 GABAA受体的体系、 包含 （如表达） 含 α2亚基的 GABAA受 体的体系和 /或包含 （如表达） 含 α3亚基的 GABAA 受体的体系，对照组 GABAA受体具 有完整的 GABAA受体功能； 且， 步骤 (2)中， 所述方法包括： 通过电生理方法检测候选 物质对 GABA通过 GABAA受体诱导电流的抑制， 如果待测物质能够显著抑制 （如抑制 20%以上， 更佳地抑制 40%以上， 更佳地抑制 50%以上）a5-GABAA受体介导的电流， 但 同时不能显著抑制含 cd或 ( 2或 (x3的 GABAA受体诱导的电流，就表明该候选物质是预 防、 改善或治疗疼痛的潜在药物。
在另一优选例中， 所述的候选物质包括 （但不限于)： 小分子化合物 (如 γ-氨基丁酸
Α的反向激动剂)、 多肽、 配体。
在另一优选例中， 所述的体系选自： 细胞体系 （或细胞培养物体系）（如背根神经节 细胞)、 亚细胞体系、 溶液体系、 组织体系、 器官体系或动物体系。
作为本发明的优选方式， 所述的方法还包括： 对获得的潜在物质进行进一步的细胞 实验和 /或动物试验， 以进一步选择和确定对于预防、 改善或治疗疼痛真正有用的物质。
在本发明的另一方面， 提供一种预防、 改善或治疗疼痛的方法， 包括给予需要的患 者安全有效量的 a5-GABAA受体的反向激动剂或其药学上可接受的盐。
在一个优选例中，所述的 a5-GABAA受体的反向激动剂或其药学上可接受的盐可以 口服、 皮下注射、 透皮贴剂、 鼻内给药或用自动注射装置。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附图说明
图 1、 在部分神经损伤 （ Spared Nerve Injury, SNI) 条件下， GABAA受体 α5亚基蛋 白在背根神经节中表达升高。
Α、 通过免疫组织化学实验测定 GABAA受体的（χ5亚基蛋白的表达情况。
Β、 通过免疫印迹实验测定 GABAA受体的 α5亚基的蛋白水平。 图 2、 在完全福氏佐剂诱导 (CFA)的炎性痛模型条件下， GABAA受体 α5亚基蛋白 在背根神经节中表达升高。
A、 通过免疫组织化学实验测定 GABAA受体的 (x5亚基蛋白的表达情况。 B、 通过免疫印迹实验测定 GABAA受体的 α5亚基的蛋白水平。 图 3、 背根神经节中的 GABAA受体 ct5亚基蛋白可以转运到坐骨神经。
A、 通过免疫组织化学实验测定 GABAA受体的 ct5亚基蛋白的表达情况。
B、 通过免疫印迹实验测定 GABAA受体的（x5亚基蛋白在坐骨神经、 背根神经节胞 体、 背根神经和脊髓背角中的蛋白水平。 图 4、 SNI诱导的疼痛模型中， 给予 MRK016或者 MRK016-M3有效地抑制神经源 性痛。
A、 SNI模型 7天后， 皮下注射 MRK016后不同时间的机械疼痛阈值。
B、 剂量依赖试验测定 MRK016-M3抑制神经源性痛的效果。
C、 加巴喷丁治疗神经源性痛的有效性以及有效剂量分析。
D、 将 SNI建立 10天的大鼠作为实验动物， 给大鼠脚掌注射 MRK016-M3 , 抑制神 经源性痛的有效性分析。
E、 鞘内注射针对 GABAA a5亚基 mRNA的 siRNA, 检测敲除实施后 a5-GABAA受 体在背根神经节中的表达情况。
F、鞘内注射特异针对 GABAA受体 α5亚基 mRNA的 siRNA可以明显提高疼痛的阈 值， 对照 siRNA没有显著地镇痛效果。
G、鞘内注射特异针对 GABAA受体 α5亚基 mRNA的 siRNA的条件下， MRK016-M3 不能明显提高疼痛的阈值； 而注射对照 siRNA的情况下， MRK016-M3能明显提高疼痛 的阈值。 图 5、 CFA诱导的炎性痛模型中， 给予 MRK016或者 MRK016-M3可以抑制大鼠的 疼痛反应。
A、 首先检测大鼠的热痛阈值和机械疼痛阈值， 然后当天注射针对 GABAA受体 ct5 亚基 mRNA的 siRNA或对照 siRNA, 第 2天给大鼠右脚掌注射 100微升的 CFA, 然后 每天检测大鼠的热痛阈值。
B、 首先检测大鼠的热痛阈值和机械疼痛阈值， 然后当天注射针对 GABAA受体 ct5 亚基 mRNA的 siRNA, 第 2天给大鼠右脚掌注射 100微升的 CFA, 然后每天检测大鼠 的机械痛阈值。
C、 在炎性痛模型 7天的时候， 腹腔注射 MRK016(2 mg/kg体重)， 发现该化合物能 有效地抑制机械疼痛。
D、 在炎性痛模型 3天的时候， 腹腔注射 MRK016 (2 mg/kg体重)， 发现该化合物能 有效地抑制热痛。
E、 在炎性痛模型建立 3天检测检测热痛， 结果显示 MRK016-M3打药后 1小时明 显抑制疼痛。
F、 在炎性痛模型建立 7天检测机械痛， 结果显示 MRK016-M3打药后 1小时明显 抑制疼痛。 图 6、 （x5-GABAA受体的配体 A5I治疗神经源性痛和炎性痛。
A、 检测 α5ΙΑ在 SNI模型中的镇痛作用。 α5ΙΑ ( 3 mg/kg体重） 可以显著抑制神经 源性痛， 吗啡 （10 mg/kg体重） 作为阳性对照。
B、 连续注射 α5ΙΑ不会产生耐受反应。 在 SNI模型建立 14天， 连续给药 6天， 分 别在给药 1-3小时后检测疼痛阈值。
C、在炎性痛模型建立 3天后，腹腔注射 A5I (3 mg/kg体重)也可以有效地治疗热痛。
D、 在炎性痛模型建立 7天后， 腹腔注射 A5I (3 mg/kg体重)也可以有效地治疗机械 痛。 图 7、 （x5-GABAA受体的不同配体 （C2、 C3禾 B C4 ) 治疗神经源性痛和炎性痛。 A、 不同配体 （C2、 C3和 C4 ) 的化学结构式。
B、 建立 SNI模型 14天后， 皮下分别注射 C2、 C3或 C4， 在 1小时后分别检测该 系列药物的镇痛效果， 该系列药物都可以有效治疗疼痛。
C、 在炎性痛模型建立 7天后， 皮下分别注射 C2、 C3或 C4， 在 1小时后分别检测 该系列药物的对机械痛的镇痛效果， 该系列药物都可以有效治疗疼痛。
D、 在炎性痛模型建立后 3天， 皮下分别注射 C2、 C3或 C4， 在 1小时后检测该系 列药物对热痛的镇痛效果， 该系列药物都可以有效治疗疼痛。 图 8、 （x5-GABAA受体的不同配体 （C5、 C6、 C7或 C8 ) 治疗神经源性痛和炎性痛。 A、 不同配体 （C5、 C6、 C7和 C8 ) 的化学结构式。
B、 建立 SNI模型 14天后， 皮下分别注射 C5、 C6、 C7或 C8， 在 1小时后检测该 系列药物的镇痛效果， 该系列药物都可以有效治疗神经源性痛。
C、 在炎性痛模型建立 7天后， 皮下注射 C5、 C6、 C7或 C8， 在 1小时后检测该系 列药物的对机械痛的镇痛效果， 该系列药物都可以有效治疗疼痛。
D、 在炎性痛模型建立后 3天后， 皮下注射化合物 C5、 C6、 C7或 C8， 在 1小时 后检测该系列药物对热痛的镇痛效果， 该系列药物都可以有效治疗疼痛。
E、 建立 SNI模型 14天后， 以 1 mg/kg或 3 mg/kg剂量分别皮下注射 C5后， 在不 同时间点检测化合物 C5的镇痛效果， 都可以有效治疗疼痛。 吗啡作为阳性对照， 以 10 mg/kg体重的剂量皮下注射。
F、 化合物 C5对戊四氮诱导的癫痫并没有作用。 在该试验中， 首先皮下注射不同剂 量的化合物 C5， 剂量分别为 1 mg/kg、 3 mg/kg, 10 mg/kg和 15 mg/kg 在 0.5小时后， 分别注射戊四氮 100 mg/kg ,试验结果证明不同剂量的化合物 C5对戊四氮诱导的惊厥发 作在反应级数和潜伏时间上没有影响。 具体实施方式
本发明人经过深入的研究， 首次揭示了一种新的治疗疼痛的方法， 用针对外周神经 a5-GABAA受体的反向激动剂治疗疼痛。
本发明披露 GABAA受体的 ct5亚基蛋白主要转运到外周的神经丛或坐骨神经，而在 SNI模型和 CFA诱导的炎性痛模型条件下该基因蛋白表达升高。本发明人假设： 那些少 量穿过或不穿过血脑屏障的 GABAA受体的反向激动剂， 就可以结合外周神经的 (x5-GABAA受体， 抑制神经源性痛和炎性痛。 考虑到 GABAA受体的 ct5亚基的主要在神 经系统表达， 此类化合物在理论上就是没有副作用或者副作用较小的镇痛药物。 术语
如本文所用， " (；包)含 a5亚基的 γ-氨基丁酸 A受体" 、 " a5-GABAA受体" 、 " (；包) 含 ct5亚基的 GABAA受体" 可互换使用。
如本文所用， " γ-氨基丁酸 A受体" 与 " GABAA受体" 可互换使用。
如本文所用， " (针对)包含 ct5 亚基的 GABAA受体的配体" 定义为能够结合于包 a5-GABAA受体的化合物， 该化合物可以调节疼痛反应。
如本文所用， "（针对)包含 ct5亚基的 GABAA受体的反向激动剂" 是指能够结合于 a5-GABAA受体的化合物， 并且该化合物能够抑制 GABA对 a5-GABAA受体的作用。
如本文所用， "治疗" 也包括预防性给药， 一旦所述病症建立后缓解或消除所述病 症。
如本文所用， "患者" 定义为任何温血动物， 例如不限于小鼠、 豚鼠、 狗、 马或人， 所述患者最好是人。
如本文所用， "急性痛" 定义为由皮肤、身体结构或内脏的损伤和 /或疾病发生的有 害刺激而引起的疼痛， 或由不产生实际组织损害的肌肉或内脏的异常功能而引起的疼 痛。
如本文所用， "慢性痛" 定义为持续超出急性疾病通常的病程或损伤治愈的合理时 间， 或与引起持续疼痛的慢性病理过程有关， 或疼痛以一定间隔复发数月或数年， 如果 在应该已经达到治愈后或超过通常的治疗过程后仍存在疼痛， 则认为是慢性痛。 在疼痛 需要经过的时间长度取决于疼痛的性质和与疼痛有关的治疗过程， 如果疼痛超过通常的 治疗过程， 则疼痛是慢性的。 慢性痛包括但不限于头痛、 面部痛、 颈痛、 肩痛、 胸痛、 腹痛、 背部痛、 腰痛、 下肢痛、 肌与骨骼疼痛、 与躯体病样精神障碍有关的疼痛、 内脏 痛、 疼痛性糖尿病性神经病、 血管疼痛、 痛风、 关节炎性痛、 癌症疼痛、 自主神经反射 性疼痛、感染性疾病 (如艾滋病和带状疱疹)导致的疼痛、 自身免疫性疾病 (风湿)导致的疼 痛、 急慢性炎症导致的疼痛、 手术后疼痛和烧伤后疼痛。
本发明揭示的药物能有效地治疗如上定义的慢性痛， 而且本发明揭示的药物可用于 治疗伴随其他病症的痛敏， 包括痛觉过敏、 异常性疼痛、 痛觉增强和疼痛记忆增强， 该 发明将改善对其疼痛的治疗。
如本文所用， "头痛" 可分为原发性头痛和继发性头痛， 原发性头痛包括紧张性头 痛、 偏头痛和丛集性头痛， 而继发性头痛是由于其他疾病引起的。 头面部的痛敏感组织 发生病变或受到刺激时， 可引起各种头痛， 这些痛敏感组织包括分布于头皮、 面部、 口 腔及咽喉等， 由于它们主要是头部的肌肉或血管， 含有丰富的神经纤维， 对疼痛比较敏 感， 所以当这些组织受到伤害时可引起头痛。
如本文所用， "面部痛" 包括但不限于三叉神经痛、 非典型面痛、 面神经麻痹和面 肌痉挛。
如本文所用， "三叉神经痛 " 是一种独特的慢性痛性疾病， 又称痛性抽搐， 是指在 三叉神经分布区域出现短暂的、 阵发性的和反复发作的电击样剧烈性疼痛， 或伴有同侧 面肌痉挛。 三叉神经痛分为原发性和继发性两种类型， 原发性三叉神经痛是指临床上未 发现神经系统体征， 检查未发现器质性病变； 继发性三叉神经痛是指在临床上有神经系 统体征， 检查发现有器质性病变， 如肿瘤和炎症等。
如本文所用， "非典型面痛" 是指由多种病因引起的疼痛。 表现为持续性烧灼样疼 痛、 无间歇性、 与特殊的动作或触发刺激无关， 疼痛多为双侧、 疼痛常常超出三叉神经 的分布范围甚至累及颈部皮肤。 病因可由鼻窦炎、 恶性肿瘤、 颌及颅底感染等原因刺激 或损伤三叉神经而引起疼痛。
如本文所用， "颈痛、 背痛、 肩痛" 是指由于急慢性肌肉劳损及骨关节的退行性变 和外伤等导致的疼痛。 引起颈、 肩及上肢疼痛的常见疾病有颈肩肌筋膜炎、 项韧带炎、 颈椎病、 肩周炎、 胸廓出口综合症、 肱骨外上髁炎等， 或由自身免疫性疾病引起的疼痛 常见于类风湿性关节炎、 强直性脊柱炎和风湿性关节炎等疾病， 其他可能引起颈痛、 背 痛、 肩痛的疾病还有颈、 肩部的肿瘤、 神经炎、 动静脉疾病和各种感染以及胸、 腹腔脏 器病变引起的牵涉痛等。
如本文所用， "胸、 腹及背部痛" 指由于胸腹部内脏、 胸腹壁组织的疾病导致的疼 痛， 包括但不限于肋间神经痛、 肋间软骨炎、 心绞痛、 腹痛 (急性腹部内脏痛)和腰背部 肌筋膜综合征。
如本文所用， "腰、 下肢痛" 是指下腰、 腰骶、 骶髂、 髋、 臀及下肢痛。 腰和下肢 痛往往并不是独立的疾病， 而是多种疾病的共有特征， 临床表现多样， 病因十分复杂， 以退行性和损伤为多， 包括但不限于腰椎间盘突出、 急性腰扭伤、 坐骨神经痛、 骨质疏 松症、 第三腰椎横突综合症、 梨状肌综合症、 膝关节骨性关节炎、 尾痛症和足根痛等涉 及的疼痛。
如本文所用， "肌与骨骼疼痛"包括但不限于肌筋膜疼痛、 创伤引发的疼痛和慢性 区域性疼痛综合症。
如本文所用， "疼痛性糖尿病" 是指糖尿病并发的神经损伤而引起的疼痛， 糖尿病 中的神经损伤至少部分是由于血流减少和高血糖引起的。 某些糖尿病患者不发生神经病 变， 而其他患者早期就发生该疾病， 糖尿病性神经病痛可以分为涉及一个或者多个病灶 部位的单神经病和全身性多神经病， 所述多神经病可以是扩散和对称的， 通常主要涉及 感觉方式 (Merrit's Textbook of Neurology, 第 9版）。 糖尿病性神经病的表现可以包括植 物神经功能障碍， 导致包括心脏、 平滑肌和腺体在内的调节障碍， 引起低血压、 腹泻、 便秘和性无能。 糖尿病神经病往往分阶段发展， 早期在神经末梢区， 植物神经病或感觉 神经病的时发生在足部， 脑神经病时发生在面部和眼周围， 出现间歇性疼痛和麻刺感， 在随后的阶段中， 疼痛更强和经常发生， 最后当某一区域痛觉丧失时， 发生为无痛性神 经病， 由于没有疼痛作为损伤的指示， 大大增加了发生严重组织损伤的风险。
如本文所用， "内脏疼痛"包括但不限于刺激性肠综合征 (IBS) , 伴有或不伴有慢性 疲劳综合征 (CFS)、 炎性肠病 (IBD)和间质性膀胱炎的疼痛。
如本文所用， "血管疼痛" 是由以下一种或多种因素产生的疼痛。 第一， 组织的灌 注不当。 引起暂时或连续的局部缺血， 诸如在运动期间发生肢体肌肉中的局部缺血。 第 二， 迟发性变化。 例如在皮肤或腹部内脏中的溃疡或坏疽。 第三， 大血管口径的突然或 加速变化。 例如动脉瘤发生的变化。 第四， 主动脉破裂。 结果是血液溢出， 刺激腹膜或 胸膜壁层中的伤害感受纤维。第五， 由于动脉内注射严重刺激动脉内皮而引起的强痉挛。 第六，静脉血回流的损害，结果是迅速扩张筋膜隔室的大量水肿 (Bonica's Management of Pain, 第一卷 (第二版))。 实例包括但不限于闭塞性动脉硬化、 闭塞性血栓脉管炎、 急性 动脉闭合、 栓塞、 先天性动静脉瘤、 血管痉挛性疾病、 Rayaud病、 手足发绀、 急性静脉 闭合、 血栓静脉炎、 静脉曲张和淋巴水肿。
如本文所用， "癌性痛" 是指恶性肿瘤在其发展过程中出现的疼痛， 癌性痛发生目 前认为有三种机制， 即： 癌症发展直接造成的疼痛、 癌症治疗后造成的疼痛和癌症患者 并发疼痛性疾病。
如本文所用， "自主神经反射性疼痛" 是指由 "反射性交感神经萎縮征" 导致的疼 痛。 反射性交感神经萎縮征是指机体遭受急慢性损伤后， 有剧烈的自发疼痛， 对触觉和 痛觉过敏， 可伴有浮肿和血行障碍， 随后可出现皮肤及肌肉骨骼的营养障碍和萎縮等症 状。
如本文所用， "术后疼痛" 是指机体对疾病本身和手术造成的组织损伤的一种复杂 的生理反应， 它表现为心理和行为上的一种不愉快的经历。
如本文所用， "关节炎性痛" 包括但不限于骨关节炎、 类风湿性关节炎、 关节强直 性脊椎炎、 牛皮癣性关节病、 痛风、 假痛风、 传染性关节炎、 腱炎、 粘液囊炎、 骨损害 和关节软组织炎症等疾病导致的疼痛。
如本文所用， "带状疱疹后的神经痛" 是指带状疱疹的皮疹愈合后， 在原来皮疹区 的皮下长期存在的剧烈疼痛。
如本文所用， "伤害性痛" 是由刺激伤害性感受器传入的组织损害过程引起的疼 痛， 或是由伤害性感受器延长的兴奋引起的疼痛。 由伤害感受器延长的兴奋引起的疼痛 可以是由于伤害感受器的持久有害刺激或其敏化或这两者共同引起， 或它们可以由这些 因素引起， 并由其持久性、 各种反射机制和其他因素而延长。
如本文所用， "痛觉 " 是指探测有害刺激的神经机制。 痛觉涉及两个步骤： 外周神 经末梢传导伤害刺激和将这些信号传递至中枢神经系统。
本文所用的术语 "烷基"指饱和的、 直链或支链的脂族烃类基团。 例如， 烷基包括 但不限于甲基、 乙基、 正丙基、 异丙基、 正丁基、 异丁基、 叔丁基。 所述的 "烷基 "较佳 地为 d_8烷基； 更佳地为 d_6烷基； 更佳地为 d_4烷基。
本文所用的术语 "烯基"指含有至少一个碳碳双键和至少 2个碳原子 (较佳地 2-6个 碳原子)的直链或支链烃基。 所述的 "烯基 "较佳地为 C2_8烯基； 更佳地为 C2_6烯基； 更 佳地为 C2_4烯基。
本文所用的术语 "块基"指含有至少一个碳碳三键和至少 2个碳原子 (较佳地 2-8个 碳原子； 更佳地为 2-6个碳原子； 更佳地为 2-4个碳原子)的直链和支链烃基。
本文所用的术语 "卤素" 指？、 Cl、 Br或 I， 特别是 F、 C1或 Br。
本文所用的术语 "烷氧基" 指的是含氧的烷基， 例如甲氧基、 乙氧基、 正丙氧基、 异丙氧基等。 较佳地其含有 2-8个碳原子； 更佳地为 2-6个碳原子； 更佳地为 2-4个碳 原子。
本文所用的术语 "环烷基" 指的是指具有 3至 7个碳原子的环烷基团， 如环丙基、 环戊基、 环己基和环庚基等。
本文所用的术语 "环烯基" 的定义如 "环烷基" ， 且含有至少一个不饱和的碳碳双 键。
本文所用的术语 "芳基"指芳族体系， 可以是单环或原本稠合的或连接在一起的多 芳环， 从而使至少一部分稠合或连接的环形成共轭的芳系。 芳基基团包括 (但不限制于): 苯基、 萘基
本文所用的术语 "杂环" 指稳定的 4-7元单环或稳定的多环杂环， 该杂环可以是饱 和的、 部分不饱和的或不饱和的， 且由碳原子和选自以下的 1-4个杂原子构成： N、 0 和 S原子。 N和 S原子可以被氧化。 杂环还可包括任何多环， 其中任一上述杂环可稠合 于芳环。
本文所用的术语 "杂芳基" 是指杂环的芳基。
本文所用的术语 "异构体"包括： 构象异构体， 光学异构体 (如对映异构体和非对映 异构体)， 几何异构体 (如顺反异构体)。 含 α5亚基的 γ-氨基丁酸 Α受体的反向激动剂及其用途 对 （x5-GABAA受体的反向激动剂的研究已有十几年了， 陆续有新的化合物合成出 来， 以往研究认为其可能治疗学习记忆 （认知） 相关的疾病。
本发明人深入研究后发现， （x5-GABAA 受体的反向激动剂可以有效抑制疼痛。 针对 (x5-GABAA受体的反向激动剂是能够结合于（x5-GABAA受体的化合物， 该化合物可结合 于 (x5-GABAA受体的苯二氮卓结合位点，该化合物可以为 (x5-GABAA受体的反向激动剂。 优选地是一些不能有效进入血脑屏障与中枢神经系统中的与 (X5-GABAA受体结合的化合 物。
GABAA受体的 α5亚基蛋白在背根神经节中表达，并且主要转运到外周；而 GABAA 受体的 ct5 亚基蛋白在 CFA诱导的炎性痛模型和 SNI模型中表达升高。 通过基因敲除 GABAA受体的 ct5亚基蛋白在背根神经节中的表达，可以明显抑制炎性痛和神经源性痛。 并且， 给 (x5-GABAA受体的反向激动剂可以明显抑制炎性痛和神经源性痛。
所述的 a5-GABAA的反向激动剂结合于 a5-GABAA受体的能力大于结合于仅包含 αΐ或 α2或 α3亚基的 GABAA受体的能力。
作为本发明的优选方式， 本发明提供了一种如结构式 (I和 Π)所示的 （x5-GABAA受 体的
Figure imgf000016_0001
其中， 各基团的定义同前。
作为本发明的另一优选方式， 本发明提供了一种如结构式 (Π)所示的 ct5-GABAA受 体的 (IV)所示的结构。
Figure imgf000016_0002
其中， 各基团的定义同前。
(1 5-GABAA受体的反向激动剂还包括在以下专利中提到的那些： WO 201 1/107812 A2 , WO 2010/1 12475 Al , WO 2010/104843 A2 , WO 2010/002451 A1 , WO 2010/127978 Al ,WO 2010/127968 Al , WO 2010/097368 Al , WO 2010/125042 Al , WO 2010/028769 Al ,WO 2010/094669 Al , WO 2010/127976 Al , WO 2010/127975 Al , WO 2009/000662 Al , WO 2009/071464 Al , WO 2009/071476 Al , WO 2009/071477 Al , WO 2008/154438 Al , WO 2008/154442 Al , WO 2008/154447 Al , WO 2007074089 Al , WO 2007/071598 Al , WO 2007/074078 A2 , WO 2010/127974 Al , WO 2007/137954 Al , WO 2007/042420 Al , WO 2007/054444 A2， WO 2007/042421 Al , WO 2007/039389 Al， WO 2007/082806
Al， WO 2006/078891 A2， WO 2006/045430 Al， WO 2006/045429 Al， WO 2006/040038-A1, WO 2006/063708 Al,WO 2005/080355 Al, WO 2005/084439 Al, WO 2005/108401 Al,WO 2004/041808 Al, WO 2004/014865 Al,WO 2004/014891 Al, WO 2004/074259 Al, WO 2004/039802, WO 2004/014865 Al， WO 2004/043930 Al， WO 2004/087137 Al , WO2004076452-A1 , WO 2004/031174 Al， WO 2004031174 Al,WO 2004/107863 Al, WO2004065388-A1 , WO 2003/093263 Al, WO 2003/086406 Al, WO 2003/093273 Al, WO 2003/087099 Al,WO 2003/048132 Al, WO 2003/066634 Al, WO 2003/008418 Al, WO 2003/097643 Al, WO 2003/044018 Al, WO 2003/006471, WO 2003/004018 Al,WO 2003/093272 Al, WO 2003/099816 Al, WO 2003/099817 Al, WO 2002/081474 Al, WO 2002/020480 Al, WO 2002/076987 Al, WO 2002/076983 Al, WO 2002/000623 A2， WO 2002/006285 Al, WO 2002/016363 Al, WO 2002/020492 Al, WO 2002/094834 Al, WO 2002/042305 Al, WO 2002/074773 Al, WO 2002/094834 Al, WO 2002/002557 A2， WO 2001/038331 A2， WO 2001/092257 Al, WO 2001/018001 Al,WO 2001/016103 Al, WO 2001/044250 Al, WO 2001/092258 Al, WO 2001/090108 Al, WO 2001/051492 Al, WO 2000/027849 A2, WO 2000/029412 Al, WO 2000/012505 A2, WO 2000/077008 A2,WO 2000/059905 Al, WO 1999/006400 Al, WO 1999/006399 Al, WO 1999/006401 Al， WO 1998/004560 Al， WO 1998/024435 Al, WO 1998/018792 Al, WO 1996/025948 Al, WO 1996/008494 Al, US 6511987, US 6297256, US 2004/0058970 Al, US 7176203, US 2010/0216752 Al， US 2008/0064748 Al, US 2002/0103371 Al， US 2008/0064748 Al, US 6395905， US 2004/0235844 Al, US 2004/0006226 Al， EP 1451161 Bl， US 2006/0040940 Al, US 2006/0058303 Al, AU 2003/276424, AU 2003/282222, US 2006/0041125 Al， US 7148222, US 6395766, AU 2002/343110, US 6H！642229, US 2003/0176449 Al, US 2004/0110778 Al， US 2003/0220348 Al, US 2008/0167324 Al, EP 1368342 Bl ， US 2010/652435 A， US2004006226-A1 US2010130481-A1 ， US2008033061-A1在本发明中"含 α5亚基的 GABAA受体的反向激动剂"包括但不限于以 上专利所述的化合物。
其中在科学文献中明确报道， 具有显著反向激动剂效果的化合物如表 1。
表 1
名称 合成方法文献或专利 结构
一 'O'
MRK-016 文献 ( Chambers MS et al" An orally bioavailable, 人
V
functionally selective inverse agonist at the
、1 benzodiazepine site of GABAA alpha5 receptors with
cognition enhancing properties. J Med Chem.2004 Nov
18; 47(24):5829-32.) 和专利 US 6355638
(Pyrazolo[l,5-d][l,2,4] triazines for enhancing
cognition) 。
Figure imgf000018_0001
Apr 22; 47(9):2176-9.)和专利 WO 9850385 Al .
Figure imgf000018_0002
containing alpha5 subunits, improves passive, but not
active, avoidance learning in rats. Brain Res. 2008 May
7;1208:150-9.
脑屏障的药物有:
Figure imgf000018_0003
有 文 献 报 道 (Jones et al.， Pharmacokinetics and metabolism studies on (3 -ier utyl-7-(5-methylisoxazol-3-yl)-2-(l -methyl l /-,2,4-triazol-5-ylmethoxy) pyrazolo[ 1 [ 1 ,2?4]triazine3 a functionally selective GABAA alpha5 inverse agonist for cognitive dysfunction. Bioorg Med Chem Lett. 2006 Feb 15;16(4):872-5.)， M K016在体内 可以代谢为 MRK016-M1、 MRK016-M2. MRK016-M3等多种结构。 而 MRK016-M3和 替换页 （细则第 26条） MRK016-M2具有明显的（x5-GABAA受体的反向激动效果， 但却不能有效地结合大脑中 的（x5-GABAA受体。 这说明， 不能有效进入血脑屏障的（x5-GABAA受体的反向激动剂可 以有效地治疗炎性痛和神经源性痛， 属于安全性很高的药物。
本发明人还发现，
Figure imgf000019_0001
可以有效地抑制 炎性痛和神经源性痛， 它们几乎不能穿过血脑屏障， 不会直接影响中枢功能， 属于中枢 副作用小且可以有效治疗疼痛的药物。 这些化合物作为本发明中优选的化合物。
本发明还包括上述所列举的各种化合物的异构体、 外消旋体、 药学上可接受的盐、 水合物或前体。
所述的 "药学上可接受的盐" 是指上述化合物与无机酸、 有机酸、 碱金属或碱土金 属等反应生成的盐。 这些盐包括 (但不限于)： （1)与如下无机酸形成的盐： 如盐酸、 氢溴 酸、 氢碘酸、 硫酸、 硝酸、 磷酸； （2)与如下有机酸形成的盐， 如乙酸、 乳酸、 柠檬酸、 琥珀酸、 延胡索酸、 葡萄糖酸、 安息香酸、 甲烷磺酸、 乙烷磺酸、 苯磺酸、 对甲苯磺酸、 草酸、 丁二酸、 酒石酸、 马来酸、 或精氨酸。 其它的盐包括与碱金属或碱土金属 (如钠、 钾、 钙或镁)形成的盐， 铵盐或水溶性的胺盐 (如 N-甲基葡糖胺盐)、低级的烷醇铵盐以及 其它药学上可接受的胺盐 (比如甲胺盐、 乙胺盐、 丙胺盐、 二甲基胺盐、 三甲基胺盐、 二 乙基胺盐、 三乙基胺盐、 叔丁基胺盐、 乙二胺盐、 羟乙胺盐、 二羟乙胺盐、 三羟乙胺盐， 以及分别由吗啉、 哌嗪、 赖氨酸形成的胺盐)， 或其它常规的 "前体药物" 的形式。 化合 物具有一个或多个不对称中心。 所以， 这些化合物可以作为外消旋的混合物、 单独的对 映异构体、 单独的非对映异构体、 非对映异构体混合物、 顺式或反式异构体存在。
所述的 "化合物的前体"指当用适当的方法服用后， 该化合物的前体在病人体内进 行代谢或化学反应而转变成结构式 I的一种化合物， 或化学结构式 I的一个化合物所组 成的盐或溶液。 化合物的前体包括但不局限于所述化合物的羧酸酯、 碳酸酯、 磷酸酯、 硝酸酯、 硫酸酯、 砜酯、 亚砜酯、 氨基化合物、 氨基甲酸盐、 偶氮化合物、 磷酰胺、 葡 萄糖苷、 醚、 乙縮醛等形式。
本发明的化合物的制备方法时本领域技术人员已知的。 例如， 文献 （Chambers MS et al" An orally bioavailable, functionally selective inverse agonist at the benzodiazepine site of GABAA alpha5 receptors with cognition enhancing properties. J Med Chem. 2004 Nov 18; 47(24):5829-32. ) 和专利 US 6355638 ( Pyrazolo[l,5-d][l,2,4] triazines for enhancing cognition) 公开了 MRK-016的化学合成方法。 文献 ( Jones P et al, Pharmacokinetics and metabolism studies on (3 -tert-butyl-7-(5 -methylisoxazo 1-3 -yl)-
2-(l -methyl- 1H-1, 2, 4-triazol-5-ylmethoxy) pyrazolo [ 1 ,5-d] [ 1 ,2,4]triazine, a functionally selective GABAA alpha5 inverse agonist for cognitive dysfunction. Bioorg Med Chem Lett. 2006 Feb 15;16(4):872-5. )公开了 MRK016-M3的化学合成方法。 文献 (Sternfeld F et al" Selective, orally active gamma- amino butyric acicU alpha5 receptor inverse agonists as cognition enhancers. J Med Chem. 2004 Apr 22; 47(9):2176-9.)禾口专禾 U WO 9850385 Al公 开了 α 5IA 的合成鉴定。 文献 (Atack JR et al" In vitro and in vivo properties of 3-tert-butyl-7-(5-methylisoxazol-3-yl)-2-(l -methyl- 1H-1, 2, 4-triazol-5-ylmethoxy)
-pyrazolo[l,5-d]-[l,2,4]triazine (MRK-016) , a GABAA receptor alpha5 subtype-selective inverse agonist. J Pharmacol Exp Ther. 2009 Nov;331(2):470-84.)公开了 MRK016、 MRK016-M3、 a5IA 均是结合于苯二氮卓结合位点选择性 a5 的反向激动剂， MRK016 (MRK-016)及其主要代谢物 (Ml、 M2、 M3)与 GABAA受体各亚型的结合亲和力 (Affinity) 和效力 (Efficacy)如表 1所示。 筛选方法
在得知了 a5-GABAA受体的反向激动剂通过选择性地结合于 (x5-GABAA受体， 发挥 抑制疼痛的作用后， 可以基于该特征来筛选结合于 (x5-GABAA受体的物质。 之后， 可从 所述的物质中找到对于抑制疼痛真正有用的药物。
因此，本发明提供筛选预防、改善或治疗疼痛的药物的方法，所述方法包括： (1) 提 供 a5-GABAA受体的体系，所述的 a5-GABAA受体具有完整的 GABAA受体功能；（2) 将 候选物质给予步骤 (1)的体系， 观察候选物质与 (x5-GABAA受体的结合情况； 若候选物质 能够与 (x5-GABAA受体受体结合， 则该候选物质是预防、 改善或治疗疼痛的潜在药物。 所述的包含 GABAA受体的 ct5亚基的体系例如可以是细胞 （如背根神经节细胞） 体系， 所述的细胞可以是内源性表达 GABAA受体的 ct5亚基的细胞；或可以是重组表达 GABAA 受体的 ct5亚基的细胞。 所述的 GABAA受体的 α5亚基的体系还可以是亚细胞体系、 溶 液体系、 组织体系、器官体系或动物体系 （如动物模型， 优选非人哺乳动物的动物模型， 如鼠、 兔、 羊、 猴等） 等。
在本发明的优选方式中， 在进行筛选时， 为了更易于观察到包含 GABAA受体的 ct5 亚基与候选物质结合情况的差异， 还可设置对照组， 所述的对照组可以是不添加所述候 选物质的 a5-GABAA受体的体系。
所述的候选物质可以包括 （但不限于)： 小分子化合物 (GABAA受体的反向激动剂)， 多肽， 配体。 较佳地， 所述的候选物质是不穿过动物体血脑屏障的物质。
作为本发明的优选方式， 所述的方法还包括： 对获得的潜在物质进行进一步的细胞 实验和 /或动物试验， 以进一步选择和确定对于预防、 改善或治疗疼痛真正有用的物质。
另一方面， 本发明还包括采用所述筛选方法获得的抑制疼痛的物质。
检测一个化合物是否与受体或受体的部分亚基结合是本领域已知的技术。 检测一个 化合物是否是针对 a5-GABAA受体的反向激动剂或者拮抗剂， 这方面的研究工作已经做 了很多， 例如在国际申请专利 WO 92/22652和 WO 94/13799中， 用 GABAA受体的 α5、 β3和 γ2组合来检测某一个化合物是否与该受体相结合； 在进行药物筛选的过程中， 通 常用 Goeders等 (Goeders NE and Kuhar MJ， Benzodiazepine binding in vivo with [3H]Ro 15-1788. Life Sci, 1985 Jul, 37(4):345-355)所述的方法。 检测一个能与 a5-GABAA受体 结合的配体是拮抗剂、 激动剂还是反向激动剂， 在这一方面的研究也很多， 可以参照文 献 (Wafford K A, Whiting P J and Kemp J A ， Differences in affinity and efficacy of benzodiazepine receptor ligands on recombinant GABAA receptor subtypes. 1993 Feb, Mol. Pharmacol 43(2)40-244)所述的方法。 而筛选药物是否进入血脑屏障的办法比较广泛， 在 文 献 (Jones et al.， Pharmacokinetics and metabolism studies on (3 -tert-butyl-7-(5 -methylisoxazo 1-3 -yl)-2-( 1 -methyl- 1 H- 1 , 2 , 4 -tr iazo 1- 5 - y lmet ho xy ) pyrazolo [ 1 ,5-<i] [ 1 ,2,4]triazine, a functionally selective GABAA alpha5 inverse agonist for cognitive dysfunction. Bioorg Med Chem Lett. 2006 Feb 15; 16(4):872-5)中报道可以检测候 选化合物抑制 (1H)Ro -15-1788 Ca5-GABAA受体标记的特异性反向激动剂)在大脑中的结 合， MRK016可以有效地抑制 (3H)Ro-15-1788在中枢的结合， 而 MRK016-M3却不能显 著的抑制 (1H)RO-15-1788在中枢的结合。 也可以通过检测药物在不同组织的方法检测， 例如检测药物在大脑和血浆中的分布比例来确定药物是否可以有效进入血脑屏障。 治疗方法
本发明通过提供治疗疼痛的方法， 满足安全和有效地治疗急性疼痛和慢性痛。 按照 本发明提供的治疗疼痛的方法， 包括给予需要这种治疗的患者有效量的 (X5-GABAA受体 的反向激动剂。
本发明特别适用于患有疼痛的老年患者、 肝功能和肾功能不好的人群。 对大多数老 年人来说， 慢性痛是常见的病症， 而疼痛往往被认为是中老年人器官老化及病变的伴随 症状。 老年人疼痛主要包括来自骨关节系统的四肢关节、 背部、 颈部疼痛、 头痛以及其 他慢性病引起的疼痛， 最常见的表现就是关节疼痛、 僵硬及活动受限， 医学上称之为退 行性骨关节病。 退行性骨关节病包括关节软骨退行性变和骨质增生 (骨刺或骨赘)， 在 50 岁以上的人群中， 发病率仅次于心脏病， 在 65岁以上的人群中发病率可达 60%〜90%。 骨关节炎是最常见的疼痛原因。 60岁以上的老年人中 80%以上有骨关节炎表现， 如关节 疼痛、 活动时有响声、 行走障碍和晨僵， 严重时可有关节红肿等。 然而老年病人往往伴 有其他慢性疾病， 如高血压、 糖尿病和心脏病等， 为治疗相应的疾病， 需要同时服用多 种不同的药物， 且老年人对药物耐受性低， 易发生不良反应。
本发明提供了一种治疗疼痛的方法， 更具体的说是治疗急性疼痛和慢性痛的方法。 该方法是用针对 a5-GABAA受体的配体作为药物治疗疼痛， 这些药物可以口服、 皮下注 射、 透皮贴剂、 鼻内给药或用自动注射装置给药。 在该方法中， 用于治疗疼痛的配体是 针对（x5-GABAA受体的反向激动剂， 是特异性地针对（x5-GABAA受体， 不能有效穿过血 脑屏障， 但同时也可是包括 αΐ或 α2或 α3的 GABAA受体的拮抗剂或反向激动剂。在实 施中， 该配体也可为 GABAA受体的拮抗剂。
在本发明中治疗疼痛的方法可以以口服和非肠道等多种给药剂型制备并给药。 因 此， 本发明所涉及的化合物可以通过注射给药， 即静脉内、 肌肉内、 皮内、 皮下、 十二 指肠内或腹腔内给药； 本发明涉及的化合物还可以通过吸入给药， 例如鼻内吸入； 此外， 本发明的化合物可以透皮给药。 组合物
本发明的化合物可以制备药物组合物， 药用载体可以是固体或液体， 固体制剂包括 散剂、 片剂、 丸剂、 胶囊、 扁囊剂、 栓剂和可分散颗粒剂， 固体载体可以是一种或多种 物质， 它们可以作为稀释剂、 矫味剂、 黏合剂、 防腐剂、 片剂崩解剂或包囊材料。 在散 剂中， 载体是微细分的固体， 本发明的化合物与微细分的活性组份存在于混合物中。 在 片剂中， 此化合物与所需的粘合载体以适当比例混合， 并压制成所需的形状和大小。 散 剂和片剂中优先含有 5至 70%活性化合物， 适宜的载体是碳酸镁、硬脂酸镁、滑石、糖、 乳糖、 果胶、 糊精、 淀粉、 明胶、 羧甲基纤维素、 羧甲基纤维素钠、 低熔点腊和可可脂 等。 同样， 扁囊剂或锭剂、 片剂、 散剂、 胶囊、 丸剂、 扁囊剂和锭剂可以是适合于口服 给药的固体剂型。
为了制备栓剂， 现将低熔点腊乳脂肪酸甘油脂或可可脂的混合物熔化， 并通过搅拌 将此活性化合物组份均匀地分散在其中， 然后， 将熔化的均匀混合物倒入大小合适的模 中， 让其冷却， 并由此固化。 溶液形式的制剂包括溶液、 悬浮剂和乳剂， 例如水或含水 丙二醇溶液。 对于非肠道注射液体制剂， 可以在含水聚乙二醇溶液中制备。 适于口服的 含水溶液可以通过将活性组份溶解于水中， 并按照需要加入适宜的着色剂、 矫味剂、 乳 化剂和增稠剂制备。
适于口服的含水混悬剂可以通过将微细分的活性组份分散于含水粘性物质中制备， 如天然或合成胶、 树脂、 甲基纤维素、 羟甲基纤维素钠及其他熟知的混悬剂。
还包括在使用前短不久转变味口服给药的液体制剂的固体形式的制剂。这些液体形 式包括溶液、 混悬剂和乳剂， 除活性组份外， 这些制剂可含有着色剂、 矫味剂、 稳定剂、 缓冲剂、合成或天然甜味剂、 分散剂、 增稠剂和助溶剂等。 此药物制剂优先是单位剂型， 在此剂型中， 此制剂被再分为含适量活性组份的单位剂量， 此单位剂量可以是包装的制 剂， 该包装含有一定量的制剂， 如包装的片剂、 胶囊和在小瓶或胶囊中的散剂。 此单位 剂型还可以是胶囊、 片剂、 扁囊剂或锭剂本身， 或其可以存在包装形式中的适当数量的 任何这些散剂。
单位剂量制剂中活性组份的量可根据具体的应用和活性组份的效力而变化， 可调节 自 0.01 mg 至约 0.1 g o 例如， 在医药用途中， 该药物可以 0.1至约 3 mg的胶囊每天给 药三次， 必要时该组合物还可以含其他相容的治疗剂。
在治疗用途中， 用于本发明的化合物以起始剂量每天 0.001 mg至 10 mg/kg体重。 但是， 这些剂量可以根据患者的需要、 被治疗病症的严重性以及使用的化合物而变化， 一般来说， 开始以小于该化合物最佳剂量的较小剂量治疗， 此后， 小量增加此剂量达到 最佳效果， 方便起见， 如果需要可将总日剂量再细分为一天内分次给药。
本发明的药物组合物还可同时与其它治疗剂或辅剂联合使用， 包括但不限于吗啡、 加巴喷丁)等。
因此， 本发明提供了一种用于治疼痛的药物， 该药物不仅有效， 而且没有明显的副 作用， 本发明的另一个目的是提供一种对于特殊病人群体， 如老人、 患有肝或肾功能衰 退、 或心脏血管性疾病的病人， 具有高度安全性的药物。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 J.萨姆布鲁克等编著的 《分子克隆实验指南》 （科学出版社， 2002年）所述的条 件， 或按照制造厂商所建议的条件。 除非另外说明， 否则百分比和份数按重量计算。
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义 相同。 此外， 任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。 文中所 述的较佳实施方法与材料仅作示范之用。
I. 实验方法
(1) 动物
雄性 SD大鼠 (200-250 g), 每 6只一组放在笼子中， 将所有动物放在 12小时亮 /循 环 (即每天包括 12小时照明和 12小时黑暗)， 任意食用食物和水。 所有试验均在观察者 对药物治疗不知情的情况下进行。
(2) 动物模型
部分神经损伤模型 （SNI): 腹腔注射 10%水合氯醛， 导致大鼠麻醉， 于大鼠后肢上 缘切开皮肤， 纵行分离肌肉， 暴露坐骨神经主干及其下的分支一胫神经、 腓总神经和腓 肠神经， 结扎并剪断胫神经和腓总神经， 保留细小的腓肠神经， 同时避免任何损伤。 将 肌肉、 皮肤逐层缝合， 然后腹腔注射抗生素作抗菌预防， 具体方法可以参考 （Decosterd I and Woo If CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. 2000 Aug; Pain 87(2): 149-58.)。
完全福式佐剂 （CFA) 诱导的炎性痛模型： 用 10%的水合氯醛腹腔注射麻醉 (0.3 ml/100 g体重)后， 双后足足趾和足掌皮下注射 CFA 200 μ1。 CFA液配制方法： 用美国 Sigma公司售的 CFA液（结核分支杆菌 (Mycobacterium tuberculosis)的生理盐水： 油悬浮 液）。
坐骨神经和背根神经结扎模型： 正常大鼠， 注射 10%水合氯醛， 导致大鼠麻醉， 于 大鼠后肢上缘切开皮肤， 纵行分离肌肉， 暴露坐骨神经主干用羊肠线结扎； 背根神经结 扎， 在臀背部剃毛后常规酒精消毒， 在腰 3和腰 4椎骨处做纵向 2 cm的切口， 钝性分 离脊骨两侧肌肉组织， 撑开器撑开肌肉和皮肤， 使手术视野暴露足够大， 用手术钳破坏 腰 3椎骨， 暴露出腰 5和腰 6的背跟神经， 用羊肠线结扎。 24小时后， 大鼠灌注， 然后 取材， 分别取背根神经节、 背根神经、 脊髓背角和坐骨神经。
戊四氮诱导的癫痫试验： 首先给大鼠注射 C5或 70%PGE400， 半小时后再向大鼠皮 下注射 100 mg/kg的戊四氮， 戊四氮溶解在盐水中， 然后观察大鼠的惊厥反应。 惊厥评 分采用 Fatholahi分级法 . 0级， 无反应； 1级， 节律性嘴和面部抽动； 2级， 躯体波动样 游走性痉挛； 3级， 全身肌阵挛、 臀部上翘； 4级， 躯体向一侧翻转； 5级， 仰翻位， 全 身强直痉挛发作。 每次给药后观察 30 分钟。 统计大鼠发作最大级数以及戊四氮注射到 诱导最大反应级数潜伏时间。
大鼠胫骨癌症痛模型， 反映临床病人转移性癌痛模型。 正常大鼠， 注射 10%水合氯 醛， 导致大鼠麻醉， 于大鼠后肢上缘切开皮肤， 纵行分离肌肉， 暴露出胫骨， 在胫骨骨 髓腔内注射同源大鼠的 3xl03 MRMT-1乳腺癌细胞， 在手术 14天后， 大鼠表现出明显 痛敏反应。 (3) 免疫组织化学和免疫印迹实验
在免疫组化实验中，不同时间点手术模型大鼠用固定液灌注，后固定 1.5 小时， 10% 蔗糖浸泡 24小时以上的组织在恒冷箱一切片机切 14 μηι的切片， 置于 -20°C冰箱保存。 切片与抗体(抗015-0 8 4受体(8& & 0¾2)山羊源性， 有效稀释比 1 : 100〜200) 孵育过 夜，然后用 0.01 M PBS洗三遍，然后上二抗，孵育 40分钟，然后用 0.01 M PBS洗三遍， 避光。 最后， 在显微镜下观察， 拍片。 免疫荧光强度用 Image-pro定量分析免疫荧光强 度。
在免疫印迹实验中， 将取材的背根神经节用 RIPAC50 mM Tris(pH7.4), 150 mM NaCl, 1% Triton X- 100， 1%去氧胆酸钠 (sodium deoxycholate), 0.1% SDS, 以及正钒酸 钠（sodium ortho vanadate), 氟化钠（sodium fluoride), EDTA, 亮抑酶肽（leupeptin)等多种 抑制剂） 裂解， 100°C煮 5分钟使蛋白变性， 待蛋白样品冷却后吸取 10-20 μΐ 样品加入 到配制后的胶孔中。 然后， 先恒电压 60伏电泳 30分钟， 待样品越过上下层胶边界时将 电压调至 90 伏电压继续电泳直到溴酚蓝至下层胶的下边缘。 电泳完毕后， 割下含有目 的条带的 SDS胶， 进行转膜操作， 即通过电转， 将带有负电荷的蛋白转移到带正电极的 PVDF膜，在含 5%的脱脂奶粉的封闭液中封闭 1小时以降低抗体的非特异结合。封闭结 束后， 将 PVDF膜浸润在含一抗的封闭液中在 4°C孵育过夜或在室温孵育 1-2小时， 结 束后， 将 PVDF膜用 TBST洗脱液清洗三次总共 1小时， 然后和带有辣根过氧化物酶的 二抗在室温孵育 1小时。 二抗孵育结束后， 又用 TBST洗脱液清洗 1小时， 最后在避光 条件下， 将 PVDFA膜和辣根过氧化酶底物 ECL-PLUS反应 5分钟， 产生荧光信号， 然 后用 X光胶片获取 PVDF膜上的荧光信号。 胶片经显影液和定影液定影。 (4) 疼痛检测方法
大鼠热痛敏定量检测法： 在大鼠足底注射 CFA液 24小时开始， 用热痛敏定量检测 法测定大鼠对热辐射刺激的縮足反应。 为了检测大鼠的热痛阈值， 将大鼠置于具有透明 玻璃板的有机玻璃盒中， 用热辐射仪 (BME-410C; CAMS) 刺激大鼠脚掌。 热痛阈值为 光源照到大鼠脚掌， 到大鼠反射抬足反应的时间长度。
大鼠机械痛敏定量检测法： 在大鼠足底注射 CFA液 24小时开始， 用机械痛敏定量 检测法测定大鼠对正常、 无害机械刺激的縮足反应。 可以参考文献 （Chaplan SR et aL, Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994
Jul;53(l):55-63. ) 所述的方法进行评估， 在该方法中， 用不同弯曲力 (相当于 1-50 g力的 范围）的 von Frey纤维施加无害机械刺激， 将大鼠置于具有金属网底板的有机玻璃盒中， 将 von Frey纤维施加在所述后肢足底表面，与足底表面垂直用 von Frey纤维施加足以引 起略微抵抗所述后肢的力， 在每只后肢足底保持 2-3秒， 如果所述后肢突然退縮， 则记 录阳性反应。可见常规给 10次刺激，取引起 50%(5-6次縮足 /10次刺激)縮足反射的重量 均值作为阈值。 CFA炎症 5-7 天后再测机械痛敏的阀值， 以确定这些动物是否表现了痛 觉过敏。
(5) 药物溶解
MRK 016、 C2、 C3、 C4、 C5、 C6、 C7、 C8 等化合物或 α5ΙΑ (A5I) 溶解在 70%(PGE400:水 =70: 30的体积比例）的 PGE400中， 剂量为 MRK 016 2 mg/kg或 A5I 3 mg/kg, C2、 C3、 C4、 C5、 C6、 C7、 C8化合物剂量 3 mg/kg 体重作为注射剂。
MRK016-M3溶解在 10% (PGE400:水 =10: 90的体积比例） 的 PGE400中， 剂量分
Figure imgf000025_0001
(PGE400:水 =30: 70的体积比例） 的 PGE400中， 剂量为 3 mg/kg， 作为注射剂。
(6) a5-GABAA基因敲除实验
按照 EX 500所述的方法学， 将 siRNA配置成可注射溶液。 然后将大鼠麻醉， 然后 将 siRNA溶液从腰 5或腰 6的位置注射进入脊髓。 siRNA注射 3天后， 取腰 5或腰 6 背根神经节， 检测 GABAA受体的 ct5亚基的蛋白表达强度。
针对 a5-GABAA受体 mRNA的 siRNA序列为： 5 'GGUGCGAACAGACAUCUAUTT 3Ό 对照 siRNA序列为： 5 'UUCUCCGAACGUGUCACGUTT 3'。
( 7) 数据统计
行为学数据统计方法为 unpaired t-test， *， p<0.05 , 代表显著性差异。 II. 实施例
实施例 1、 GABAA受体的 α5亚基在 SNI模型条件下表达升高
在 SNI模型中， 通过免疫组织化学实验和免疫印迹实验测定 GABAA受体的 α5亚 基蛋白的表达情况， 结果如图 1所示。
免疫组织化学实验显示， SNI模型可以诱导 GABAA受体的 α5亚基蛋白表达升高。 在免疫组织化学实验中， SNI模型制备 14天后 GABAA受体的 ct5亚基蛋白表达升高， 对照为正常大鼠的背根神经节切片 (正常)， 如图 1A所示。 结果显示： GABAA受体的 ct5 亚基在背根神经节中细胞表达升高， 更有趣的是， 在小细胞中， GABAA受体的 ct5亚基 蛋白荧光信号强度升高尤为明显。
免疫印迹实验显示， 在 SNI 模型中， 随着时间的增加， GABAA受体 ct5亚基的蛋 白总量明显升高， 如图 1B所示。 实施例 2、 GABAA受体的 α5亚基蛋白在炎性痛模型下表达升高
在 CFA诱导的炎性痛模型中，通过免疫组织化学实验和免疫印迹实验测定 GABAA 受体的 cc5亚基蛋白的表达情况， 结果如图 2所示。
免疫组织化学实验显示， 在注射 CFA 2天后， 在背根神经节中， CFA诱导的炎性 痛模型可以促进 GABAA受体 ct5亚基蛋白表达升高， 如图 2A所示。
免疫组织化学实验中， 发现 GABAA受体 ct5亚基蛋白在背根神经节中小细胞表达 升高。统计显示 GABAA受体 ct5亚基在背根神经节中细胞表达比例升高。而免疫印记实 验显示， GABAA受体 ct5亚基的蛋白量在炎性痛模型中明显增加（图 2B)， 甘油醛 -3-磷酸 脱氢酶 (GAPDH)作为阳性对照。 实施例 3、 GABAA受体的 α5亚基蛋白在背根神经节中表达， 并且转运到坐骨神经 在坐骨神经和背根神经结扎模型中， 观察 GABAA受体 ct5亚基蛋白量的情况， 结 果如图 3所示。
图 3Α显示， GABAA受体 ct5亚基蛋白可以转运到坐骨神经， 而几乎不转运到背根 神经。 CGRP (降钙素基因相关肽 Μ乍为一种可以沿坐骨神经和背根神经传输的多肽， 作为 实验的阳性对照。 免疫印迹结果表明坐骨神经、 背根神经节胞体、 背根神经束和脊髓背 角中 GABAA受体 ct5亚基的蛋白量明显增加。
图 3B显示， 坐骨神经中的 GABAA受体 ct5亚基蛋白量明显高于背根神经， 而在脊 髓中 GABAA受体 ct5亚基的蛋白量更少，甘油醛 -3-磷酸脱氢酶 （GAPDH)作为阳性对照。 实施例 4、 a5-GABAA受体的特异反向激动剂可以有效地抑制神经源性痛
SNI 模型模拟了与神经损伤相关的神经源性痛， 该损伤可以由外伤和压伤直接导 致， 或可以由一系列疾病而间接导致， 其中所说的疾病如感染、 癌症、 代谢疾病、 毒素、 营养缺乏、 免疫功能障碍以及肌骨骼改变 （musculoskeletal changes)。神经源性痛在当前 临床是最难治疗的疼痛之一， 严重影响了病人的生活质量。 加巴喷丁是当前治疗神经源 性痛的一线药物， 然而加巴喷丁可以导致病人嗜睡、 眩晕、 行走不稳和疲劳感等副作 用。
本发明人发现， 化合物 MRK016和它的代谢物 MRK016-M3可以有效地抑制疼痛。 在建立 SNI模型 Ί天后， 皮下注射 MRK016可以有效地抑制神经源性痛， 结果显示在打 药 1小时后可以明显抑制疼痛， 如图 4A所示所示。
本发明人检测了 MRK016-M3是否可以抑制神经源性痛。 通过剂量依赖试验， 发现 MRK016-M3也可以有效地抑制神经源性痛， 如图 4B所示。 在该实验中， 本发明人用建 立 SNI模型 10天到 20天的大鼠作为实验动物， 发现用 10 mg/kg、 3 mg/kg和 1 mg/kg 的剂量， 在口服给药 1小时后都能显著抑制机械疼痛阈值。
本发明人同时检测了加巴喷丁在治疗神经源性痛的效果， SNI模型 1 1天的大鼠作为 实验动物， 发现加巴喷丁可以有效地治疗神经源性痛， 如图 4C所示； 但是 MRK016-M3 的用药剂量远远小于加巴喷丁的用药剂量， 治疗疼痛的效果相似。
为了进一步确认 MRK016-M3 是通过外周神经起作用， 本发明人将 SNI模型建立 10天的大鼠作为实验动物， 局部给药， 给大鼠脚掌注射 MRK016-M3(1 mg/ml) 100微升， 结果发现同样可以明显抑制神经源性痛， 如图 4D所示。
为了证明该药物是通过 （x5-GABAA 受体起作用， 通过基因敲除实验检测了 a5-GABAA受体在 SNI模型中的作用。本发明人通过鞘内注射针对 GABAA受体 α5亚基 mRNA的 siRNA, 统计发现该方法可以明显地敲除 GABAA受体 α5亚基蛋白在背根神 经节中的表达， 如图 4Ε所示。
通过免疫印迹实验， 发现在注射特异 siRNA后可以明显降低 GABAA受体 α5亚基 蛋白在背根神经节中的表达， 对照 siRNA对 GABAA受体 ct5亚基蛋白无明显改变。 甘 油醛 -3-磷酸脱氢酶 (GAPDH)作为阳性对照。 在 SNI 模型中， 发现鞘内注射特异针对 GABAA受体 ct5亚基的 siRNA可以明显提高疼痛的阈值， 非特异 siRNA对疼痛没有显 著影响， 如图 4F所示。 在该实验中， 本发明人在 SNI模型建立 7天后， 向大鼠脊髓注 射针对 GABAA受体 α5亚基的 siRNA，然后在 SNI模型 9天、 1 1天、 13天、 15天和 17 天分别检测大鼠的疼痛阈值。 为了检测进一步检测 MRK016-M3是通过受体 a5-GABAA 受体抑制慢性痛， 在建立 SNI模型 4天后， 检测大鼠的疼痛阈值， 然后注射对照 siRNA 和针对 GABAA受体 α5亚基的 siRNA,在 4天后检测 MRK016-M3对大鼠疼痛阈值的影 响， 本发明人的结果显示在注射对照 siRNA的大鼠中， MRK016-M3C口服， 3 mg/kg)可 以明显地抑制神经源性痛； 而在注射针对 GABAA受体 ct5亚基的特异 siRNA的大鼠中，
MRK016-M3(口服， 3 mg/kg)则没有显著效果， 如图 4G所示。
根据上述机制， 所属技术领域的人员可以理解： 只要能够阻止 GABA 通过
(X5-GABAA受体发挥生物学功能的物质均为预防和治疗疼痛的潜在物质。
文 献 ( Jones P et al" Pharmacokinetics and metabolism studies on
(3 -tert-butyl-7-(5 -methylisoxazo 1-3 -yl)- 2-( 1 -methyl- 1 H- 1 ,2,4-triazol-5-ylmethoxy) pyrazolo
[1,5-d] [l,2,4]triazine, a functionally selective GABAA alpha5 inverse agonist for cognitive dysfunction. Bioorg Med Chem Lett. 2006 Feb 15;16(4):872-5.)报道， MRK016和它的代谢 物 MRK016-M3具有类似的针对 a5-GABAA受体的反向激动效果，然而 MRK016在大脑 中富集，而 MRK016-M3却很难进入大脑与 a5-GABAA受体相结合。这提示 MRK016-M3 不能有效与中枢表达的 a5-GABAA受体相结合， 不会导致中枢系统的副作用， 而可以有 效地抑制疼痛反应， 是一个潜在的临床镇痛药物。
同时该实验也同样证明：作用于外周神经 (x5-GABAA受体的反向激动剂也可以治疗 神经源性痛。 上述结论说明： 可以结合 (X5-GABAA受体但是不穿过血脑屏障的特异反向 激动剂可以有效地抑制神经源性痛， 为筛选潜在镇痛临床药物提供了新的筛选方法。 实施例 5、 a5-GABAA受体的反向激动剂治疗炎性痛
炎性痛一般是由于组织或器官因病变发生炎症而导致的疼痛，存在有急性疼痛和慢 性痛。 在动物试验中， 组织损伤性病理痛模型是与临床炎性痛相似的模型， 其中 CFA 诱导的炎性痛模型反映了临床上慢性炎性痛的各项指标。 在此模型中， 注射 CFA后 2-3 小时注射部位皮肤开始出现炎症反应， 6-24小时达到高峰， 痛敏和水肿可以持续 1-2周 (Ladarola et al" Differential activation of spinal cord dynorphin and enkephalin neurons during hyperalgesia: evidence using cDNA hybridization." Brain Res. 1988， vol.455， No.2, pp:205-12), 该模型炎性痛特点显著， 持续时间长。 本发明人发现， 化合物 MRK016和 它的代谢物 MRK016-M3可以有效地抑制炎性痛。
脊髓注射针对 GABAA受体 α5亚基的 siRNA，抑制 GABAA受体的 α5亚基在背根神 经节表达， 也可以有效地抑制炎性痛的发生， 如图 5Α和 Β所示。 在该实验中， 本发明 人首先检测大鼠的热痛阈值和机械疼痛阈值，然后当天注射针对 GABAA受体 ct5亚基的 siRNA, 第 2天给大鼠右脚掌注射 100微升的 CFA， 然后每天检测大鼠的热痛和机械痛 阈值。 结果显示， 抑制 GABAA受体的 ct5亚基在背根神经节表达可以明显抑制炎性痛。
根据上述结果，所属技术领域的人员可以理解，只要能够阻止 GABA通过 (x5-GABAA 受体发挥生物学功能的物质均为预防和治疗疼痛的潜在物质。
腹腔注射 MRK016(2 mg/kg体重）能有效地抑制炎性痛。 该实验中， 在炎性痛模型 7 天的时候， 是机械痛比较敏感的时期， 本发明人腹腔注射 MRK016(2 mg/kg体重)， 发现 该化合物能有效地抑制机械疼痛 (图 5C); 在炎性痛模型 3天的时候， 是热疼比较敏感的 时期， 腹腔注射 MRK016(2 mg/kg体重)， 发现该化合物能有效地抑制热痛（图 5D); 实验 结果发现 MRK016可以在打药后 1小时明显抑制炎性痛。
口服给药 MRK016-M3也可以有效地抑制炎性痛， 本发明人分别在炎性痛模型建立 3天检测热痛和炎性痛模型 7天检测机械痛， 结果显示 MRK016-M3打药后 1小时明显 抑制炎性痛， 如图 5E和 F所示。
同时， 该实验也同样证明： 作用于外周神经 （x5-GABAA受体的反向激动剂也可以 治疗炎性痛。 该实验说明： 可以结合 a5-GABAA受体但是不穿过血脑屏障的特异反向激 动剂可以有效地抑制炎性痛， 为筛选潜在镇痛临床药物提供了新的筛选方法。 实施例 6、 a5-GABAA受体的反向激动剂 A5I治疗神经源性痛和炎性痛
α5ΙΑ (Α5Ι) 是默沙东 （Merck) 公司开发的一个特异针对 a5-GABAA受体的反向激 动剂。 在临床 Π期实验中， 发现该药物由于水溶性的问题， 造成了一定的毒性问题。
在文献 (Munro G et al.， A question of blance-positive versus negative allosteric modulation of GABAA receptor subtypes as drive of analgesi efficacy in rat model of inflammatory and neuropathic pain. Neuropharmacology. 201 1 Jul- Aug;61(l-2): 121-32.) 报 道该化合物治疗疼痛没有效果。在这里本发明人检测了 A5I在 SNI和炎性痛模型中的镇 痛作用。 数据显示： 腹腔注射 A5I (3 mg/kg 体重)可以有效地治疗神经源性痛， 见图 6A 所示。 在该实验中， SNI模型建立 14天后， 皮下注射 A5I (3 mg/kg), 打药后 0.5小时、 1小时、 2小时、 3小时和 24小时， 检测大鼠的疼痛阈值， 结果证明该化合物可以有效 抑制神经源性痛。
A5I连续给药不会产生耐受。 在该实验中， SNI模型建立 14天后， 连续给药 6天， 分别在给药 1-3小时后检测疼痛阈值， 发现在连续给药 6天后， 仍然可以有效地治疗疼 痛， 见图 6B所示。
在 CFA诱导的炎性痛模型中， 本发明人发现腹腔注射 A5I (3 mg/kg 体重)也可以有 效地治疗炎性痛， 如图 6C和 D所示。 该实验中， 在炎性痛模型建立 3天， 检测 A5I对 热痛的影响， 打药后 0.5、 1.0、 2.0和 3.0小时检测疼痛阈值， 而在炎性痛模型建立后 7 天， 检测 A5I对机械痛疼痛阈值的影响， 打药后 0.5、 1.0、 2.0和 24小时检测机械疼痛 阈值。 实施例 7、 筛选药物
(1) 细胞水平筛选
将 GABAA受体的不同亚基表达在细胞系中， 包括人肾上皮细胞系 293 细胞系或 L(tk-)细胞系中。 将所述细胞培养于培养基中， 将该种细胞作为用于筛选抑制疼痛的药 物的细胞模型。 一个 et亚基、 β亚基和 γ亚基对形成一个完整的功能型 GABAA受体是 必不可少的。 在该实施例中， 本发明人建立了以下细胞模型：
(a) 用 α5亚基（蛋白序列见 GenBank登录号 NM_001165037.1)、 β3亚基（蛋白序列见 GenBank登录号 NM_021912.4)和 γ2亚基（蛋白序列见 GenBank登录号： NM_198904.2) 同时表达在 293细胞系中， 组成含 a5-GABAA受体；
(b) 用 αΐ亚基 (GenBank登录号： NM_001 127648.1)、 β3亚基和 γ2亚基同时表达在
293细胞系中， 组成含 αΐ亚基的 GABAA受体；
(c) 用 α2亚基 (蛋白序列见 GenBank登录号 NM_000813.2)、 β3亚基和 γ2亚基同时 表达在 293细胞系中， 组成包含 α2亚基的 GABAA受体；
(d) 用 α3亚基 (蛋白序列见 GenBank登录号 ΝΜ_000808.3)、 β3亚基和 γ2亚基同时 表达在 293细胞系中， 组成含 α3亚基的 GABAA受体。
上述 (a)-(d)均能形成具有完整功能的 GABAA受体。
然后提取细胞膜和检测蛋白浓度， 分别检测待测物质与放射性标记化合物 (例如 1 nM [1H]Ro-0151788) 的竞争结合，计算待测物质的 Ki值。上述方法可参照文献 （Ballard TM et al.， R04938581, a novel cognitive enhancer acting at GABAA alpha5 subunit-containing receptors. Psychopharmacology (Berl). 2009 Jan; 202(1-3): 207-23) 报道 的方法。如果测试组中候选物质与 (x5-GABAA受体的结合抑制常数 Ki (nM) 小于含其他 et亚基 (cd亚基、 （x2亚基或 (x3亚基） 受体的 Ki， 就表明该候选物是预防、 改善或治疗疼 痛的潜在药物。所述技术领域人员了解许多方法可以用于评价含 a5-GABAA受体的配体 结合于 (x5-GABAA受体的能力， 包括但不限于上述的方法。
本发明人通过电生理的方法检测待测物质的反向激动效率。 该方法可参照文献
(Ballard TM et al.， R04938581, a novel cognitive enhancer acting at GABAA alpha5 subunit-containing receptors. Psychopharmacology (Berl). 2009 Jan; 202 (1-3): 207-23)报道 的方法。如果待测物质能够显著抑制 GABA通过 a5-GABAA受体诱导电流，但同时不能 明显抑制 GABA引起的含 cd或（x2或（x3的 GABAA受体诱导的电流， 就表明该候选物 是预防、 改善或治疗疼痛的在药物。 所述技术领域人员了解许多方法可以用于评价 a5-GABAA受体的配体反向激动 a5-GABAA受体的效率，包括但不限于上述的方法。
通过对体外血脑屏障模型 (blood-brain barrier model, BBBM)检测待测物质是否有效 通过血脑屏障。 在该实验中， 将脑微血管内皮细胞 （Brain microvessl endothelial cell)和 星形胶质细胞共同培养， 然后检测待测物质是否可以有效穿过细胞膜， 具体方法参考相 关文献 ( Culot M et al.， An in vitro blood-brain barrier model for high throughput (HTS) toxico logical screening. Toxicol In Vitro. 2008 Apr; 22(3):799-811. ) 。
采用 MRK016、 MRK016-M3、 a5IA作为候选物质进行了测试， 结果显示在表 2中， MRK016、 MRK016-M3和 α5ΙΑ对各亚型 GABAA受体的亲和力相似， 但对 a5-GABAA 受体的反向激动效率 (Efficacy)远高于对含 αΐ或 α2或 α3 的 GABAA受体的反向激动效 率， 从而它们是对于抑制哺乳动物疼痛有用的物质。 优选的， MRK016-M3 不能显著穿 过血脑屏障， 理论上是一个无副作用的有效镇痛药物。
因此， 如果测试组中候选物质与 γ-氨基丁酸 A的 ct5亚基的结合系数 Ki (nM) 小于 其他亚基 (cd亚基、 （x2亚基或（x3亚基)， 就表明该候选物是预防、 改善或治疗疼痛的潜 在药物。 或者， 如果待测物质能够显著抑制 GABA引起的含 ct5亚基 GABAA受体诱导 的电流， 但同时不能明显抑制 GABA引起的含 cd或（x2或（x3的 GABAA受体诱导的电 流， 就表明该候选物是预防、 改善或治疗疼痛的在药物， 或者兼而有之。 以上均表明该 候选物是预防、 改善或治疗疼痛的在药物。
表 2
Figure imgf000031_0001
反向激动效率为候选物质对 GABA通过 a5-GABAA受体诱导的电流的抑制效率 = (候选物质对 GABA通过 a5-GABAA受体的电流强度-缺少候选物质存在时 GABA通过 a5-GABAA受体诱导的电流强度 )/缺少候选物质存在时 GABA通过 a5-GABAA受体诱导 的电流强度。
(2) 动物水平筛选
用候选物质皮下注射给予腹腔注射 （或通过口服） 动物， 在处理后适当时间， 测定 候选物质在动物体内的分布。 可通过以下两种方法检测待测化合物与中枢 a5-GABAA受 体的结合效率。
第一种方法， 检测待测物质在动物体内的药物分布。 在该方法中， 通过检测待测物 质是否可竞争或抑制同位素标记物质例如 (3H)Ro- 15- 1788 在大脑海马组织中的结合， 抑 制效率不能超过 60%。实验动物在给予实验动物一定剂量的候选物质和 (1H)Ro- 15- 1788， 然后检测该候选物质是否抑制已知 (x5-GABAA受体的特异配体在中枢的分布， 如果候选 物质抑制 (1H)Ro- 15- 1788在中枢的分布小于 60%， 就表明该候选物是预防、 改善或治疗 疼痛的潜在药物。 就表明该候选物是副作用较小治疗疼痛的潜在药物。 结果发现，
Figure imgf000032_0001
(MRK016-M3), 不能有效抑制 (3H)Ro-15-1788在中枢的结合， 抑制效率为 12% 左 右 (Jones P et al., Pharmacokinetics and metabolism studies on (3 -tert-butyl-7-(5-methylisoxazol-3 -yl)-2-( 1 -methyl- 1 H- 1 ,2,4 -triazol-5-ylmethoxy) pyrazolo [ 1 ,5 -d] [ 1 ,2,4]triazine, a functionally selective GABAA alpha5 inverse agonist for cognitive dysfunction. Bioorg Med Chem Lett. 2006 Feb 15;16(4):872-5)。
第二种方法，检测待测化合物在动物内的组织分布。首先， 将待测物质注射 (或口服) 到动物体内， 然后在注射 0.25-2.0 小时后提取大脑组织和血浆组织， 用色谱-质谱联用 (L-MS) 的方法检测药物的浓度， 如果化合物在大脑组织和血浆组织的比例 (brain/plasma r ， 就表明该候选物是 的潜在药
物。 结果发现，
Figure imgf000032_0002
(少量地跨血脑屏障)， 障)这两个 化合物在大脑组织和血浆组织的比例小于 0.5， 就表明该候选物是副作用较小且可以治 疗疼痛的潜在药物。 实施例 8、 制备 (3-(3-叔丁基 -2-((2-甲基 -2 氢 -1,2,4-三唑 -3-基）甲基） 吡唑并
[1,5-d] [1,2,4] 三嗪 -7-基） 异恶唑 -5-基)甲醇及其类似物
中间体 1 : 4-叔丁基 甲酰 -1氢-吡唑 -4-甲苯磺酸
(a) 3-叔丁基 -4-羟基 -2(5H)-呋喃酮 (3 -tert-butyl-4-hydroxyfuran- 2(5H)-one)
Figure imgf000032_0003
将 羟基 -2(5H)-呋喃酮 (4.0 g, 40 mmol) 和 2-甲基 -2-丙醇 (3.0 g, 40 mmol) 在 45。C 搅泮 15分钟， 然后将该混合液用浓硫酸在 45 °C快速处理一下， 然后将该溶液在 45Γ继续 搅拌 48小时。 然后将混合液用水 (10 mL)稀释， 用乙醚萃取 （10 mL x 3)， 有机层用硫酸 镁 (MgS04 )干燥。蒸发溶剂并将该残余物在硅胶上进行层析，用己垸： 乙酸乙酯= 20: 1 to 10: 1 , 得到产物 3-叔丁基 -4-羟基 -2(5H)-呋喃酮 1.85克， 为一种白色固体。
4- 叔 丁 基 羟 甲 基 ） -1H-
(4 -tert-butyl - 3 -(hydroxymethyl) - 1 H-pyrazol- 5 -o 1)
Figure imgf000032_0004
替换页 （细则第 26条） 将 3-叔丁基 -4-羟基 -2(5H)-呋喃酮 （2.2克， 14 mmol) 在常温下溶解于乙醇 (22 mL) 中， 然后加入水合肼 (4.1 g, 70 mmol)， 在 78°C的条件下搅拌 72小时。 将反应溶液蒸发， 然后用水 (20 mL)冲洗， 然后用乙酸乙酯提取 (20 mL x 3)， 有机层用 MgS04干燥， 低压 蒸发得到 2克产物 （4-叔丁基 -3- (羟甲基） -1氢 -吡唑 -5-醇） ， 为一种白色固体。
(c) 4- 叔 丁 基 -3- ( 羟 甲 基 ） -1 氢 - 吡 唑 -4- 甲 苯 磺 酸
4-methylbenzenesulfonate)
Figure imgf000033_0001
将 4-叔丁基 -3- (羟甲基） -1氢 -吡唑 -5-醇（2.0克， 12 mmol)在 DCM (40 mL)在常温 下搅拌，然后加入对甲苯磺酰氯（TsCl) (2.7 g, 14 mmol)o将三乙胺 Et3N (1.55 g， 2.12 mL) 逐滴加入。 然后在常温下搅拌 20小时。 然后将混合液用卤水 (20 mL)冲洗， 用 DCM提 取 （10 mL X 3) , 有机层用硫酸镁 (MgS04 )干燥。 将该残余物在硅胶上进行层析， 用 DCM： MeOH = 30:l , 得到产物 4-叔丁基 -3- (羟甲基） -1氢 -吡唑 -4-甲苯磺酸 2.7克， 为 黄色固体。
4-叔丁基 -3-甲酰 -1 氢 - P比唑 -4-甲苯磺酸（4-tert-butyl-3-formyl-lH-pyrazol-5-yl 4-methylbenzenesulfonate)
Figure imgf000033_0002
将 4-叔丁基 -3- (羟甲基） -1氢 -吡唑 -4-甲苯磺酸 (100 mg, 0.31 mmol) 在常温下溶解 在 CHC13 (2 mL)， 然后加入 Mn02 (70 mg, 0.8 mmol)。 将该溶液在 70°C搅拌 48小时。 再加 Mn02 (34 mg, 0.4 mmol)。 然后在 70°C搅拌 6小时。 冷却到室温， 将所述混合物过 滤并蒸发滤液， 将该残余物在硅胶上进行层析， 用 DCM : MeOH = 50: l洗脱， 得到产物 4-叔丁基 -3-甲酰 -1氢 -吡唑 -4-甲苯磺酸 30 mg, 为黄色固体。
中间体 2: 5-氯甲基 - 1-甲基 -1氢 -1,2,4-三唑
(a) ( 2-甲基 -2氢 -1,2,4-三唑 -3-基） 甲醇[(2-1^11^1-211-1,2，4-^&201-3 1)0^11 01 ]
I I
Formalin ,N-
1-甲基 -1氢 -1,2,4-三唑 (200 mg, 1.8 mmol) 和福尔马林（1 mL) 在 135°C条件下在封 闭容器中混匀过夜。 冷却到室温， 负压蒸发滤液， 将该残余物在硅胶上进行层析， 用 DCM : MeOH = 20:l洗脱， 得到产物（2-甲基 -2氢 -1，2，4-三唑 -3-基） 甲醇 140 mg， 为白 色固体。
(b) 5-氯甲基- 1-甲基 -1氢 -1,2,4-三唑 [5-(chloromethyl)-l-methyl-lH-l,2，4- triazole] ί J
Λ . SOCi， - ,N、、，
N」J 85%
将 （2-甲基 -2氢 -1,2,4-三唑 -3-基） 甲醇 (300 mg，2.7 mmol)加入到冰上放置的惰性气 替换页 （细则第 26条) 体氩气条件下的亚硫酰氯 SOCl2 (5 mL)。 混合液在 80°C加热 1小时。 负压蒸发， 然后将 残留物用叔丁基甲基醚（TBME)研磨并且过滤， 得到产物 5-氯甲基 - 1-甲基 -1氢 -1,2,4- 三唑 380 mg， 为白色固体。 中间体 3
(a)5-羟 carboxylate)
Figure imgf000034_0001
将氯代肟基乙酸乙脂 (8.0 g， 53 mmol)溶于氯仿 (80 mL)中， 然后在常温下加入丙炔醇 (9.0 g, 158 mmol) 15分钟后， 加入三乙胺（10.7 g， 105.6 mmol), 在 45°C 条件下搅拌 48 小吋。 蒸发反应溶液， 将该残余物在硅胶上进行层析， 用(HeXane : EtOAc = 3: l洗脱， 得到产物 5-羟甲基-异恶唑 -3-羧酸乙酯 4g，为白色油状物。
(b)5- (羟甲基)异恶唑 -3-碳酰肼 5-(hydroxymethyl)wasoxazole-3-carbohydrazide
Figure imgf000034_0002
将 5-羟甲基-异恶唑 -3-羧酸乙酯 (4.3 g， 25 mmol)溶于乙醇 （43 mL)中， 然后在常温下 加入水合肼 (3.7 g， 63 mmol)。 在室温下搅拌 3 h。 反应溶液过滤， 用乙醇 (10 mL)清洗， 通过模式泵干燥， 得到产物 5- (羟甲基)异恶唑 -3-碳酰肼 3.4克，产物为白色固体。
(c) 5- ( (叔丁基二甲基硅氧垸基） 甲基） 异恶唑 -3-碳酰肼 [5-((tert- butyldimethylsilyloxy)
Figure imgf000034_0003
将 5- (羟甲基） 异恶唑 -3-碳酰肼 (550 mg, 3.5 mmol)溶解在干燥 DMF (15 mL)中， 然后加入咪唑 （380 mg， 5.6 mmol)。 把 t-BuMe2SiCl (630 mg， 4.2 mmol)溶解在 DMF中， 在 -10°C 条件下滴加到上述溶液。 在室温条件下将反应溶液混勾过夜。 反应分别用冷水 和乙酸乙酯终止。 水层用乙酸乙酯 (50 mL x 2)萃取。 这有机层用卤水清洗 (20 mL x 2)， 然后用硫酸钠干燥， 过滤并蒸发得到 0.9 g淡黄色固体， 5- ( (叔丁基二甲基硅氧烷基） 甲基） 异恶唑 -3-碳酰肼。
合成步骤 1
(a) 3-叔丁基 -7- ( 5- ( (叔丁基二甲基硅氧烷基） 甲基） 异恶唑 -3-基） 吡唑并 [l,5-d][l,2,4] 三 嗪 -2- 基 -4- 对 甲 苯 磺 酸 3-tert-butyl-7-(5-((tert-butyldimethylsilyloxy)methyl)isoxazol-3-yl)pyrazolo[l,5-d][l,2,4]tria zin-2-yl 4-methylbenzenesulfonate
替换页 （细则第 26条)
Figure imgf000035_0001
5- ( (叔丁基二甲基硅氧垸基） 甲基）异恶唑 -3-碳酰肼 (850 mg, 3.1 mmol)和 4-叔丁 基 -3-甲酰 -1氢 -吡唑 -4-甲苯磺酸 （1.1 g, 2.8 mmol) 在二甲苯 (20 mL)加热在氩气条件下 回流 72 小时。 蒸发得到残余物， 用热甲醇研磨残余物， 然后得到沉淀物， 固体过滤收 集， 真空干燥得到 750 mg 目标中间体， 一种棕色固体。 母液浓缩， 残留物用硅胶上进 行层析， 用 DCM : MeOH = 200: l洗脱， 得到较纯的中间产物， 为棕色泡沫状物质， 200 mg。 -
(b) 3-叔丁基 -7- ( 5- (羟甲基） 异恶唑 -3-基） 卩比挫并 [l,5-d][l，2,4] 三嗪 -2-基 3 -tert-butyl-7-(5-(hy in-2-ol
Figure imgf000035_0002
3-叔丁基 -7- ( 5- ( (叔丁基二甲基硅氧垸基）甲基） 恶唑 -3-基）吡唑并 [l，5-d][l,2,4] 三嗪 -2-基 -4-对甲苯磺酸化合物 （800 mg， 1.4 mmol) 溶解于甲醇 (20 mL)中，然后加入 4 N aq. NaOH (l mL)。 混合反应溶液氩气条件下在 60°C条件下反应 2小时， 冷却到室温。加 入 2 N aq. HC1 (2.5 mL)。将所述混合物过滤并蒸发滤液，将该残余物在硅胶上进行层析， 用 DCM : MeOH = 20:l洗脱， 得到产物。 混合有机物用卤水洗 2次， 用硫酸钠干燥， 过 滤然后得到 360 mg 产物， 直接用于下一步反应。
(c) (3-(3-叔丁基 -2-((2-甲基 -2 氢 -1,2,4-三唑 -3-基）甲基） 吡唑并 [l,5-d][l,2，4] 三嗪
-7- 基 ） 异 恶 唑 -5- 基 ） 甲 醇 （3-(3-tert-butyl -2-
((2-methyl-2H-l,2,4-triazol-3-yl)methoxy)pyrazolo
[l,5-d][l,2,4]triazin-7-yl)isoxazol-5-yl)methanol
Figure imgf000035_0003
3-叔丁基 -7- ( 5- (羟甲基)异恶唑 -3-基）吡唑并 [l,5-d][l，2,4] 三嗪 -2-基 (360 mg， 1.2 mmol)溶解于 DMF (10 mL)中， 加入 5-氯甲基- 1-甲基 -1氢 -1，2,4-三唑盐酸盐 (250 mg, 1.5 mmol), 再加入碳酸钾 (1 g，7.5 mmol)。 反应溶液在氩气条件下 70°C加热 5小时。 蒸干 溶剂与甲苯共沸， 并将该残余物在乙酸己脂 （EtOAc) 和水之间分配， 分离出有机相。 水层用乙酸己脂 (30 mL x 2)萃取。 混合有机物用卤水洗， 用硫酸钠干燥， 蒸发得到固体 物质。将该残余物在硅胶上进行层析，用 DCM： MeOH = 50:1洗脱，得到产物 260 mg, 为 黄色粉末固体。 IH NMR (400 MHz, DMSO-d6) δ: 9.60 (s, IH), 7.94 (s, IH), 7.22 (s， IH), 替换页 （细则第 26条） 5.85 (m， 1H)， 5.62 (s, 2H), 4.74 (s, 2H), 3.91(s, 3H), 1.45 (s, 9H)， -0.02 (s, 6H); ESI-MS: m/z 385 [M+H] +. 用上述的方法可以合成下列化合物：
用 3-叔丁基 -7- ( 5- (羟甲基) 恶唑 -3-基） 吡唑并 [l，5-d][l，2，4] 三嗪 -2-基与 2-氯 甲基吡啶盐酸盐用上述类似的方法可以合成下列化合物 （C2)(3-(3-叔丁基 -2-(2-吡啶基 甲氧基 )-3，3a-2 氢 -B比唑并 [l，5-d][l，2，4]三嗪 -7-基） ^恶唑 -5-基）甲醇，如下图所示： m/z 381
Figure imgf000036_0001
同例也可以合成下列化合物， 化合物 3 (C3) (3-(3-叔丁基 -2-(3-吡啶基甲氧基 )-3，3a-2 氢-吡唑并 [l，5-d][l，2，4] 三嗪 -7-基 z 381 [M+H]+.
Figure imgf000036_0002
化合物 4 (C4) (3-(3-叔丁基 -2-(4-吡啶基甲氧基 )-3，3a-2氢 -B比唑并 [l，5-d][l，2，4] 嗪 -7-基） ^恶唑 -5-基）甲醇：
Figure imgf000036_0003
实施例 9、 C2、 C3、 C4化合物可以抑制神经源性痛和炎性痛
将 C2、 C3、 C4 化合物分别溶解在 70%(PGE400:水 =70:30 的体积比例）的 PGE400 中， 剂量分别为 3 mg/kg， 作为注射剂。
在这里本发明人检测了 C2、 C3、 C4(图 7A)化合物在 SNI模型中和炎性痛模型中的 治疗镇痛作用。
图 7A显示了化合物 C2、 C3、 C4的化学结构式。
图 7B显示： 腹腔注射 C2、 C3、 C4化合物可以有效地治疗神经源性痛。 在该实验 中， SNI模型建立 14天后， 皮下分别注射 C2、 C3、 C4化合物 (3 mg/kg体重)， 打药后 1 小时， 检测大鼠的疼痛阈值， 结果证明该化合物可以有效抑制神经源性痛。
在 CFA诱导的炎性痛模型中， 本发明人在建模 3或 7天后， 腹腔注射 C2、 C3或 C4化合物 (3 mg/kg体重)， 然后在 1小时后检测注射药物对热痛或机械痛的影响， 如图 7C和 D， 该系列化合物均可以有效抑制热痛或机械痛反应。 实施例 10、 制备 {3-[6- (吡啶 -2-甲基) -[1， 2， 4】三唑 [3， 4-a] 酞嗪 -3-基 异恶唑 -5- 基)甲醇
Figure imgf000037_0001
1、 制备中间体 4: 1-(1-氯酞嗪 -4-基)
中间体 1 : 1-(1-氯酞嗪 -4-基）
( a) 1,4-酞嗪二
Figure imgf000037_0002
将异苯并呋喃 (5.92 g, 0.04 mol) 溶解于醋酸 (22 mL) 中， 将肼 (2.8 mL, 0.044 mol) 滴加， 回流 4小时.冷却到室温,过滤,乙醇清洗， 然后在负压下蒸发， 得到产物 2,3-二氢 -1 ,4-酞嗪二酮 (2,3-dihydrophathalazine-l，4-dione, 6.13 g， 产率为 94·6%)， 为白色固体。将 该产物溶解在 POC13 (50 mL), 在 1 10°C搅拌混匀 3小时，冷却到室温， 然后慢慢倾倒到 冷水中。 过滤， 并用水清洗， THF条件下重新结晶得到 1 ,4-酞嗪二氯， 为白色固体。
(b) 1 -(1-氯酞嗪 -4-基)肼 l-(l- razine
Figure imgf000037_0003
肼一水 (5.6 mL, 0.17 mol)在乙醇 (150 mL)中煮沸，然后加入 1 ,4-酞嗪二氯 (3.0 g, 0.015 mol) , 加热回流 0.5小时。 混合液冷却到室温， 过滤收集固体物， 用乙醚清洗。 石油醚 / 乙酸乙酯条件下重新结晶， 得到产物 1-(1 -氯酞嗪 -4-基)肼， 为淡黄色固体。
中间体 2: 5-羟甲基 -carboxylic acid
Figure imgf000037_0004
(a) 5-羟甲基-异恶唑 -3-羧酸乙酯的获得方法可以参考实施例 8。 将 5-羟甲基-异恶唑 -3-羧酸乙酯 （参考实施例 8， 1.5 g; 8.76 mmol)和 1 M 氢氧化钠 （ 18 mL， 18 mmol)在 常温下搅拌 1.5小时。 卤水 （40 mL ) 加入， 用 6N 盐酸将 pH值调到 2。 用醋酸乙酯 ( 6X 60 mL )萃取。然后用硫酸镁干燥， 蒸发得到产物 5-羟甲基-异恶唑 -3-碳酸（ 1.2 g， 产率 95% ) ， 为白色固体。
步骤 1: 替换页 （细则第 26条) (a) Ν，-(μ 氯 苯 酞 -4- 基 ）-5-( 羟 甲 基 ） 异 恶 唑 -3- 碳 酰 肼 N'-(l-chloro ohydrazide
Figure imgf000038_0001
向干燥二氯甲垸中溶解 5-羟甲基-异恶唑 -3-碳酸 (770 mg, 5.4 mmol)和三乙基胺 (1.1 g, 10.8 mmol, 2 eq.) 在干燥 CH2C12 (100 mL), 在 0°C温度条件下， 加入双 (2-氧代 -3-恶 唑垸基)次磷酰氯 （BOP-C1, 1.47 g, 5.4 mmol, leq.). 在 0°C温度条件下搅拌 0.5小时。 再加入 1-(1-氯酞嗪 -4-基)肼 (943 mg, 4.86 mmol, 0.9 eq.)。 混合液在 0°C搅拌 2小时， 再 在室温下搅拌过夜。 蒸发， 固体残余物用水和乙醚清洗， 得到产物 N，-(l-氯苯酞 -4- 基) -5- (羟甲基） 异恶唑 -3-羧酸乙酯 (900 mg， 产率: 60%)， 为黄色固体。
(b) (3-(6- 氯 -[1，2，4] 三 唑 [3，4-a] 酞 嗉 -3- 基 ） 异 恶 唑 -5- 基 ） 甲 醇 (3-(6-chloro-[l,2,4]triazo l
Figure imgf000038_0002
N，-(l-氯苯酞 -4-基) -5- (羟甲基） 异恶唑 -3-羧酸乙酯 （ 900 mg， 2.82 mmol)和乙基胺喹纳 定 （154 mg，0.13 mmol, 0.4 eq.)在二甲苯 (60 mL)回流 3小时。 DCM (CH2C12)加入到悬浮液， 得到均匀混合液。 有机相用水清洗， 硫酸镁干燥， 过滤， 然后蒸发得到产物 (3-(6-氯 -[1，2，4] 三唑 [3,4-a] 酞嗪 -3-基） 异恶唑 -5-基)甲醇 (300 mg，产率为 35%)， 为黄色固体。
(c){3-[6- (吡啶 -2-甲基） -[1， 2， 4]三唑 [3， 4-a] 酞嗪 -3-基] - 异恶唑 -5-基）甲醇
{
Figure imgf000038_0003
吡啶 -2-基 -甲醇 （300 mg， 1 mmol) 在 DMF (30 mL) 中，在室温下加入 NaH (80 mg, 2 mmol, 2eq.) o 混合液在室温下搅拌 15分钟。 （3-(6-氯 -[1,2,4]三唑 [3,4-a] 酞嗪 -3-基） 异恶 唑 -5-基）甲醇 （164 mg， 1.5 mmol, 1.5 eq.) 加入， 室温搅拌 1小时。 冷水终止反应， 并且 浓缩。残留物用硅胶上进行层析， 得到产物 C5(50 mg， yield: 13.4%)。 1HNMR (400 MHz, DMSO-d6) 6:8.64(d,lH),8.63-8.60(d,lH), 8.53-8.33(d,lH), 8.13(t,lH) 7.99 (t, 1H),7.87 (t,lH),7.76(d,lH), 7.41-7.39(t,lH), 7.15(s,lH) ，5.87(t,lH),5.71 (s,2H),4.74-4.73(d,2H). m/z(ESI+)375(M+H+).
用上述类似的方法， 合成下列化合物：
化合物 6(C6): {3-[6- (吡啶 -3-甲基) -[1， 2, 4]三唑 [3， 4-a] 酞嗪 -3-基] - 异恶唑 -5-基) 甲醇 m/z(ESI+)375(M+H+). 替换页 （细则第 26条)
Figure imgf000039_0001
化合物 7(C7): {3-[6- (吡啶 -4-甲基) -[1， 2， 4]三唑 [3， 4-a] 酞嗪 -3-基]- 异恶唑 -5-基)
Figure imgf000039_0002
化合物 8(C8): {3-[6-(2-溴-苯 )-[1， 2， 4]三唑 [3， 4-a] 酞嗪 -3-基]- 异恶唑 -5-基）甲醇 1HNMR (400 MHz, DMSO-d6) δ： 8.60(d,lH),8.27(d, 1H)， 8.13(t, lH),7.99(d, 1H)， 7.85(d, 1H), 7.50(t, 1H)， 7.47(t, 1H)， 7.41(t, 1H)， 7.21(s, lH)，5.85(t， lH),5.71(s, 2H),4.75(d, 2H)
Figure imgf000039_0003
实施例 11、 C5、 C6、 C7、 C8化合物可以抑制神经源性痛和炎性痛
将上述实施例合成的化合物 C5、 C6、 C7和 C8化合物分别溶解在 70% PGE400中， 剂量分别为 3 mg/kg 体重， 作为注射剂。
本发明人检测了 C5、 C6、 C7和 C8化合物在 SNI模型中和炎性痛模型中的镇痛作 用。
图 8A显示了化合物 C5、 C6、 C7和 C8的化学结构式。
图 8B显示： 腹腔注射 C5、 C6、 C7和 C8化合物可以有效地治疗神经源性痛。 在该 实验中， SNI模型建立 14天后，皮下分别注射 C5、 C6、 C7和 C8化合物 (3 mg/kg 体重)， 打药后 1小时， 检测大鼠的疼痛阈值， 结果证明该化合物可以有效抑制神经源性痛。
在 CFA诱导的炎性痛模型中， 本发明人在建模 3或 7天后， 腹腔注射 C5、 C6、 C7 和 C8化合物 (3 mg/kg 体重)， 然后在 1 小时后检测药物对热痛或机械痛的影响， 如图 8C和 D所示， 该系列化合物均可以有效抑制热痛和机械痛反应。
C5在行为学检测中， 镇痛效果最为理想。 图 8E中， 皮下注射 C5分别以 1 mg/kg 体 重和 3 mg/kg体重的剂量， 然后打药后 0.5小时、 1小时、 2小时， 3小时、 6小时和 12 小时， 检测大鼠的疼痛阈值， 结果证明该化合物可以有效抑制神经源性痛。在该试验中， 吗啡作为阳性对照， 皮下注射， 剂量为 10 mg/kg。 同时， 本发明人也检测了 C5是否影 响大脑中枢反应，我们检测了 C5是否潜在诱导癫痫反应，在图 8F中，发现 C5在 1 mg/kg 、 3 mg/kg, 10 mg/kg和 15 mg/kg的剂量对戊四氮诱导的癫痫并没有作用。 这提示 C5 可以有效地治疗疼痛， 并且没有潜在的中枢副作用。 实施例 12、 一类 a5-GABAA受体的反向激动剂的镇痛效果验证
(x5-GABAA受体的反向激动剂广泛应用
切有限公司申请了一系列相关化合物， 并且
Figure imgf000040_0001
在药物， 进行临床 I期试验。 在这里本发明人按照文献 (； Knust H et al., The discovery and unique pharmacological profile of R04938581 and R04882224 as potent and selective GABAA alpha5 inverse agonists for the treatment of cognitive dysfunction. Bioorg Med Chem letter. 2009 Oct 15;19(20):5940-4)和美国专利（US 2006/0084642 Al ， US 2007/0082890 Al等)合成了下表中所示的化合物。 在大鼠慢性痛模型中， 检测了这一类 化合物在疼痛中的作用。 如下图所示， 将下列化合物分别溶解在 PGE400:水 =(30: 70的 体积比例）的 PGE400中， 剂量为 3 mg/kg体重， 作为注射剂。
在 SNI模型中和炎性痛模型中的治疗镇痛作用。 行为数据显示： 腹腔注射下列化合 物 (3 mg/kg 体重)可以有效地治疗神经源性痛。 在该实验中， SNI建立 14天后， 皮下注 射下列化合物 (3 mg/kg体重)， 打药后 1小时， 检测大鼠的疼痛阈值， 结果证明该系列化 合物可以有效抑制神经源性痛。 在 CFA诱导的炎性痛模型中， 我们在建模 3或 7天后， 腹腔注射下列化合物 (3 mg/kg体重)， 然后在 1小时后检测药物对热痛或机械痛的影响， 结果如下所示， 该系列化合物均可以有效抑制热痛或机械痛反应。
同时， 本发明人检测了该类型化合物在在大鼠组织中的分布。 首先将待测物质皮下 注射到动物体内， 然后在注射 1 小时后提取大脑组织和血浆组织， 用色谱-质谱联用 (L-MS) 的方法检测药物的浓度， 如果化合物在大脑组织和血浆组织的比例 (brain/plasma ratio) 小于 0.1， 就表明该候选物是副作用较小且可以治疗疼痛的潜在药
物。
Figure imgf000040_0002
表 3
化合物 大脑 /血液 CFA诱导的炎性 CFA诱导的炎性 神经源性痛 含量比例 痛模型 (热痛） 痛模型 (机械痛） ( SNI 模型）
<0.2 镇痛效果显著 镇痛效果显著 镇痛效果显著
Figure imgf000041_0001
可以有效地抑制 CFA诱导的炎性痛， 并且也可以抑制 SNI诱导的神经源 性痛。 这三个化合物几乎不能穿过血脑屏障， 不会直接影响中枢功能， 属于中枢副作用 小， 且可以有效治疗疼痛的药物。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员 可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定 的范围。

Claims

权 利 要 求
1. 含 ( 5亚基的 γ-氨基丁酸 A受体的反向激动剂或其药学上可接受的盐的用途， 用 于制备预防、 改善或治疗疼痛的药物。
2. 如权利要求 1所述的用途， 其特征在于， 所述的含 ( 5亚基的 γ-氨基丁酸 A受体 的反向激动剂是选择性结合于该受体的苯二氮卓结合位点的配体。
3. 如权利要求 1所述的用途， 其特征在于， 所述的 ct5亚基的 γ-氨基丁酸 A受体的 反向激动剂是具有式 (I)或 （Π) 结构的化合物或其药学上可接受的盐：
Figure imgf000042_0001
( II) ，
其中， R'a表示卤素； 或 d_6烷基、 C3_7环烷基、 C4_7环烯基、 C6_8二环烷基、 C6_10 芳基、 C3_7杂环烷基、 定义为或者含有 6个原子， 其中 1， 2或 3个为氮， 或者含有 5个 原子； 其中 1、 2或 3个独立选自氧、 氮或硫而且其中氧或硫不超过一个的芳环的杂芳 基， 或二 (d_6) 烷基氨基， 这些基团中的任意一个可以用一个或多个选自以下的取代基 任选取代： 卤素、 R3、 OR3、 OC(0)R3、 NR4R5、 NR4R5(C1 -6)烷基、 NR4R5C(0)、 NR4R5C(0) (Cw)烷基、 CN、 氰基 (Cw)烷基或 R6。
Rlb表示 d_6烷基、 C3_7环烷基、 C4_7环烯基、芳基、 C3_7杂环烷基、杂环芳基、二 (d_6)
R3表示 C2_6烷基、 ^_6链烯基、 C2_6块基、 ^_6环烷基、 C3_6环烷基 （d_6)烷基、 氰 基 (d_6)烷基、 羟基 （d_6)烷基且 R3任选为 1、 2或 3氟代的。
R4和 R5每个独立为氢、 d_6烷基、 〔2_6链烯基、 C2_6块基、 _6环烷基或 CF3， 或 R4和 R5与它们共同连接的氮原子一起形成 4-7元杂脂肪环， 该脂肪环含有所述氮原予 和一个任选自 0、 N和 S的其它杂原子， 所述环可以被一个或多个 R3基团任选取代。
R6为 C6_1Q芳基、 C6_1Q芳基 (C1-6)烷基、 杂芳基或杂芳基 (d_6)烷基. 其中杂芳基如上 定义， 而且 R6任选被一个、 两个或三个独立选自以下的取代基取代： 卤原子和 d_4烷 基、 C2_4链烯基、 C2_4块基、 C1-4烷氧基、 C2_4链烯氧基和 C2_4块氧基基团，其中每个基 团是未取代的或被一个、 两个或三个卤原子取代。
X'为 NR4R5; 或 X'为含有 1、 2、 3或 4个独立选自氧、 氮和硫的杂原子的 5元杂芳 环， 杂原子中最多有一个为氧或硫， 或为含有 1、 2或 3个氮原子的 6元杂芳环， 所述 5 或 6元杂芳环任选稠合于苯环或吡啶环上并且该杂芳环任选被 RW和 /或 Ry和 /或 Rz取代， 其中 Rw为卤素、 R3、 OR3、 OC(0) R3、 C(0)OR3、 NR4R5、 NR4C(0)R5、 OH、 三 (C1 -6烷 基）甲硅烷基 Cw烷氧基 Cw烷基、 CN或 R6， Ry为卤素、 R3、 OR3、 OC(0) R3、 NR4R5、 NR4C(0) R5、 、 NR4 R5(Cw)烷基或 CN而 Rz为 R3、 OR3、 OC(0)R3， 前提是当 X'为吡 啶衍生物时， 该吡啶环任选为 N-氧化物形式， 并且前提是 X'为四唑衍生物时， 它被 d_4 垸基基团保护；或 X'为由一个、两个或三个独立选自以下的基团任选取代的苯基'： 卤橐、 氰基、 d.6垸基、 C2.6链烯基、 C2— 6炔基和 环垸基。
Z'表示含有 1、 2或 3个独立选自氧、 氮和硫的杂原子的 5元杂芳环， 最多有 1个杂 原子为氧或硫， 并且前提是当 1个原子为氧或硫时至少也存在 1个氮原子， 或为含有 2 或 3个氮原子的除吡嗪之外的 6元杂芳环， 所述环中的任何 1个可以由 1个或多个选自 以下的取代基任选取代：卤素、 R3、 OR3、 OC(0)R3、 NR4R5、 NI^R^C^)烷基、 NR4R5C(0)、 NE^RS OXC^)垸基、 CN、 氰基 (d.6)垸基或 R6。
4. 如权利要求 3所述的用途， 其特征在于， 所述的 α5亚基的 γ-氨基丁酸 A受体的 反向激动剂是具有式 (II 的盐：
其中， Rle表示溴、
Figure imgf000043_0001
d.6垸基、 C3.7 环烷基； 1为含有 1、 2或 3氮原子的 5元杂环， 杂原子中最多有一个为氧或硫， 或为含 有 1、 2或 3个氮原子的 6元杂芳环， 所述 5或 6元杂芳环所述环中的任何 1个可以由 1 个或多个选自以下的取代基任选取代： d.6垸基、 氨基、 吡啶基、 CF3、 芳基 (d.6)垸基、 吡啶基 (d.6)烷基、 卤素、 氰基、 氰基 (d.6)垸基、 羟甲基、 羟基或其酮基互变异构体。 Z1表示羟基、 卤素、 羟基或氨基取代的 d-C4垸基、 C2-C4链烯基、 C2-C4链炔基、 d-C4
5. 如权利要求 3所述的用途， 其特征在于， 所述的含 α5亚基的 γ-氨基丁酸 A的反 向
Figure imgf000043_0002
6. 如权利要求 3所述的用途， 其特征在于， 所述的含 α5亚基的 γ-氨基丁酸 A的反 向激动剂为下列化合物或其药学 该化合物为：
Figure imgf000043_0003
替换页 （细则第 26条)
7. 如权利要求 1所述的用途， 其特征在于， 所述的 α5亚基的 γ-氨基丁酸 A受体的 反向激动剂是具有式 (VI) ) 结构的化合物或其药学上可接受的盐-
Figure imgf000044_0001
！^是卤素、 d.7垸基、 C2.7链块基、 环垸基、 d.7烷氧基、 OCF3、 -NHR、 -NHC(0)R 或 -NHS02R; R是氢、 C1-7垸基、 被卤素取代的 C1-7烷基、 杂芳基、 -(CH^O-Cw垸基、 -NH-d.7垸基； R2是卤素、 氢、 -C(0)0(CM)垸基、 C(0)NHCH2CCH、 C(0)NHCH2CH2、 S02CH2CH3、 _7烷基、 被卤素取代的 d.7垸基、 氰基、 C3.6环垸基； n是 0、 1、 2 或 3。
8. 如权利要求 7所述的用途， 其特征在于， 所述的含 α5亚基的 γ-氨基丁酸 A的反 向
Figure imgf000044_0002
9. 如权利要求 1所述的用途， 其特征在于， 所述的含 α5亚基的 γ-氨基丁酸 Α的 反向
Figure imgf000044_0003
10. 如权利要求 1所述的反向激动剂， 其特征在于， 所述的含 (x5亚基的 γ-氨基丁 酸 Α受体的反向激动剂是结合外周神经表达的含 α5亚基的 γ-氨基丁酸 Α受体， 但不能 有效与中枢表达的含 α5亚基的 γ-氨基丁酸 Α受体相结合。
1 1. 如权利要求 1所述的用途，其特征在于，所述的疼痛是外周神经相关的慢性痛。
12. 如权利要求 1所述的用途， 其特征在于， 所述的慢性痛是神经源性痛、 炎性痛 和癌性痛。
13. 如权利要求 1所述的用途， 其特征在于， 所述的疼痛病包括： 头痛、 面部痛、 颈痛、 肩痛、 背痛、 胸痛、 腹痛、 背部痛、 腰痛、 下肢痛、 肌肉与骨骼疼痛、 躯体疼痛、 血管疼痛、 痛风、 关节炎疼痛、 与躯体病样精神障碍有关的疼痛、 内脏疼痛、 感染性疾 替换页 （细则第 26条） 病导致的疼痛、 多骨疼痛、 镰刀细胞贫血、 自身免疫性疾病、 多发性硬化或炎症有关的 疼痛、 急慢性炎症疼痛、 癌性疼痛、 神经源性痛、 损伤或手术引起的疼痛、 癌性痛、 伤 害感受性疼痛、 糖尿病、 外周神经病、 疱疹后神经痛、 三叉神经痛、 腰部或子宫颈神经 根病、 舌咽神经痛， 自主神经反射性疼痛、 反射性交感神经营养不良、 神经根撕脱、 癌 症、 化学损伤、 毒素、 营养缺乏、 病毒或细菌感染、 退行性骨关节病或它们的联合形式。
14. 一种筛选预防、 改善或治疗疼痛的药物的方法， 所述方法包括：
(1) 提供一包含含 α5亚基的 γ-氨基丁酸 Α受体的体系，所述的含 ct5亚基的 γ-氨基 丁酸 Α受体具有完整的 γ-氨基丁酸 Α受体功能；
(2) 将候选物质给予步骤 (1)的体系， 观察候选物质与含 ct5亚基的 γ-氨基丁酸 A受 体的结合情况； 若候选物质能够与含 α5亚基的 γ-氨基丁酸 A受体结合， 则该候选物质 是预防、 改善或治疗疼痛的潜在药物。
15.如权利要求 14所述的方法，其特征在于，步骤 (1)中，还包括设置对照组的步骤， 所述的对照组选自： 包含含 cd亚基的 γ-氨基丁酸 A受体的体系、 包含含（x2亚基的 γ- 氨基丁酸 Α受体的体系和 /或包含含 ( 3亚基的 γ-氨基丁酸 Α受体的体系，对照组 γ-氨基 丁酸 Α受体具有完整的 γ-氨基丁酸 Α受体功能。
在步骤 (2)中， 所述方法包括： 在包含含 ct5亚基的 γ-氨基丁酸 A受体的体系中， 检 测候选物质与含 α5亚基的 γ-氨基丁酸 A受体的结合情况， 并与对照组比较， 若候选物 质与含 ct5亚基的 γ-氨基丁酸 Α受体的结合抑制常数 Ki在统计学上小于其与含 cd、 α2 或 (χ3亚基的 γ-氨基丁酸 Α受体的结合抑制常数 Ki， 就表明该候选物是预防、 改善或治 疗疼痛的潜在药物。
16.如权利要求 14所述的方法，其特征在于，步骤 (1)中，还包括设置对照组的步骤， 所述的对照组选自： 包含含 cd亚基的 γ-氨基丁酸 Α受体的体系、 包含含 o2亚基的 γ- 氨基丁酸 Α受体的体系和 /或包含含 ( 3亚基的 γ-氨基丁酸 Α受体的体系，对照组 γ-氨基 丁酸 Α受体具有完整的 γ-氨基丁酸 Α受体功能。
步骤 (2)中， 所述方法包括： 通过电生理方法检测候选物质对 γ-氨基丁酸通过 γ-氨 基丁酸 Α受体诱导的电流的抑制， 如果待测物质能够显著抑制含 ct5亚基 γ-氨基丁酸 A 受体介导的电流， 但同时不能显著抑制含 cd或 ( 2或 (x3的 γ-氨基丁酸 A受体介导的电 流， 就表明该候选物质是预防、 改善或治疗疼痛的潜在药物。
17. 一种预防、 改善或治疗疼痛的方法， 包括给予需要的患者安全有效量的含 ct5 亚基的 γ-氨基丁酸 Α受体的反向激动剂或其药学上可接受的盐。
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