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Definitions

• Nociceptors are specialized primary afferent neurons and the first cells in a series of neurons that lead to the sensation of pain.
• the receptors in these cells can be activated by different noxious chemical or physical stimuli.
• the essential functions of nociceptors include the transduction of noxious stimuli into depolarizations that trigger action potentials, conduction of action potentials from primary sensory sites to synapses in the central nervous system, and conversion of action potentials into neurotransmitter release at presynaptic terminals, all of which depend on ion channels.
• TRP channel protein of particular interest is the vanilloid receptor.
• the vanilloid receptor is a non-selective cation channel which is activated or sensitized by a series of different stimuli including capsaicin, heat and acid stimulation and products of lipid bilayer metabolism (anandamide), and lipoxygenase metabolites. See, for example Smith, et al., Nature, 418:186-190 (2002).
• Compounds, such as those of the present invention, which interact with the vanilloid receptor can thus play a role in treating or preventing or ameliorating these conditions.
• 02/16319 suggest that compounds having a high affinity for the vanilloid receptor are useful for treating stomach- duodenal ulcers.
• Ureidopyrrolidines that are said to exhibit analgesic, central nervous system, and pyschopharmacologic activities. These patents specifically disclose the compounds l-(l-phenyl-3-pyrrolidinyl)-3- ⁇ henyl urea and l-(l-phenyl-3- pyrrolidinyl)-3-(4-methoxyphenyl) urea respectively.
• International Patent Applications, Publication Numbers WO 01/62737 and WO 00/69849 disclose a series of pyrazole derivatives which are stated to be useful in the treatment of disorders and diseases associated with the NPY receptor subtype Y5, such as obesity.
• German Patent Application Number 2502588 describes a series of piperazine derivatives. This application specifically discloses the compound N-[3-[2-(diethylamino) ethyl]- l,2-dihydro-4-methyl-2-oxo-7- quinolinyl]-4-phenyl- 1 -piperazinecarboxamide.
• compositions having the compounds of the present invention as active ingredients and to their use to treat, prevent or ameliorate a range of conditions in mammals such as but not limited to pain of various genesis or etiology, for example acute, chronic, inflammatory and neuropathic pain, dental pain and headache (such as migraine, cluster headache and tension headache).
• pain of various genesis or etiology for example acute, chronic, inflammatory and neuropathic pain, dental pain and headache (such as migraine, cluster headache and tension headache).
• L is substituted or unsubstituted cycloalkyl
• each of W, Z, Y and X is independently N or CR 4 ;
• R 3 is halo, Cj-C 6 alkyl, halo Ci-C 6 alkyl, or hydroxy Cj-C 6 alkyl; each R 4 is independently hydrogen, C 1 -C 6 alkyl, hydroxyl C 1 -C 6 alkyl, Ci-C 6 alkylamino, Cj-C 6 alkoxy, amino C 1 -C 6 alkoxy, substituted amino Ci-C 6 alkoxy, di C 1 -C 6 alkylamino C 1 -C 6 alkoxy, cycloalkyl C 1 -C 6 alkoxy, Ci-C 6 alkoxycarbonyl, C 1 -C 6 alkylarylamino, aryl Ci-C 6 alkyloxy, amino, aryl, aryl C 1 -C 6 alkyl, sulfoxide, sulfone, sulfanyl, aminosulfonyl, arylsulfonyl, sulfuric acid, sulfuric acid ester, azido
• L is cycloalkyl.
• L is substituted or unsubstiruted cyclopropyl.
• L is cyclopropyl substituted with H, halo, Cj-C 6 alkyl, halo Cj-C 6 alkyl, or hydroxy C r C 6 alkyl.
• R 6 is independently H; and each of R 3 and R 3 is Cl.
• R 5 and R 6 is independently H; and R 3 is independently selected from halo, C 1 -C 6 alkyl, halo C 1 -C 6 alkyl, or hydroxy
• R 5 and R 6 is independently H; and R 3 is independently selected t-Bu and CF 3 .
• R 1 is substituted or unsubstituted aryl, heteroaryl, bicycloaryl or bicycloheteroaryl.
• R 1 is substituted or unsubstituted phenyl or pyridyl; and the substitution is selected from H, halo, C 1 -C 6 alkyl, halo C 1 -C 6 alkyl, hydroxy C 1 -C 6 , cyano,
• R 1 may be substituted or unsubstituted
• each of A 1 , A 2 , A 3 , A 4 , B ! and B 2 is independently CR 4 and N; and each of R 4 is independently H, substituted or unsubstituted lower alkyl, halo, hydroxyl, alkoxy, substituted alkoxy, amino, substituted amino, or hydroxyalkyl. In another embodiment, each of R 4 is independently H, C 1 -C 6 alkyl, halo, or hydroxy CpC 6 alkyl. [0030] In yet another particular embodiment, with respect to the compounds of formula I and IA, R 1 may be substituted or unsubstituted
• each of A 5 and A 8 is independently CR 4 R 4' , NR 4' , O 5 S, SO or SO 2 ; each of A 6 and A 7 is independently CR 4' , NR 4' , CR 4' R 4' or CO; each of B 3 and B 4 is independently; when R 4' is attached to C, each R 4 is independently H, Cj-C 6 alkyl, halo, or hydroxy C 1 -C 6 alkyl, and when R 4 is attached to N, each R 4 is independently H or C r C 6 alkyl; and the dotted bond represents a single or a double bond.
• R 4' is Cj-C 6 alkyl or hydroxy C 1 -C 6 alkyl.
• R 1 may be substituted or unsubstituted
• each of A 9 , A 10 and A 11 is independently CR 4' , CR 4 R 4' , CO, CS, N, NR 4' , O, S, SO or SO 2 ; each of B 5 and
• B 6 is independently CR 4 ; when R 4 is attached to C, each R 4 is independently H, C 1 -C 6 alkyl, halo, or hydroxy C 1 -C 6 alkyl, and when R 4 is attached to N, each R 4 is independently H, or C 1 -C 6 alkyl; and each of the dotted bonds independently represents a single or a double bond.
• R 4 is C 1 -C 6 alkyl or hydroxy C 1 -C 6 alkyl.
• R 1 may be substituted or unsubstituted:
• the ring may be further substituted with R 4 , and R is as described in the preceding paragraphs; and when feasible, the ring N can further be substituted with H or C 1 -C 6 alkyl.
• R 1 may be substituted or unsubstituted:
• R 1 may be substituted or unsubstituted:
• each of A 5 and A 8 is independently CH 2 , CHMe, NH, NMe, O, S, SO or SO 2 ; and R 4' is C 1 -C 6 alkyl or hydroxy C 1 -C 6 alkyl.
• R 1 may be substituted or unsubstituted:
• each of A 9 , A 10 and A 11 is independently CH, CH 2 , N, NH, O, or S; each R 4' is independently H, Ci-C 6 alkyl or hydroxy C r C 6 alkyl; and each of the dotted bonds independently represents a single or a double bond.
• R 1 may be
• R 1 is as described in the preceding paragraphs and R 4' is alkyl or substituted alkyl. In yet another embodiment R 4' is substituted alkyl. In yet another particular embodiment R 4 is hydroxy alkyl. In yet another particular embodiment R is hydroxymethyl, hydroxylethyl or hydroxypropyl. In yet another particular embodiment R is hydroxymethyl. [0038] In one particular embodiment, with respect to the compounds of formula I and IA, R 1 is as described in the preceding paragraphs and R 4' is alkyl or substituted alkyl. In yet another embodiment R 4' is substituted alkyl. In yet another particular embodiment R 4 is hydroxy alkyl. In yet another particular embodiment R is hydroxymethyl, hydroxylethyl or hydroxypropyl. In yet another particular embodiment R is hydroxymethyl. [0038] In one particular embodiment, with respect to the compounds of formula I and IA, R 1 is
• R 1 may be substituted or unsubstituted:
• R 1 may be:
• W 3 Z, X and Y may for example each represent CR 4 especially
• X may represent N and W, Z and Y may each represent CR .
• each of X, Y and Z represents CR 4 especially CH.
• W is N.
• Y is N.
• each of W, X and Z represents CR 4 especially
• CH and Y represents CR 4 .
• R 4 may for example represent substituted alkyl, halo, sulfone, alkoxy, or amino. Particularly, R 4 may represent substituted alkyl or halo. More particularly, R 4 may be methyl, chloro, trifluoromethyl or fluoro.
• each of W and X represents CR 4 especially CH and each of Y and Z represent CR 4 .
• each R 4 may for example represent substituted alkyl, halo, alkoxy, or amino.
• R 4' may represent substituted alkyl or halo. More particularly, R 4 may be methyl, trifluoromethyl, chloro or fluoro.
• the present invention provides amide compounds according to formula
• each of W, Z, Y and X is independently N or CR 4 ; each of A 1 , A 2 , A 3 , A 4 , B 1 and B 2 is independently N or CR 4 ; R 3 is t-Bu or CF 3 ; each R 4 is as described for formula I; and R 4 is Ci-Cg alkyl or hydroxyl CrC ⁇ alkyl.
• each of B 5 and B 6 is independently CH and N.
• the present invention provides amide compounds according to formula
• each of A 5 and A 8 is independently CR 4 R 4' , NR 4' , O, S, SO or SO 2 ; each of A 6 and A 7 is independently CR 4' , NR 4' , CR 4 R 4 or CO; each of B 3 and B 4 is independently CR 4 ; when R 4' is attached to C, each of R 4 is independently H, CpCe alkyl, halo, or hydroxy Ci-Cg alkyl, and when R 4 is attached to N, each of R is independently H or Ci-C 6 alkyl; and the dotted bond represents a single or a double bond; R 3 is t-Bu or CF 3 ; each R is as described for formula I; and R 4 is Q-C 6 alkyl or hydroxyl Ci-C 6 alkyl.
• the present invention provides amide compounds according to formula
• R 3 is t-Bu, or CF 3 ;
• X and W are independently C-H;
• Z and Y are independently C-H, C-F, C-Cl, C-Me or C-OMe;
• B 3 and B 4 are independently CR 4 or N; and wherein each of A 5 and A 8 is independently CR 4 R 4 , NR 4 , O, S, SO or SO 2 ;
• each of A 6 and A 7 is independently CR 4' , NR 4' , CR 4 R 4' or CO;
• each of R 4' is independently H, substituted or unsubstituted alkyl or aryl; and the dotted bond represents a single or a double bond.
• the present invention provides amide compounds according to formula
• R 3 is t-Bu, or CF 3 ;
• X and W are independently C-H ;
• Z and Y are independently C-H, C-F, C-Cl, C-Me or C-OMe;
• B 3 and B 4 are independently CR 4 ; and
• a 5 and A 8 are independently O or NH.
• each of B 3 and B 4 is independently CH and N.
• a 5 and A 8 both may be O.
• a 5 and A 8 both may be NH.
• a 5 may be O and A 8 may be NH. In one particular embodiment, with respect to the compounds of formula IV or IVA, A 5 may be NH and A 8 may be O. [0049] In yet another embodiment, the present invention provides amide compounds according to formula
• each ofA 9 , A 10 and A n is independently CH, CH 2 , N, NH, O, or S; each of B 5 and B 6 is independently CR 4 ; each R 4' is independently H, Ci-C 6 alkyl or hydroxy Ci-C 6 alkyl; and each of the dotted bonds independently represents a single or a double bond; R 3 is t-Bu or CF 3 ; each R 4 is as described for formula I; and R 4 is C)-C 6 alkyl or hydroxyl CpC 6 alkyl.
• each of B 5 and B is independently CH and N.
• the present invention provides amide compounds according to formula
• each ofW, Z, Y and X is independently N or CR 4 ; each of B 7 , B 8 and B 9 is independently CR 4 ; R 3 is t-Bu or CF 3 ; each R 4 is as described for formula I; and R 4 is CpC 6 alkyl or hydroxyl CpQ alkyl.
• each of B 7 , B 8 and B 9 is independently CH and N.
• Y and Z both may be C-H.
• Y is C-H and Z is C-F or C-Cl.
• Y is C-H and Z is C-F.
• Y is C-H and Z is C-Cl.
• Y is C-H and Z is C-Me or C-OMe.
• Y and Z both may be C-F.
• Y and Z both may be C-Cl.
• W, Z, X and Y may for example each represent CR 4 , especially CH.
• X may represent N and W
• Z and Y may each represent CR 4 .
• each of X, Y and Z represents CR , especially CH.
• W is N.
• Y is N.
• each of W, X and Z represents CR 4 especially CH and Y represents CR 4 .
• R 4 may for example represent substituted alkyl, halo, alkoxy, or amino.
• R 4' may represent substituted alkyl or halo. More particularly, R 4 may be methyl, chloro or fluoro.
• each of W and X represents CR 4 especially CH and each of Y and Z represent CR 4 .
• each R 4 may for example represent substituted alkyl, halo, alkoxy, or amino.
• R 4 " may represent substituted alkyl or halo. More particularly,
• R 4 may be methyl, chloro or fluoro.
• each of W and X is N or CR 4
• each of Y and Z is N or CR 4 and each R 4 is independently selected from hydrogen, alkyl, trihaloalkyl and halo.
• each R 4 is independently H, CH 3 , CF 3 , Cl, or F. In certain embodiments, each R 4 is H.
• each of W, X, and Z is N or CH, and Y is C-
• R 4 is hydroxyl substituted alkyl.
• R 4 is -(C ⁇ ) n - OH wherein n is selected from 1-6.
• R 4 is CH 2 OH.
• the compounds of the invention are set forth and may be selected from a comprehensive listing of such compounds, set forth later on herein in Table 1.
• Table 1 contains in
• the compounds of the present invention are useful for the treatment of inflammatory pain and associated hyperalgesia and allodynia. They are also useful for the treatment of neuropathic pain and associated hyperalgesis and allodynia (e.g. trigeminal or herpetic neuralgia, diabetic neuropathy, causalgia, sympathetically maintained pain and deafferentation syndromes such as brachial plexus avulsion).
• neuropathic pain and associated hyperalgesis and allodynia e.g. trigeminal or herpetic neuralgia, diabetic neuropathy, causalgia, sympathetically maintained pain and deafferentation syndromes such as brachial plexus avulsion.
• the compounds of the present invention are also useful as anti-inflammatory agents for the treatment of arthritis, and as agents to treat Parkinson's
• Alzheimer's Disease Alzheimer's Disease, stroke, uveitis, asthma, myocardial infarction, traumatic brain injury, spinal cord injury, neurodegenerative disorders, alopecia (hair loss), inflammatory bowel disease and autoimmune disorders, renal disorders, obesity, eating disorders, cancer, schizophrenia, epilepsy, sleeping disorders, cognition, depression, anxiety, blood pressure, lipid disorders, and atherosclerosis.
• this invention provides compounds which are capable of modifying ion channels, in vivo.
• Representative ion channels so modified include voltage-gated channels and ligand-gated channels, including cation channels such as vanilloid channels.
• the present invention provides pharmaceutical compositions comprising a compound of the invention, and a pharmaceutical carrier, excipient or diluent.
• the pharmaceutical composition can comprise one or more of the compounds described herein.
• a method for treating mammals, including humans, as well as lower mammalian species, susceptible to or afflicted with a condition from among those listed herein, and particularly, such condition as may be associated with e.g. arthritis, uveitis, asthma, myocardial infarction, traumatic brain injury, acute spinal cord injury, alopecia (hair loss), inflammatory bowel disease and autoimmune disorders, which method comprises administering an effective amount of one or more of the pharmaceutical compositions just described.
• this invention provides a method of treating a mammal susceptible to or afflicted with a condition that gives rise to pain responses or that relates to imbalances in the maintenance of basal activity of sensory nerves.
• Compounds have use as analgesics for the treatment of pain of various geneses or etiology, for example acute, inflammatory pain (such as pain associated with osteoarthritis and rheumatoid arthritis); various neuropathic pain syndromes (such as post-herpetic neuralgia, trigeminal neuralgia, reflex sympathetic dystrophy, diabetic neuropathy, Guillian Barre syndrome, fibromyalgia, phantom limb pain, post- masectomy pain, peripheral neuropathy, HIV neuropathy, and chemotherapy-induced and other iatrogenic neuropathies); visceral pain, (such as that associated with gastroesophageal reflex disease, irritable bowel syndrome, inflammatory bowel disease, pancreatitis, and various gyn
• this invention provides methods of treating a mammal susceptible to or afflicted with neurodegenerative diseases and disorders such as, for example Parkinson's disease, Alzheimer's disease and multiple sclerosis; diseases and disorders which are mediated by or result in neuroinf ⁇ ammation such as, for example traumatic brain injury, stroke, and encephalitis; centrally-mediated neuropsychiatric diseases and disorders such as, for example depression mania, bipolar disease, anxiety, schizophrenia, eating disorders, sleep disorders and cognition disorders; epilepsy and seizure disorders; prostate, bladder and bowel dysfunction such as, for example urinary incontinence, urinary hesitancy, rectal hypersensitivity, fecal incontinence, benign prostatic hypertrophy and inflammatory bowel disease; irritable bowel syndrome, over active bladder, respiratory and airway disease and disorders such as, for example, allergic rhinitis, asthma and reactive airway disease and chronic obstructive pulmonary disease; diseases and disorders which are mediated by or result in inflammation such
• this invention provides methods for synthesizing the compounds of the invention, with representative synthetic protocols and pathways disclosed later on herein.
• Figure 1 Graph depicts IC 50 curve for Compound 7 mXenopus oocytes. The IC 50 calculated is
• halo such as fluoro, chloro, bromo
• -CN -CF 3 , -OH, -OCF 3
• C 2 -C 6 alkenyl C 3 -C 6 alkynyl, C 1 -C 6 alkoxy, aryl and di- C 1 -C 6 alkylamino.
• Acyl refers to a radical -C(O)R, where R is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein.
• Acylamino refers to a radical -NRO(O)R, where R' is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl and R is hydrogen, alkyl, alkoxy, cycloalkyl, cycloheteroalkyl, aiyl, arylalkyl, heteroalkyl, heteroaryl or heteroarylalkyl, as defined herein.
• Acyloxy refers to the group -OC(O)R where R is hydrogen, alkyl, aryl or cycloalkyl.
• Substituted alkenyl includes those groups recited in the definition of "substituted” herein, and particularly refers to an alkenyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)-, aryl— S(O)-, alkyl-S
• Alkoxy refers to the group -OR where R is alkyl. Particular alkoxy groups include, by way of example, tnethoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2- dimethylbutoxy, and the like.
• Substituted alkoxy includes those groups recited in the definition of "substituted” herein, and particularly refers to an alkoxy group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, heteroaryl, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)-
• aryl-S(O)- alkyl-S(O) 2 - and aryl-S(O) 2 -.
• Alkoxycarbonylamino refers to the group -NRC(O)OR' where R is hydrogen, alkyl, aryl or cycloalkyl, and R' is alkyl or cycloalkyl.
• Aliphatics refers to bydrocarbyl organic compounds or groups characterized by a straight, branched or cyclic arrangement of the constituent carbon atoms and an absence of aromatic unsaturation. Aliphatics include, without limitation, alkyl, alkylene, alkenyl, alkenylene, alkynyl and alkynylene. Aliphatic groups typically have from 1 or 2 to about 12 carbon atoms.
• Alkyl refers to monovalent saturated aliphatic hydrocarbyl groups particularly having up to about 11 carbon atoms, more particularly as a lower alkyl, from 1 to 8 carbon atoms and still more particularly, from
• the hydrocarbon chain may be either straight-chained or branched. This term is exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, ? ⁇ -butyl, jso-butyl, tert-butyl, n-hexyl, H-octyl, /ert-octyl and the like.
• the term "lower alkyl” refers to aikyl groups having 1 to 6 carbon atoms.
• alkyl also includes
• Substituted alkyl includes those groups recited in the definition of "substituted” herein, and particularly refers to an alkyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, heteroaryl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)- , aryl-S(O)-, alkyl group having 1 or
• Alkylene refers to divalent saturated aliphatic hydrocarbyl groups particularly having up to about 11 carbon atoms and more particularly 1 to 6 carbon atoms which can be straight-chained or branched. This term is exemplified by groups such as methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), the propylene isomers (e.g., - CH 2 CH 2 CH 2 - and -CH(CH 3 )CH 2 -) and the like.
• Substituted alkylene includes those groups recited in the definition of "substituted” herein, and particularly refers to an alkylene group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)-, aryl-S(O)-, alkyl-S(O) 2 - and aryl-S(O) 2 -
• alkenyl refers to monovalent olefinically unsaturated hydrocarbyl groups preferably having up to about 11 carbon atoms, particularly, from 2 to 8 carbon atoms, and more particularly, from 2 to 6 carbon atoms, which can be straight-chained or branched and having at least 1 and particularly from 1 to 2 sites of olefinic unsaturation.
• Alkynyl refers to acetylenically unsaturated hydrocarbyl groups particularly having up to about
• alkynyl groups include acetylenic, ethynyl (-CsCH), propargyl (-CH 2 C ⁇ CH), and the like.
• Substituted alkynyl includes those groups recited in the definition of "substituted” herein, and particularly refers to an alkynyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)-, aryl-
• Alkanoyl or “acyl” as used herein refers to the group R-C(O)-, where R is hydrogen or alkyl as defined above.
• Aryl refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
• Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fiuorene, hexacene, hexaphene, hexalene, ⁇ s-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, ⁇ enta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like.
• Substituted Aryl includes those groups recited in the definition of "substituted” herein, and particularly refers to an aryl group that may optionally be substituted with 1 or more substituents, for instance from 1 to 5 substituents, particularly 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, alkoxycarbonyl, alkyl, substituted alkyl, alkynyl, substituted alkynyl, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thiol, alkyl-
• alkyl or arylalkyl refers to an alkyl group, as defined above, substituted with one or more aryl groups, as defined above.
• Arylamino refers to the group aryl- NR'R", wherein each of R' and R" are independently selected from hydrogen, aryl and heteroaryl.
• Alkoxyamino refers to a radical -N(H)OR where R represents an alkyl or cycloalkyl group as defined herein.
• R' is an aryl as defined herein.
• Alkylsulfinyl refers to a radical -S(O)R where R is an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methylsulfinyl, ethylsulf ⁇ nyl, propylsulfinyl, butylsulfinyl and the like.
• Alkylthio refers to a radical -SR where R is an alkyl or cycloalkyl group as defined herein that may be optionally substituted as defined herein. Representative examples include, but are not limited to, methylthio, ethylthio, propylthio, butylthio, and the like.
• amino refers to the radical -NH 2 .
• substituted amino includes those groups recited in the definition of “substituted” herein, and particularly refers to the group -N(R) 2 where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, cycloalkyl, substituted cycloalkyl, and where both R groups are joined to form an alkylene group. When both R groups are hydrogen, -N(R) 2 is an amino group.
• Aminocarbonyl refers to the group -C(O)NRR where each R is independently hydrogen, alkyl, aryl and cycloalkyl, or where the R groups are joined to form an alkylene group.
• Aminocarbonylamino refers to the group -NRC(O)NRR where each R is independently hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form an alkylene group.
• Aminocarbonyloxy refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, aryl or cycloalky, or where the R groups are joined to form an alkylene group.
• Arylalkyloxy refers to an -O-arylalkyl radical where arylalkyl is as defined herein.
• Arylamino means a radical -NHR where R represents an aryl group as defined herein.
• Arylsulfonyl refers to a radical -S(O) 2 R where R is an aryl or heteroaryl group as defined herein.
• Bicycloaryl refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent bicycloaromatic ring system.
• Typical bicycloaryl groups include, but are not limited to, groups derived from indane, indene, naphthalene, tetrahydronaphthalene, and the like.
• an aryl group comprises from 8 to 11 carbon atoms.
• Bicycloheteroaryl refers to a monovalent bicycloheteroaromatic group derived by the removal of one hydrogen atom from a single atom of a parent bicycloheteroaromatic ring system.
• Typical bicycloheteroaryl groups include, but are not limited to, groups derived from benzofuran, benzimidazole, benzindazole, benzdioxane, chromene, chromane, cinnoline, phthalazine, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, benzothiazole, benzoxazole, naphthyridine, benzoxadiazole, pteridine, purine, benzopyran, benzpyrazine, pyridopyrimidine, quinazoline, quinoline, quinolizine, quinoxaline, benzomorphan, tetrahydroisoquinoline, tetrahydroquinoline, and the like.
• the bicycloheteroaryl group is between 9-11 membered bicycloheteroaryl, with 5-10 membered heteroaryl being particularly preferred.
• Particlar bicycloheteroaryl groups are those derived from benzothiophene, benzofuran, benzothiazole, indole, quinoline, isoquinoline, benzimidazole, benzoxazole and benzdioxane.
• Carbamoyl refers to the radical -C(O)N(R) 2 where each R group is independently hydrogen, alkyl, cycloalkyl or aryl, as defined herein, which may be optionally substituted as defined herein.
• Carboxy refers to the radical -C(O)OH.
• Cycloalkenyl refers to cyclic hydrocarbyl groups having from 3 to 10 carbon atoms and having a single cyclic ring or multiple condensed rings, including fused and bridged ring systems and having at least one and particularly from 1 to 2 sites of olefrnic unsaturation.
• Such cycloalkenyl groups include, by way of example, single ring structures such as cyclohexenyl, cyclopentenyl, cyclopropenyl, and the like.
• Cyclone refers to the radical -OCN.
• Dialkylamino means a radical -NRR' where R and R' independently represent an alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, or substituted heteroaryl group as defined herein.
• Halo or "halogen” refers to fluoro, chloro, bromo and iodo. Preferred halo groups are either fluoro or chloro.
• Niro refers to the radical -NO 2 .
• Substituted refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s). Typical substituents include, but are not limited to, -X, -R 14 , -O " ,
• R 6' and R 7' may be hydrogen and at least one of R 6 and R 7 is each independently selected from alkyl, alkenyl, alkynyl, cycloheteroalkyl, alkanoyl, alkoxy, aryloxy, heteroaryloxy, alkylamino, arylamino, heteroarylamino, NR 10 COR 11 , NR 10 SOR 1 ⁇ NR 10 SO 2 R 14 , COOalkyl, COOaryl, CONR 10 R 11 , CONR 10 OR 11 , NR 10 R 11 , SO 2 NR 10 R 11 , S-alkyl, S-alkyl, SOalkyl, SO 2 alkyl, Saryl, SOaryl, SO 2 aryl; or R 6' and R 7' may be joined to form a cyclic ring (saturated or unsaturated) from 5 to 8 atoms, optionally containing one or more heteroatoms selected from the group N, O or S
• Hetero when used to describe a compound or a group present on a compound means that one or more carbon atoms in the compound or group have been replaced by a nitrogen, oxygen, or sulfur heteroatom. Hetero may be applied to any of the hydrocarbyl groups described above such as alkyl, e.g. heteroalkyl, cycloalkyl, e.g. cycloheteroalkyl, aryl, e.g. heteroaryl, cycloalkenyl, cycloheteroalkenyl, and the like having from 1 to 5, and especially from 1 to 3 heteroatoms.
• Heteroaryl refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system.
• Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,
• the heteroaryl group is between 5-20 membered heteroaryl, with 5-10 membered heteroaryl being particularly preferred.
• Particlar heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzoforan, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.
• Examples of representative heteroaryls include the following:
• each Y is selected from carbonyl, N, NR 4 , 0, and S.
• each X is selected from CR 4 2 , NR 4 , O and S; and each Y is selected from NR 4 , O and S, and where R 6 is R 2 .
• Examples of representative cycloheteroalkenyls include the following:
• each X is selected from CR 4 , NR 4 , O and S; and each Y is selected from carbonyl, N, NR 4 , O and S.
• Examples of representative aryl having hetero atoms containing substitution include the following:
• Hetero substituent refers to a halo, O, S or N atom-containing functionality that may be present as an R 4 in a R 4 C group present as substituents directly on A, B, W, X, Y or Z of the compounds of this invention or may be present as a substituent in the "substituted" aryl and aliphatic groups present in the compounds.
• hetero substituents examples include:
• each R is independently an aryl or aliphatic, optionally with substitution.
• hetero substituents containing R groups preference is given to those materials having aryl and alkyl R groups as defined herein. Preferred hetero substituents are those listed above.
• Substituting groups include carbonyl or thiocarbonyl which provide, for example, lactam and urea derivatives.
• M is CR 7 , NR 2 , O, or S;
• Q is O, NR 2 or S.
• R 7 and R 8 are independently selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)-, aryl-S(O)-, alkyl-S(O) 2 - and aryl- S(O) 2 -.
• Dihydroxyphosphoryl refers to the radical -PO(OH) 2 .
• Substituted dihydroxyphosphoryl includes those groups recited in the definition of "substituted” herein, and particularly refers to a dihydroxyphosphoryl radical wherein one or both of the hydroxyl groups are substituted. Suitable substituents are described in detail below.
• Aminohydroxyphosphoryl refers to the radical -PO(OH)NH 2 .
• substituted herein, and particularly refers to an aminohydroxyphosphoryl wherein the amino group is substituted with one or two substituents. Suitable substituents are described in detail below. In certain embodiments, the hydroxyl group can also be substituted.
• Sulfone refers to the group -SO 2 R.
• R is selected from H, lower alkyl, alkyl, aryl and heteroaryl.
• Thioaryloxy refers to the group -SR where R is aryl.
• Thiol refers to the group -SH.
• “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
• Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, r ⁇ aleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2- hydroxyemanesulf
• Preventing refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).
• Prodrugs refers to compounds, including derivatives of the compounds of the invention,which have cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention which are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.
• Subject includes humans.
• the terms “human,” “patient” and “subject” are used interchangeably herein.
• Prodrugs include acid derivatives well know to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant on the compounds of this invention are preferred prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. Preferred are the C 1 to C 8 alkyl, C 2 -C 8 alkenyl, aryl, C 7 -C 12 substituted aryl, and C 7 -C 12 arylalkyl esters of the compounds of the invention.
• Tautomers refer to compounds that are interchangeable forms of a particular compound structure, and that vary hi the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of ⁇ electrons and an atom (usually H).
• enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base.
• Another example of tautomerism is the aci- and nitro- forms of phenylnitromethane, that are likewise formed by treatment with acid or base. Representative enol — keto structures and equilibrium are illustrated below:
• Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.
• the compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)- stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art.
• the compounds of the present invention are useful for preventing and/or treating a broad range of conditions, among them, arthritis, Parkinson's disease, Alzheimer's disease, stroke, uveitis, asthma, myocardial infarction, the treatment and prophylaxis of pain syndromes (acute and chronic or neuropathic), traumatic brain injury, acute spinal cord injury, neurodegenerative disorders, alopecia (hair loss), inflammatory bowel disease and autoimmune disorders or conditions in mammals.
• the present invention provides prodrugs and derivatives of the compounds according to the formulae above.
• Prodrugs are derivatives of the compounds of the invention, which have cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention, which are pharmaceutically active, in vivo.
• Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.
• Prodrugs include acid derivatives well know to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides.
• Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant on the compounds of this invention are preferred prodrugs.
• double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters.
• Preferred are the Ci to C 8 alkyl, C 2 -C$ alkenyl, aryl, C 7 -Ci 2 substituted aryl, and C 7 -Ci 2 arylalkyl esters of the compounds of the invention.
• CCI Model Chronic Constriction Injury Model
• mice Male Sprague-Dawley rats (270-300 g; B.W., Charles River, Tsukuba, Japan) are used.
• the chronic constriction injury (CCI) operation is performed according to the method described by Bennett and Xie (Bennett, GJ. and Xie, Y.K. Pain, 33:87-107, 1988). Briefly, animals are anesthetized with sodium pentobarbital (64.8 mg/kg, i.p.) and the left common sciatic nerve is exposed at the level of the middle of the thigh by blunt dissection through the biceps femoris.
• sodium pentobarbital (64.8 mg/kg, i.p.
• VFHs von Frey hairs
• Caco-2 cells are grown on filter supports (Falcon HTS multiwell insert system) for 14 days.
• Culture medium is removed from both the apical and basolateral compartments and the monolayers are preincubated with pre-warmed 0.3 ml apical buffer and 1.0 ml basolateral buffer for 0.75 hour at 37°C in a shaker water bath at 50 cycles/min.
• the apical buffer consists of Hanks Balanced Salt Solution, 25 mM D-glucose monohydrate, 20 mM MES Biological Buffer, 1.25 mM CaCl 2 and 0.5 mM MgCl 2 (pH 6.5).
• the basolateral buffer consists of Hanks Balanced Salt Solution, 25 mM D-glucose monohydrate, 20 mM HEPES Biological Buffer, 1.25 mM CaCl 2 and 0.5 mM MgC12 (pH 7.4).
• test compound solution (lO ⁇ M) in buffer is added to the apical compartment.
• the inserts are moved to wells containing fresh basolateral buffer and incubated for 1 hr. Drug concentration in the buffer is measured by LC/MS analysis.
• Cell paste of HEK-293 cells expressing the HERG product can be suspended in 10-fold volume of
• the cells are homogenized using a Polytron homogenizer (at the maximum power for 20 seconds) and centrifuged at 48,00Og for 20 minutes at 4°C. The pellet is resuspended, homogenized and centrifuged once more in the same manner. The resultant supernatant is discarded and the final pellet is resuspended (10-fold volume of 50 mM Tris buffer) and homogenized at the maximum power for 20 seconds.
• the membrane homogenate is aliquoted and stored at -8O 0 C until use.
• compounds are diluted in 96 well polypropylene plates as 4- ⁇ oint dilutions in semi-log format. All dilutions are performed in DMSO first and then transferred into 50 mM Tris buffer (pH 7.5 at 25°C) containing 1 mM MgCl 2 , 10 mM KCl so that the final DMSO concentration becomes equal to 1%.
• Compounds are dispensed in triplicate in assay plates (4 ⁇ l). Total binding and nonspecific binding wells are set up in 6 wells as vehicle and 10 ⁇ M dofetilide at final concentration, respectively.
• the radioligand is prepared at 5.6x final concentration and this solution is added to each well (36 ⁇ l).
• the assay is initiated by addition of YSi poly-L- lysine Scintillation Proximity Assay (SPA) beads (50 ⁇ l, 1 mg/well) and membranes (110 ⁇ l, 20 ⁇ g/well). Incubation is continued for 60 min at room temperature. Plates are incubated for a further 3 hours at room temperature for beads to settle. Receptor-bound radioactivity is quantified by counting Wallac MicroBeta plate counter.
• SPA YSi poly-L- lysine Scintillation Proximity Assay
• HEK 293 cells which stably express the HERG potassium channel are used for electrophysiological study.
• the methodology for stable transfection of this channel in HEK cells can be found elsewhere (Z.Zhou et al., 1998, Biophysical Journal, 74, pp230-241).
• MEM Minimum Essential Medium
• FCS Fetal Calf Serum
• HERG currents are studied using standard patch clamp techniques in the whole-cell mode.
• the cells are supervised with a standard external solution of the following composition (mM); NaCl, 130; KCl, 4; CaCl 2 , 2; MgCl 2 , 1; Glucose, 10; HEPES, 5; pH 7.4 with NaOH.
• Whole-cell recordings are made using a patch clamp amplifier and patch pipettes which have a resistance of l-3MOhm when filled with the standard internal solution of the following composition (mM); KCl, 130; MgATP, 5; MgCl 2 , 1.0; HEPES, 10; EGTA 5, pH 7.2 with KOH.
• the voltage protocol is applied to a cell continuously throughout the experiment every 4 seconds (0.25Hz).
• vehicle (0.5% DMSO in the standard external solution) is applied for 10-20 min by a peristalic pump.
• the test compound of either 0.3, 1, 3, 1OmM is applied for a 10 min period.
• the 10 min period includes the time during which supplying solution is passing through the tube from solution reservoir to the recording chamber via the pump. Exposure time of cells to the compound solution is more than 5 min after the drug concentration in the chamber well reaches the intended concentration. There is a subsequent wash period of 10-20min to assess reversibility. Finally, the cells are exposed to high dose of dofetilide (5mM), a specific IKr blocker, to evaluate the insensitive endogenous current.
• dofetilide 5mM
• IKr blocker a specific IKr blocker
• Test compounds (1 ⁇ M) are incubated with 3.3 mM MgCl 2 and 0.78 mg/mL HLM (HLlOl) in 100 mM potassium phosphate buffer (pH 7.4) at 37°C on the 96-deep well plate.
• the reaction mixture is split into two groups, a non-P450 and a P450 group.
• NADPH is only added to the reaction mixture of the P450 group.
• An aliquot of samples of the P450 group is collected at 0, 10, 30, and 60 min time points, where the 0 min time point indicates the time when NADPH is added into the reaction mixture of P450 group.
• An aliquot of samples of a non-P450 group is collected at -10 and 65 min time points.
• the half-life value is obtained by plotting the natural logarithm of the peak area ratio of compounds/ internal standard versus time. The slope of the line of best fit through the points yields the rate of metabolism (k). This is converted to a half-life value using following equations: / k Mono-Iodoacetate (MIA)-induced OA model [00186] Male 6-weeks-old Sprague-Dawley (SD, Japan SLC or Charles River Japan) rats are anesthetized with pentobarbital. The injection site (knee) of MIA is shaved and cleaned with 70% ethanol. Twenty-five ml of MIA solution or saline is injected in the right knee joint using a 29G needle.
• the effect of joint damage on the weight distribution through the right (damaged) and left (untreated) knee is assessed using an incapacitance tester (Linton Instrumentation, Norfolk, UK). The force exerted by each hind limb is measured in grams.
• the weight- bearing (WB) deficit is determined by a difference of weight loaded on each paw. Rats are trained to measure the WB once a week until 20 days post MIA-injection. Analgesic effects of compounds are measured at 21 days after the MIA injection. Before the compound administration, the "pre value" of WB deficit is measured. After the administration of compounds, attenuation of WB deficits is determined as analgesic effects.
• CFA Complete Freund's adjuvant
• Tuberculosis H37RA (Difco, MI) in 100 ⁇ L of liquid paraffin (Wako, Osaka, Japan)) is injected into the plantar surface of a hind paw of the rats.
• thermal hyperalgesia is determined by method described previously (Hargreaves et al., 1988) using the plantar test apparatus (Ugo-Basil, Varese, Italy). Rats are adapted to the testing environment for at least 15 minutes prior to any stimulation. Radiant heat is applied to the plantar surface of a hind paw and paw withdrawal latencies (PWL, seconds) are determined. The intensity of radiant heat is adjusted to produce the stable PWL of 10 to 15 seconds.
• the test compound is administered in a volume of 0.5 mL per 100 g body weight. PWL are measured after 1, 3 or 5 hours after drag administration.
• Mechanical hyperalgesia is administered in a volume of 0.5 mL per 100 g body weight. PWL are measured after 1, 3 or 5 hours after drag administration. Mechanical hyper
• MI in 100 ⁇ L of liquid paraffin (Wako, Osaka, Japan)) is injected into the plantar surface of a hind paw of the rats.
• mechanical hyperalgesia is tested by measuring paw withdrawal threshold (PWT, grams) to pressure using the analgesy-Meter (Ugo-Basile, Varese, Italy). The animals are gently restrained, and steadily increasing pressure is applied to the dorsal surface of a hind paw via a plastic tip. The pressure required to elicit paw withdrawal is determined.
• the test compound is administered in a volume of 0.5 mL per 100 g body weight. PWT are measured after 1, 3 or 5 hours after drag administration.
• FDSS 6000 (Hamamatsu Photonics, Japan), a fluorometric imaging system.
• the cell suspension in resting buffer (HBSS supplemented with 1OmM HEPES, pH 7.4) is pre-incubated with varying concentrations of the test compounds or resting buffer (buffer control) for 15 minutes at room temperature under dark conditions.
• the cells are automatically added the stimulating solution (HBSS supplemented with MES, final assay buffer ⁇ H5.8) by the FDSS 6000.
• the IC 50 values of VRl antagonists are determined from the half of the increase demonstrated by buffer control samples after acidic stimulation.
• compounds of this invention are dissolved (2 mg/niL) in a mixture of 5% of 10% Tween 80 in water (v/v) and 95% of 0.5 % methyl cellulose in water (w/v). The animals are weighed before dosing. The determined body weight is used to calculate the dose volume for each animal.
• Dose volume (mL/kg) 1 mg/kg/formulation concentration (mg/mL)
• the dosing volume is about 2 mL/kg.
• PO rats are typically dosed through oral gavage at 2.5 mL/kg to achieve a dose level of 5 mg/kg.
• blood samples are collected (using a pre-heparinized syringe) via the jugular vein catheter at 2, 5, 15, 30, 60, 120, 180, 300, 480, and 1440 minutes post dosing.
• blood samples are collected (using a pre- heparinized syringe) via the jugular vein catheter before dosing and at 5, 15, 30, 60, 120, 180, 300, 480, and 1440 minutes post dosing.
• the %F is reported as the mean %F of all oral dosed animals.
• VRl protein is a heat-gated cation channel that exchanges approximately ten calcium ions for every sodium ion resulting in neuronal membrane depolarization and elevated intracellular calcium levels.
• the functional activity of compounds at the VRl receptor may be determined by measuring changes in intracellular calcium levels in neurons such as the dorsal root ganglion.
• DRG neurons are grown on PDL coated 96-well black-walled plates, in the presence of DMEM medium containing 5% Penstrep, 5% Glutamax, 200 ⁇ g/ml hygromycin, 5 ⁇ g/ml blasticide and 10% heat inactivated
• the plated neurons are transferred into a chamber on the stage of a Nikon eclipse TE300 microscope after which neurons are allowed to attain a stable fluorescence for about 10 minutes before beginning the experiment.
• the assay consists of two stages, a pretreatment phase followed by a treatment phase. First, a solution of the test compound is added from a multivalve perfusion system to the cells for 1 minute (pretreatment).
• capsaicin 250 nM is added in the presence of the test compound (treatment) for a specific period between 20 and 60 seconds.
• Fura2 is excited at 340 and 380 nM to indicate relative calcium ion concentration. Changes in wavelength measurements are made throughout the course of the experiment. The fluorescence ratio is calculated by dividing fluorescence measured at 340 nM by that at 380 nM. Data are collected using Intelligent Imaging's Slidebook software. All compounds that inhibit capsaicin induced calcium influx greater than 75% are considered positives.
• DRG neurons may be recovered from either neonatal or adult rats and plated onto poly-D-lysine coated glass coverslips. The plated neurons are transferred into a chamber to allow drug solutions to be added to the cells using a computer-controlled solenoid-valve based perfusion system. The cells are imaged using standard DIC optics. Cells are patched using finely-pulled glass electrodes. Voltage-clamp electrophysiology experiments are carried out using an Axon Instruments Multiclamp amplified controlled by pCLAMP8 software.
• the cells are placed into a whole-cell voltage clamp and held at a voltage of -8OmV while monitoring the membrane current in gap-free recording mode.
• 50OnM capsaicin is added for 30 seconds as a control. Test compounds at various concentrations are added to the cells for 1 minute prior to a 30 second capsaicin application. Differences between control experiments and drug positive capsaicin experiments are used to determine the efficacy of each test compound. All compounds that inhibit capsaicin induced current greater than 50% are considered positives.
• the amide compounds of this invention are typically administered in the form of a pharmaceutical composition.
• Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
• the compounds of this invention are administered in a pharmaceutically effective amount.
• the amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
• compositions of this invention can be administered by a variety of routes including by way of non limiting example, oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and intranasal.
• routes including by way of non limiting example, oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and intranasal.
• the compounds of this invention are preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration.
• compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing.
• unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
• Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.
• the furansulfonic acid compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
• Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like.
• Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
• a lubricant such as magnesium stearate
• a glidant such as colloidal silicon dioxide
• a sweetening agent such as sucrose or saccharin
• Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art.
• the active compound in such compositions is typically a minor component, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like.
• Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.
• the active ingredients When formulated as a ointment, the active ingredients will typically be combined with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with, for example an oil-in-water cream base.
• Such transdermal formulations are well-known in the art and generally include additional ingredients to enhance the dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope of this invention.
• transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety.
• the compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems.
• sustained release materials A description of representative sustained release materials can be found in
• a compound of formula I is admixed as a dry powder with a dry gelatin binder in an approximate
• a compound of formula I is admixed as a dry powder with a starch diluent in an approximate 1:1 weight ratio. The mixture is filled into 250 mg capsules (125 mg of active compound per capsule). Formulation 3 - Liquid
• a compound of formula I (125 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with, a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (11:89, 50 mg) in water.
• Sodium benzoate (10 mg) flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL.
• the compound of formula I is admixed as a dry powder with a dry gelatin binder in an approximate 1 :2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of active compound) in a tablet press.
• the compound of formula I is dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of approximately 5 mg/ml.
• Stearyl alcohol (250 g) and a white petrolatum (250 g) are melted at about 75°C and then a mixture of a compound of formula I (50 g) methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate (10 g), and propylene glycol (120 g) dissolved in water (about 370 g) is added and the resulting mixture is stirred until it congeals.
• the present compounds are used as therapeutic agents for the treatment of conditions in mammals.
• the compounds and pharmaceutical compositions of this invention find use as therapeutics for preventing and/or treating neurodegenerative, autoimmune and inflammatory conditions in mammals including humans.
• this invention provides a method of treating a mammal susceptible to or afflicted with a condition associated with arthritis, uveitis, asthma, myocardial infarction, traumatic brain injury, acute spinal cord injury, alopecia (hair loss), inflammatory bowel disease and autoimmune disorders, which method comprises administering an effective amount of one or more of the pharmaceutical compositions just described.
• this invention provides methods of treating a mammal susceptible to or afflicted with neurodegenerative diseases and disorders such as, for example Parkinson's disease, Alzheimer's disease and multiple sclerosis; diseases and disorders which are mediated by or result in neuroinflammation such as, for example traumatic brain injury, stroke, and encephalitis; centrally-mediated neuropsychiatric diseases and disorders such as, for example depression mania, bipolar disease, anxiety, schizophrenia, eating disorders, sleep disorders and cognition disorders; epilepsy and seizure disorders; prostate, bladder and bowel dysfunction such as, for example urinary incontinence, urinary hesitancy, rectal hypersensitivity, fecal incontinence, benign prostatic hypertrophy and inflammatory bowel disease; respiratory and airway disease and disorders such as, for example, allergic rhinitis, asthma and reactive airway disease and chronic obstructive pulmonary disease; diseases and disorders which are mediated by or result in inflammation such as, for example rheumatoid arthritis
• a preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more may also be administered to achieve adequate steady state levels.
• the maximum total dose is not expected to exceed about 2 g/day for a 40 to 80 kg human patient.
• each dose provides from about 0.01 to about 20 mg/kg of the compound or its derivative, with preferred doses each providing from about 0.1 to about 10 mg/kg and especially about 1 to about 5 mg/kg.
• Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses.
• the compounds or thier derivatives of this invention When used to prevent the onset of a neurodegenerative, autoimmune or inflammatory condition, the compounds or thier derivatives of this invention will be administered to a patient at risk for developing the condition, typically on the advice and under the supervision of a physician, at the dosage levels described above.
• Patients at risk for developing a particular condition generally include those that have a family history of the condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition.
• the compounds of this invention can be administered as the sole active agent or they can be administered in combination with other agents, including other active derivatives.
• a VRl antagonist may be usefully combined with another pharmacologically active compound, or with two or more other pharmacologically active compounds, particularly in the treatment of pain.
• a VRl antagonist particularly a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as defined above, may be administered simultaneously, sequentially or separately in combination with one or more agents selected from: • an opioid analgesic, e.g.
• morphine heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine or pentazocine;
• NSAID nonsteroidal antiinflammatory drug
• diclofenac difhisinal, etodolac
• fenbufen fenoprofen
• flufenisal flurbiprofen
• ibuprofen indomethacin
• ketoprofen ketorolac
• meclofenamic acid mefenamic acid
• meloxicam nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin or zomepirac
• NSAID nonsteroidal antiinflammatory drug
• a barbiturate sedative e.g. amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobartital, secobarbital, talbutal, tlieamylal or thiopental;
• a benzodiazepine having a sedative action e.g. chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam or triazolam;
• an Hl antagonist having a sedative action e.g. diphenhydramine, pyrilamine, promethazine, chlorpheniramine or chlorcyclizine;
• a sedative such as glutethimide, meprobamate, methaqualone or dichloralphenazone
• a skeletal muscle relaxant e.g. baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine;
• an NMDA receptor antagonist e.g. dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmor ⁇ hinan), ketamine, memantine, pyrroloquinoline quinine, cis-4-
• dextromethorphan (+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmor ⁇ hinan)
• ketamine memantine
• pyrroloquinoline quinine cis-4-
• an alpha-adrenergic e.g. doxazosin, tamsulosin, clonidine, guanfacine, dexmetatomidine, modaf ⁇ nil, or 4-amino-
• a tricyclic antidepressant e.g. desipramine, imipramine, amitriptyline or nortriptyline;
• an anticonvulsant e.g. carbamazepine, lamotrigine, topiratmate or valproate;
• a tachykinin (NK) antagonist particularly an NK-3, NK-2 or NK-I antagonist, e.g. (aR,9R)-7-[3,5- bis(trifluoromethyl)benzyl]-8,9,10,l l-tetrahydro-9-methyl-5-(4-methyl ⁇ henyl)-7H-[l,4]diazocino[2,l-g][l,7]- naphthyridine-6-13-dione (TAK-637), 5-[[(2R,3S)-2-[(lR)-l-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4- fluorophenyl)-4-mo ⁇ holinyl]-methyl]-l,2-dihydro-3H-l,2,4-triazol-3-one (MK-869), aprepitant, lanepitant, dapitant or 3-[[2-methoxy-5-(trifluoromethoxy)
• a muscarinic antagonist e.g oxybutynin, tolterodine, propiverine, tropsium chloride, darifenacin, solifenacin, temiverine and ipratropium;
• COX-2 selective inhibitor e.g. celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib;
• a neuroleptic such as droperidol, chlorpromazine, haloperidol, perphenazine, thioridazine, mesoridazine, trifluoperazine, fluphenazine, clozapine, olanzapine, risperidone, ziprasidone, quetiapine, sertindole, aripiprazole, sonepiprazole, blonanserin, iloperidone, perospirone, raclopride, zotepine, bifeprunox, asenapine, lurasidone, amisulpride, balaperidone, pahndore, eplivanserin, osanetant, rimonabant, meclinertant, Miraxion® or sarizotan;
• ⁇ a beta-adrenergic such as propranolol
• a corticosteroid such as dexamethasone
• a 5-HT receptor agonist or antagonist particularly a 5-HT1B/1D agonist such as eletriptan, sumatriptan, naratriptan, zolmitriptan or rizatriptan;
• a 5-HT2A receptor antagonist such as R(+)-alpha-(2,3-dimethoxy-phenyl)-l-[2-(4-fluorophenylethyl)]-4- piperidinemethanol (MDL- 100907); • a cholinergic (nicotinic) analgesic, such as ispronicline (TC-1734), (E)-N-methyl-4-(3-pyridinyl)-3-buten-l-amine
• a PDEV inhibitor such as 5-[2-ethoxy-5-(4-methyl-l-piperazinyl-sul ⁇ honyl)phenyl]-l-methyl-3-n-pro ⁇ yl-l,6- dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil), (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4- methylenedioxyphenyl)-pyrazino[2',r:6,l]-pyrido[3,4-b]indole-l,4-dione (IC-351 or tadalaf ⁇ l), 2-[2-ethoxy-5-(4- ethyl-piperazin-l-yl-l-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,l-f][l,2,4]triazin-4-one (var
• an alpha-2-delta ligand such as gabapentin, pregabalin, 3-methylgabapentin, (la,3a,5a)(3-amino-methyl- bicyclo[3.2.0]hept-3-yl)-acetic acid, (3S,5R)-3—aminomethyl-5_methyl-heptanoic acid, (3S,5R)-3_amino-
• a serotonin reuptake inhibitor such as sertraline, sertraline metabolite demethylsertraline, fluoxetine, norfluoxetine
• fluoxetine desmethyl metabolite
• fluvoxamine paroxetine
• citalopram citalopram metabolite desmethylcitalopram
• escitalopram d,l-fenfluramine
• femoxetine ifoxetine
• cyanodothiepin litoxetine
• nefazodone cericlamine and trazodone
• a noradrenaline (norepinephrine) reuptake inhibitor such as maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine, tomoxetine, mianserin, buproprion, buproprion metabolite hydroxybuproprion, nomifensine and viloxazine (Vivalan®), especially a selective noradrenaline reuptake inhibitor such as reboxetine, in particular (S, S)- reboxetine;
• a dual serotonin-noradrenaline reuptake inhibitor such as venlafaxine, venlafaxine metabolite O- desmethylvenlafaxine, clomipramine, clomipramine metabolite desmethylclomipramine, duloxetine, milnacipran and imipramine;
• an inducible nitric oxide synthase (iNOS) inhibitor such as S-[2-[(l-iminoethyl)amino]ethyl]-L-homocysteine, S-[2-
• a prostaglandin E2 subtype 4 (EP4) antagonist such as N-[( ⁇ 2-[4-(2-ethyl-4,6-dimethyl-lH-imidazo[4,5-c]pyridin-l- yl)phenyl]ethyl ⁇ amino)-carbonyl]-4-methylbenzenesulfonamide or 4-[(lS)-l-( ⁇ [5-chloro-2-(3- fluorophenoxy)pyridin-3-yl]carbonyl ⁇ amino)ethyl]benzoic acid;
• a leukotriene B4 antagonist such as l-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylic acid (CP-105696), 5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E- hexenyl]oxyphenoxy] -valeric acid (ONO-
• a 5-lipoxygenase inhibitor such as zileuton, 6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H- ⁇ yran-4-yl])phenoxy- methyl]-l-methyl-2-quinolone (ZD-2138), or 2,3,5-trimethyl-6-(3-pyridylmethyl),l,4-benzoquinone (CV-6504);
• a sodium channel blocker such as lidocaine
• a 5-HT3 antagonist such as ondansetron
• pharmaceutically acceptable salts and solvates thereof • a 5-HT3 antagonist, such as ondansetron; and the pharmaceutically acceptable salts and solvates thereof.
• compositions may conveniently be combined in the form of a kit suitable for coadministration of the compositions.
• the compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
• the target compounds are synthesized by known reactions outlined in the following schemes.
• the products are isolated and purified by known standard procedures. Such procedures include (but are not limited to) recrystallization, column chromatography or HPLC.
• This compound was purchased from a commercial source.
• the reaction vessel was sealed with a teflon topper and the mixture stirred from 0 0 C to rt and stirred at rt for approximately 3 hours.
• the mixture was then partitioned in a separating funnel and the ether layer containing the product used directly in the next step without further purification.
• Geometrical isomer A (more polar isomer)
• geometrical isomer A is the pure cis- or pure trans- geometrical isomer. It should also be noted that geometrical isomer A is a racemic mixture containing two enantiomers. 1 H NMR (CDCl 3 ;
• Geometrical isomer B (least polar isomer)
• geometrical isomer B is the pure trans- or pure cis- geometrical isomer. It should also be noted that geometrical isomer B is a racemic mixture containing two enantiomers. 1 H NMR (CDCl 3 ;
• 2-methyl-4-(2-(trifluoromethyl)cyclo ⁇ ropyl)benzoic acid geometrical isomer B is a racemic mixture and contains two enantiomers.
• 2-methyl-4-(2-(trifluoromethyl)cyclopropyl)benzoic acid geometrical isomer A is a racemic mixture and contains two enantiomers.
• the mixture was concentrated under vacuum (to ca. 1 mL) and then purified by preparative thin-layer chromatography.
• 2-methyl-4-(2-(trifluoromethyl)cyclopro ⁇ yl)benzoic acid geometrical isomer B note: this compound is a racemic mixture and contains two enantiomers; 16 mg, 0.07 mmol), 1-hydroxybenzotriazole (15 mg, 0.08 mmol) andiV-(3-dimethylaminopropyl)-iV'-ethylcarbodiimide hydrochloride (15 mg, 0.08 mmol) were dissolved in CH 2 Cl 2 tlieniV,7V-di-z5O-propylethylamine (30 ⁇ L, 0.2 mmol) was added.
• the mixture was stirred at room temperature for 15 min then the appropraite amine (2.0 eq based on acid starting material) was added and the mixture stirred overnight.
• the mixture was concentrated under vacuum (to ca. 1 mL) and then purified by preparative thin-layer chromatography.
• Trimethylaluminum (1.0 M in hexane; 2.3 mL) was added over 2-3 min to a stirred solution of 3- quinolinamine (330 mg, 2.3 mmol) in toluene (20 mL) at room temperature under nitrogen. The mixture was stirred at room temperature overnight (reactions turns red in color), then a solution of methyl cis- and trans- methyl 2- methyl-4-(2-(trifluoromethyl)cyclo ⁇ ropyl)benzoate (300 mg, 1.2 mmol) in toluene (10 mL) was added in one portion and the mixture heated to reflux and stirred for 3 hours.
• Method D A Representative Synthesis Of Benzamides Using An Automated Parallel Synthesis Method [00313]
• the appropriate benzoic acid (2 mmol) is dissolved or suspended in 15ml of chloroform and treated with 20 mmol of thionyl chloride.
• the reaction mixture is refluxed for fifteen minutes and the solvents are removed under vacuum.
• the residue is dissolved in 4ml of anhydrous chloroform and 60 ⁇ l (30 ⁇ vole) of this solution is added to each well of the 96 well glass plates.
• Appropriate amine is then added to the corresponding well (60 ⁇ mole), followed by ⁇ iV-diisopropylethylamine (120 ⁇ mole).
• the plate is then heated at 65 ° Cfor 15 minutes.
• the solvents are removed using an HT- 12 Genevac centrifugal evacuator and 100 ⁇ l of DMSO is added to each well and the compounds are transferred to a 96-well polypropylene reaction plate.
• TFFH fluoro-iV, ⁇ N' ⁇ V'- tetramethylformamidinium hexafluorophosphate;12mg, 45 ⁇ mol)
• diisopropylethylamine 6.0mg, 45 ⁇ mol
• the appropriate amine 60 ⁇ mol
• iV,N-Diisopropylethylamine (1 eq) is added in one portion to a stirred mixture of 2-methyl-4-(3,3- dimethylbut-l-ynyl)benzoic acid (leq) and ⁇ N ⁇ V ⁇ -tetramethyl-C>-(7-azabenzotriazol-l-yl)uronium hexafluorophosphate (1.05 eq) in A ⁇ N-dimethylformamide (ca. 3 mL per 0.5 mmol of starting acid) at room temperature. The mixture is stirred at room temperature for approx. 2 hours then a solution of the appropriate amine
• DIPEA (0.92 mmol) is added to the solution of appropriate acid (0.46 mmol), appropriate amine
• DIPEA (0.92 mmol) is added to the solution of appropriate acid (4.0 mmol), appropriate amine
• the chromatographic method employed was 10-100% gradient of acetonitrile to water over 8 minutes at a flow rate of 6 ml per minute.
• the column used was a 10X50mm YMC C 18 and the compounds were collected using a Gilson 204 fraction collector.
• Functional activity of compounds against the VRl receptor was determined by measuring changes in intracellular calcium in HEK 293 cells expressing hVRl. Compounds were examined for their ability to inhibit agonist-induced calcium influx. Dual wavelength ratiometric dye, Fura2, was used as an indicator of relative levels of [Ca 2+ ] in a 96-well format using a Flex Station ® , Molecular Devices.
• hVRl was cloned into a pcDNA5/TO vector from Invitrogen and stably transformed into T-REx
• the agonist EC 50 was determined at the start of the assay and compound IC 5 O experiments were run using an agonist concentration equal to its EC 50 as stimulus.
• the assay consists of two stages: a pre-treatment phase followed by a treatment phase. 50 ⁇ l of a compound solution was added to the cells (Pre-treatment). In some instances, following pre-treatment, 50 ⁇ l of the test compound in a saline solution at pH 5.1 was added (Treatment).
• Oocytes are prepared by surgically removing Xenopus ovaries obtained from NASCO. The oocytes are isolated by enzymatic dissociation using collagenase (Worthington, 2mg/ml). Oocytes are then individually injected with HsVRl RNA. Each oocyte receives 64 nl of RNA solution in water at a concentration of 0.5 ⁇ g/ ⁇ l. Injected oocytes are stored in standard oocyte incubation solution, ND96, containing (in niM) 96 NaCl, 2 KCl, 1 MgCl 2 , 1.8 CaCl 2 and 50 ⁇ g/ml Gentamicin at 16°C. Capsaicin induced VRl current is observed in oocytes 4-5 days after injection.
• oocytes are placed in the recording chambers. Each oocyte is impaled by 2 glass electrodes having resistances of 0.5 to 1 MOhm when filled with a 3 M KCl solution. Electrode advancement and oocyte impalement are under software control (OpusXpress® 1.1 Molecular Devices Corporation). [00339] The solutions are prepared in 96 well plates and robotically pipetted into the oocyte recording chambers by an 8 channel pipettor. Test solution delivery to the oocytes during the experiment is also under software control.
• a set of plates with wells containing 250 nM capsaicin are used initially to verify VRl expression.
• Capsaicin induced VRl current is modulated in a calcium dependent manner.
• the oocytes are exposed to several 250 ⁇ l applications of 250 nM capsaicin until a stable current amplitude is obtained with each application.
• a set of 96 well plates containing the test solutions is prepared so that the sequence of solution application to the oocyte is as follows: 250 ⁇ ls of 250 nM capsaicin is followed by a several minute wash with standard oocyte saline. 1 ml of the test compound is then added at a particular test concentration, followed immediately by 250 ⁇ l of the compound at the same concentration plus 250 nM capsaicin.
• the capsaicin induced VRl current is recorded in the absence and presence of the test compound for each concentration.
• the standard test concentrations for the compounds tested here usually range from 0.3 to 2000 nM.

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