Patent Application
20080214537
The present invention provides in certain embodiments compounds having the general structure of formula I as shown below:
The invention additionally provides pharmaceutically acceptable salts, esters, solvates, stereoisomers, or racemates of compounds of Formula I.
In the compounds of Formula I, the moiety B is a bond or a 1, 2, 3, or 4-carbon atom chain which completes a four to eight-membered saturated, singly unsaturated, or doubly unsaturated carbocyclic ring, such as, for instance and without limitation, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, and cyclooctyl moieties which are fused with the piperidinyl ring of Formula I. Singly unsaturated carbocyclic rings refer to rings having a single double bond and doubly unsaturated carbocyclic rings refer to rings having two non-adjacent double bonds. Each of the ring atoms in B may be optionally substituted with one or more substituents independently selected from H, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, fluoro, chloro, cyano, hydroxyl, C1-C4 hydroxy alkyl. Additionally, in those embodiments where B completes a seven or eight-membered ring, two nonadjacent carbon atoms in the chain B can be bridged by a methylene or an ethylene moiety.
The R1 group is H, C1-C5 straight, branched or cyclic alkyl, —COR13, —SO2R13, benzyl, or alkyl or halo substituted benzyl and R13 is —OH, C1-C4 alkyl, which can be a straight chain or branched.
R2a is -L-R14 such that the optional linker L is —Xt(CO)mXp(CH2)nYq— and X is O or NH, Y is O or S, m is 0or 1, n is 0, 1, or 2, p is 0 or 1, q is 0 or 1, and t is 0 or 1; wherein, if p and t are both 1, then m is 1, q is zero and X is NH. R14 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted saturated heterocyclyl, optionally substituted straight or branched C1-C8 alkyl, or optionally substituted straight or branched C1-C8 heteroalkyl. In certain embodiments, when R14 comprises aryl or heteroaryl, then the aryl or heteroaryl moiety is either a single 5 or 6-membered ring or a fused bicyclic moiety. R2b is H or methyl.
In certain embodiments R3a and R8a complete a bridge across the ring completed by B in Formula I, such that either R3a and R8a taken together form a methylene or ethylene bridge or one of R3a and R8a is H and the other forms a methylene or ethylene bridge to a carbon atom in the chain B. The bridge carbons of such a bridge may be unsubstituted or substituted with methyl, chloro, or fluoro; in certain embodiments a bridge carbon may be geminal dimethyl substituted.
In other embodiments R3a and R8a do not complete a bridge and instead are each selected independently from the group consisting of H, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, fluoro, chloro, cyano hydroxyl, C1-C4 hydroxy alkyl. In other embodiments one or both of R3a and R8a is a bond forming a second ring bond with an adjacent ring carbon atom thereby completing a double bond in the ring, such as, for instance, the double bond in a cyclopentenyl moiety.
Rhd 3b and R8b are each independently selected from the group consisting of H and methyl.
R9, R11, and R12 are each independently selected from the group consisting of H, halo, nitro, cyano, hydroxyl, amino, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkoxy, C1-4 haloalkyl, trihalomethoxy, trihalomethyl, and cyclopropyl.
R10 is a group having an optional methylene, ethylene or propylene linker and a carbonyl, ester, thionyl, amine, amide, reverse amide, thioamide, reverse thioamide, sulfonyl, sulfoxyl, thioether, sulfonamide, reverse sulfonamide, urea, or a thiourea moiety as a linker to R15 or, alternatively, having a formula chosen from the following: —(CH2)r—CO—(CH2)rR15, —(CH2)r—COO—(CH2)rR15, —(CH2)r—CS—(CH2)rR15, —(CH2)rN(R16)((CH2)rR17), —(CH2)r—CO—N(R16)((CH2)rR17), —(CH2)rNH—CO—(CH2)rR15, —CH2)r—CS—N(R16)((CH2)rR17), —(CH2)rNH—CS—(CH2)rR15, —(CH2)rSO2—(CH2)rR15, —(CH2)rSO—(CH2)rR15, —(CH2)rS—(CH2)rR15, —(CH2)rSO2—N(R16)((CH2)rR17), —(CH2)rNH—SO2—(CH2)rR15, —(CH2)rNH—CO—N(R16)((CH2)rR17), and —(CH2)rNH—CS—N(R16)((CH2)rR17). Additionally R10 can be a group having an optional methylene, ethylene or propylene linker coupled to a 5-6 membered heteroaryl ring optionally substituted with R15. In R10 each r is independently selected from 0, 1, 2, and 3; R15 is H or optionally substituted straight or branched C1-C8 alkyl, optionally substituted straight or branched C1-C8 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted 5-7 membered cycloheteroalkyl, optionally substituted aryl, or optionally substituted 5-6 membered or bicyclic heteroaryl.
R16 and R17 may be taken together to form an optionally substituted saturated heterocyclyl with the N to which they are bonded in which case r is zero, such that the optional alkylene linker between such N atom and R17 is null. Alternatively, R16 and R17 can be each independently selected from the group consisting of substituted straight chain or branched C1-C8 alkyl, optionally substituted straight or branched C1-C8 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted 5-7 membered cycloheteroalkyl, optionally substituted aryl, optionally substituted 5-6 membered and bicyclic heteroaryl.
In certain embodiments of Formula I, when R2a is unsubstituted phenyl, when R1, R2b, R3b, R8b, R9, R11, R12 are all H, when R2a, R8a and B taken together complete an unsubstituted norbornanyl moiety, and when R10 is —CO—N R16R17, then R16 is —(CH2)sR18 wherein R18 is optionally substituted aryl or cyclopropyl, and s is 1, 2, or 3 and R17 is H.
In certain embodiments of Formula I when R10 is —COOH, and R1, R2b, R3b, R8b, R9, R11, R12 are all H, wherein R2a, R8a and B taken together complete an unsubstituted norbornanyl moiety, and wherein m, n, p, q, and t are all zero, then R14 is not phenyl.
In certain embodiments of Formula I, R3a, R8a and B are taken together to complete an optionally substituted, bridged 6, 7 or 8-membered ring. In certain of such embodiments the bridge is a methylene moiety; in other such embodiments the bridge is an ethylene moiety. In certain embodiments of Formula I, R3a, R8a and B taken together complete an optionally substituted norbornanyl moiety.
In certain other embodiments of Formula I, R3a and R8a are H and do not form a bridge and B completes a cyclopentenyl moiety.
In certain embodiments, the ring nitrogen of Formula I is unsubstituted, i.e., R1 is H. In certain embodiments, the ring nitrogen of Formula I is substituted with C1-C5 alkyl. In certain embodiments, the ring nitrogen of Formula I is substituted with a carbonyl moiety —COR13. In certain other embodiments, the ring nitrogen of Formula I is substituted with a sulfonyl moiety —SO2R13.
In certain embodiments of Formula I, the phenyl ring substituents R9, R11, and R12 are all H. In other embodiments of Formula I, the linker L in R2a is null or alkylene such that m, p, q, and t are zero. In particular embodiments, n is an integer. In other particular embodiments n is zero.
In certain other embodiments of Formula I, R14 is optionally substituted aryl or optionally substituted heteroaryl. In still other embodiments of Formula I, R14 is optionally substituted phenyl. In alternative embodiments of Formula I, R14 is optionally substituted heteroaryl. In other alternative embodiments of Formula I, R14 is optionally substituted 5 or 6-membered heteroaryl, which in certain particular embodiments is optionally substituted pyrimidinyl, pyridinyl, pyrazinyl, thiazolyl, furanyl, thiophenyl, oxazolyl, or imidazolyl. In certain other embodiments of Formula I, R14 is optionally substituted straight or branched C1-C6 alkyl.
In particular embodiments of Formula I all integers designated as “r”0 in R10 are zero such that R10 does not contain fully saturated alkylene linkers. In other embodiments of Formula I, at least one r is not zero; for example when r is 1 or 2 so as to provide a methylene or an ethylene linker, respectively, in R10 linking to R15 or R16.
In certain embodiments of Formula I, R10 is a sulfonyl-linked R15, of the formula —SO2R15 or a “reverse” amide of the formula —NH—CO—R15.
In certain other embodiments of Formula I, R10 is a sulfonamide of the formula —SO2—NR16R17.
In yet other embodiments of Formula I, R10 is a reverse sulfonamide of the formula —(CH2)rNH—SO2R15. In still other certain embodiments of Formula I, R10 is a urea of the formula —(CH2)rNH—CO—NR16R17.
In particular embodiments of Formula I, R10 is an optional C1-C3 alkylene linker coupled to a 5-6 membered heteroaryl ring which can be substituted with R15.
Also provided in the present invention are compounds having the structure of formula II:
and pharmaceutically acceptable salts, esters, solvates, stereoisomers and racemates thereof, wherein R1 is chosen from H, C1-C5 straight, branched or cyclic alkyl, —COR13, or —SO2R13, wherein R13 is C1-C3 straight or branched alkyl; R2a is -L-R14 where L is —(NH)p(CH2)rYq— and Y is carbonyl or thionyl, p and q are each independently 0 or 1; wherein R14 is chosen from optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted straight or branched C1-C4 alkyl, or optionally substituted straight or branched C1-C4 heteroalkyl; and when R14 includes aryl or heterocyclyl moiety, such aryl or heterocyclyl moiety is either a single 5- or 6-membered ring or a fused bicyclic moiety.
In Formula II, R2b is H or methyl; R3 and R8 are each independently chosen from hydrogen and C1-C3 alkyl; R9, R11 and R12 are each independently chosen from hydrogen, halo, cyano, hydroxyl, amino, and C1-4 alkyl; R10 is chosen from —(CH2)r—COO—(CH2)r—R15, —(CH2)rN(R16)((CH2)r—R17), —(CH2)rR16—(CH2)r—R17, —(CH2)r—CO—N(R16)((CH2)r—R17), —(CH2)rNH—CO—(CH2)r—R15, —(CH2)rSO2—(CH2)r—R15, —(CH2)rSO—(CH2)r—R15, —(CH2)rSO2—N(R16)((CH2)rR17), —(CH2)rSO2—NR16—CO—((CH2)r—R17), —(CH2)rNH—SO2—(CH2)r—R15, —(CH2)rNH—CO—N(R16)((CH2)r—R17), —(CH2)rNH—CS—N(R16)((CH2)r—R17), (CH2)rN—(SO2R16)(SO2R17), —CO—NH—(CH2)r—(C3-C6 cycloalkyl) and —CO—NH—(CH2)r-(aryl); wherein each r is independently chosen from 0, 1, 2, and 3; and wherein R15 is chosen from hydrogen, optionally substituted straight or branched C1-C6 alkyl, optionally substituted straight or branched C1-C6 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted 5-7 membered heterocyclyl, optionally substituted aryl and optionally substituted 5-6 membered or bicyclic heteroaryl. The moieties R16 and R17 are either taken together to form an optionally substituted heterocyclyl or are each independently chosen from hydrogen, substituted straight or branched C1-C6 alkyl, optionally substituted straight or branched C1-C6 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted 5, 6 or 7-membered heterocyclyl, optionally substituted aryl and optionally substituted 5 or 6-membered heteroaryl.
In Formula II Rio cannot be a carboxylate moiety. Further, when R2a is unsubstituted phenyl, and R1, R2b, R3b, R8b, R9, R11 and R12 are all hydrogen, and R10 is —CO—NHR16, then the moiety R16 is —(CH2)sR18, wherein R18 is optionally substituted aryl or cyclopropyl and s is 1, 2, or 3.
In certain embodiments of Formula II when, R1 is hydrogen or —SO2R13: then R2b, R3, R8, R9, R11 and R12 are each H; and R10 is chosen from —(CH2)r—COO—(CH2)r—R15, —(CH2)rN(R16)((CH2)r—R17), —(CH2)rR16—(CH2)r—R17, (CH2)r—CO—N(R16)((CH2)r—R17), —(CH2)rNH—CO—(CH2)r—R15, —(CH2)rSO2—(CH2)r—R15, —(CH2)rSO2—N(R16)((CH2)r—R17), —(CH2)rSO2—N(R16)—CO—((CH2)r—R17), —(CH2)rNH—SO2—(CH2)r—R15, —(CH2)rNH—CO—N(R16)((CH2)r—R17), —(CH2)rNH—CS—N(R16)((CH2)r—R17), —CO—NH—(CH2)r—C3-C6 cycloalkyl and —CO—NH—(CH2)r-aryl; and R14 is chosen from optionally substituted straight or branched C1-C3 alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl.
In certain other embodiments of Formula II the operators in, p, q and t are each 0. In certain other particular embodiments of Formula II the operator n is 1 or 2; in certain other particular embodiments n is 0. In other particular embodiments, each instance of the operator, r is zero.
In particular embodiments of Formula II, R14 is optionally substituted aryl. In other particular embodiments R14 is optionally substituted 5 or 6-membered heteroaryl. In still other particular embodiments R10 is the moiety —(CH2)rSO2—N(R16)((CH2)r—R17) or the moiety —(CH2)rNH—CO—(CH2)r—R15. In alternative embodiments, R10 is chosen from —(CH2)—COO—(CH2)r—R15, —(CH2)rN(R16)((CH2)r—R17), —(C2)rR16—(CH2)r—R17, —(CH2)r—CO—N(R16)((CH2)r—R17), —(CH2)rSO2—(CH2)r—R15, and —(CH2)rSO2—N(R16)—CO—((CH2)r—R17). In alternative embodiments, R10 is chosen from —(CH2)rNH—SO2—(CH2)r—R15, —(CH2)rNH—CO—N(R16)((CH2)r—R17), —(CH2)rNH—CS—N(R16)((CH2)r—R17), (CH2)rN—(SO2R16(SO2R17), —CO—NH—(CH2)r—(C3-C6 cycloalkyl) and —CO—NH—(CH2)r-(aryl).
The present invention also provides compounds having the structure of Formula II, wherein in certain embodiments the compounds have an EC50 of less than about 1 μM for a mammalian CB2 receptor. In other embodiments, the compounds have an EC50 of less than about 500 μM for a mammalian CB2 receptor. In still other embodiments, the compounds have an EC50 of less than about 100 nM for a mammalian CB2 receptor. In other embodiments, the compounds have an EC50 of less than about 75 nM for a mammalian CB2 receptor. In yet other embodiments, the compounds have an EC50 of less than about 20 nM for a mammalian CB2 receptor. In certain other embodiments, the compounds have an EC50 of less than about 10 nM for a mammalian CB2 receptor. In certain embodiments, the compounds have an EC50 of less than about 1.0 nM for a mammalian CB2 receptor. The mammalian CB2 receptor can be any mammalian CB2, such as for instance and without limitation, a mouse CB2 receptor, a rat CB2 receptor or in a particular aspect, the mammalian CB2 receptor can be a CB2 receptor of a primate, such as a human.
The present invention also provides compounds of the structure of Formula II, which in certain embodiments have an EC50 of greater than about 100 nM for a mammalian CB1 receptor. In other embodiments, the compounds have an EC50 of greater than about 1.0 μM for a mammalian CB1 receptor. In still other embodiments, the compounds have an EC50 of greater than about 10 μM for a mammalian CB1 receptor. In still other embodiments, the compounds have an EC50 of greater than about 100 μM for a mammalian CB1 receptor. The mammalian CB1 receptor can be any mammalian CB1, such as for instance and without limitation, a mouse CB1 receptor, a rat CB1 receptor or in a particular aspect, the mammalian CB1 receptor can be a CB1 receptor of a primate, such as a human.
DEFINITIONS: As used in this specification the following terms have the definitions listed below:
“Halo” means fluoro, chloro, bromo or iodo. Fluoro and chloro groups are preferred halo substituents. Halo alkyl and halo alkoxy means an alkyl or alkoxy group having one or more H atoms replaced with a halo group and includes perhalo substituted groups.
“Alkyl” means a linear or branched carbon chain, such as, but without limitation, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, and their branched chain isomers. The term “alkenyl” means a linear or branched carbon chain with one or more double bonds, such as, but without limitation, vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl. “Alkynyl” means a linear or branched carbon chain with one or more triple bonds, such as for instance, ethynyl, propynyl, 3-methyl-1-pentynyl and 2-heptynyl. As used herein “cycloalkyl” means a monocyclic, bicyclic or bridged saturated carbocyclic ring, each ring having from 3-8 carbon atoms. Norbornane and adamantane are examples of such cycloalkyl moieties that are bicyclic or bridged saturated carbocyclic rings. The term “cycloalkyl” also includes a monocyclic ring fused to an aryl or heteroalkyl substituent. Cycloalkyl substituents include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and tetrahydronaphthyl.
“Heteroalkyl” means a linear or branched chain comprising carbon atoms and at least one heteroatom (N, S, or O) such as ethers, thioethers and amines. Cycloheteroalkyl means a monocyclic or bicyclic ring having at least one ring heteroatom.
“Alkylene” means an alkyl diradical bonded to other moieties in two locations such as methylene (—CH2—), ethylene (—CH2CH2—), etc. Alkylene moieties can form linkers interposed between two moieties attached at any two carbon atoms of the alkylene moiety.
“Alkoxy” means an alkyl moiety having an ether linkage such as methoxy, ethoxy, etc. Hydroxy alkyl means an alkyl moiety substituted with one or more hydroxyl groups, e.g., 2-hydroxy ethyl.
Where a term such as “alkyl”, “alkoxy”, or “cycloalkyl”, etc. is modified with a specified number of carbon atoms or a range of number of carbon atoms, e.g., C1-C4 alkyl, such moiety has a number of carbon atoms within the specified range, i.e., in this example 1-4, corresponding to methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, or t-butyl.
“Aryl” includes a monocyclic or bicyclic aromatic ring including only carbon atoms in the ring. “Aryl” also includes an aryl group fused to a monocyclic cycloalkyl or monocyclic cycloheteroalkyl group. Aryl groups include phenyl, naphthyl, quinolinyl, tetrahydroquinolinyl, benzofuranyl and dihydrobenzopyranyl.
“Heteroaryl” means a monocyclic, bicyclic or tricyclic aromatic ring containing at least one heteroatom with each ring containing 5 or 6 atoms. The heteroatom(s) can be independently selected from N, O or S. Examples of monocyclic heteroaryl groups include thiophenyl, pyrolyl, pyrolinyl, furanyl, axazolyl, thiazolyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, triazolyl, thiadiazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, prazinyl and triazinyl. Examples of bicyclic heteroaryl groups include indolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, purinyl, quinolinyl, isoquinolinyl, quinazolinyl and pteridinyl. Examples of tricyclic heteroaryl groups include carbazolyl, phenothiazinyl and phenoxazinyl.
“Heterocyclyl” means a group of one or more fused rings, wherein at least one ring includes at least one heteroatom. The heterocyclyl group can, but need not include one or more unsaturated bonds, and can in certain embodiments include one or more aromatic rings. Thus, as used in this specification, the term “heterocyclyl” includes “heteroaryl” groups as well as unsaturated non-aromatic ring-containing groups and fully saturated cyclic groups having at least one heteroatom.
Where a moiety is herein described as optionally substituted, such moiety may be unsubstituted, singly substituted or multiply substituted with the same or different substituents. Optional substituents can be, for example, an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkaryl, aralkyl, heteroaryl, heteroaralkyl, cycloheteroalkyl, haloalkyl, perhaloalkyl, alkoxy, carboxy, amino, amide, sulfonyl, sulfoxyl, sulfonamidyl, hydroxyl, halo, nitro, cyano, thio, or alkylthio group.
The term “bridged” compound as used herein refers to compounds having an alkylene bridge across the carbocycle formed by B in formula I. Thus, the compound “methylene bridged N-(cyclopropylmethyl)-6-phenyl-5,6,6a,7,8,9,10,10a-octahydro phenanthridine-2-carboxamide” refers to the compound having a methylene bridge across the saturated carbocycle of the phenanthridine (See Example 2d).
Compounds of the invention can have one or more asymmetric centers called chiral centers. In certain embodiments, the carbon atoms at the 2, 3 and 4 positions of the nitrogen-containing ring of compounds of formula I or of formula II can be chiral centers. These compounds can occur as racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers with respect to all or any subset of chiral centers of such compounds. All sterioisomeric forms of the compounds of formula I or of formula II are contemplated within the scope of the present invention.
In certain embodiments the compounds of the invention are agonists for a mammalian G protein-coupled receptor (GPCR), particularly a human GPCR. In particular embodiments, the compounds of the invention are agonists for a mammalian cannabinoid CB2 receptor, such as for instance, the human CB2 receptor. In certain embodiments, the compounds of the invention are full agonists for the human CB2 receptor. That is, when contacted with the human CB2 receptor at sufficiently high concentrations above the EC50, these compounds are capable of fully inducing the activity of the receptor.
In other particular embodiments, the compounds of the invention are partial agonists for the human CB2 receptor. When contacted with the human CB2 receptor at concentrations above the EC50, the maximum level of activity induced by these compounds is significantly below the maximal activity of the receptor induced by the full agonists. As used herein, compounds that showed induction of less than 80% of the maximal range of activity induced by CP55940 are designated as partial agonists.
In some embodiments, the compounds of the invention are selective agonists for a mammalian CB2 receptor and do not function as agonists for the CB1 receptor of that mammal. In particular embodiments, the compounds are selective agonists for the human CB2 receptor while having little or no agonist activity for the human CB1 receptor. In a particular embodiment, the compound of the invention is a salt, solvate, ester, stereoisomer, mixture of one or more stereoisomers, or a racemate of a compound having the structure of formula I. In another particular embodiment, the invention provides a pharmaceutically acceptable salt, solvate, ester, stereoisomer, mixture of one or more stereoisomers, or racemate of a compound having the formula of formula II.
Without wishing to be bound by theory, the compounds of the present invention are believed to be ligands for a mammalian CB2 receptor. In certain embodiments, the compounds of the present invention have an EC50 of less than about 1 μM for a mammalian CB2 receptor. In other embodiments the compounds have an EC50 of less than about 500 μM, while in other embodiments the compounds have an EC50 of less than about 100 μM for a mammalian CB2 receptor, particularly the human CB2 receptor. In other embodiments, the compounds have an EC50 of less than about 75 nM for the human CB2 receptor. In still other embodiments, the compounds have an EC50 of less than about 50 nM for the human CB2 receptor. In yet other embodiments, the compounds have an EC50 of less than about 20 nM for the human CB2 receptor. In still other embodiments, the compounds have an EC50 of less than about 10 nM for the human CB2 receptor. In other embodiments, the compounds have an EC50 of less than about 5 nM for the human CB2 receptor. In other particular embodiments, the compounds have an EC50 of less than about 1.0 nM for the human CB2 receptor.
In one embodiment, the invention provides a pharmaceutical composition that includes a pharmaceutically effective amount of a compound of the invention. In a particular embodiment, the invention provides a pharmaceutical composition of the invention is effective for treating a disease condition or state addressable by modulating the activity of a cannabinoid CB2 receptor in a human or an animal in need thereof. In one aspect the disease condition or state is addressable by increasing the activity of the CB2 receptor with an agonist. In another aspect the disease condition or state is addressable by reducing the activity of the CB2 receptor with an antagonist.
In view of their ability to modulate the activity of the CB2 receptor, the compounds of the present invention can be used in the treatment of conditions and diseases or disorders that include, but are not limited to, inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, psoriasis, eczema, multiple sclerosis, diabetes and thyroiditis.
Certain compounds of the invention can also be used in the treatment of disorders such as, but are not limited to, pain (e.g. inflammatory pain, visceral pain, postoperative pain, cancer pain, neuropathic pain, musculoskeletal pain, dysmenorrhea, menstrual pain, migraine and headache).
Certain compounds of the invention can also be used in the treatment of skin disorders (e.g. sunburn, dermatitis, pruritis); lung disorders (e.g. chronic obstructive pulmonary disease, cough, asthma, bronchitis); ophthalmic disorders (e.g. glaucoma, retinitis, reinopathies, uveitis, conjunctivitis); gastrointestinal disorders (e.g. ulcerative colitis, irritable bowel syndrome, coeliac disease, inflammatory bowel disease, gastroesophageal reflux disease, organ transplant, nausea, emesis); cardiovascular disorders (e.g. stroke, cardiac arrest, atherosclerosis, myocardial ischemia); neurodegenerative, neuroinflammatory or psychiatric disorders (e.g. senile dementia, Alzheimer's disease, vascular dementia, amyotrophic lateral sclerosis, neuroinflammation, tinnitus); bladder disorders (e.g. bladder hyper-reflexia, cystitis) and cancer, such as for instance, lymphoblastic leukemia and lymphoma, acute myelogenous leukemia, chronic lymphocytic leukemia, glioma, skin cancer, breast cancer, prostate cancer, liver cancer, kidney cancer, lung cancer, pancreatic cancer.
In addition, certain compounds of the invention can be used to modulate bone formation and/or resorption for treating conditions including, but not limited to, ankylosing spondylitis, gout, arthritis associated with gout, osteoarthritis and osteoporosis.
Certain compounds of the invention can also be used for the treatment of neuropathic pain including, but not limited to diabetic neuropathy, fibromyalgia, lower back pain, sciatica, pain from physical trauma, cancer, amputation, toxins or chronic inflammatory conditions.
Compounds of the invention and their pharmaceutically acceptable salts can be administered in a standard manner, for example orally, parenterally, sublingually, dermally, transdermally, rectally, or via inhalation, or by buccal, nasal, ocular or otic administration.
All reactions involving moisture sensitive compounds were carried out under an anhydrous nitrogen or argon atmosphere. All reagents were purchased from commercial sources and used without further purification. Normal phase chromatography was done on an ISCO CombiFlash Companion and reverse phase chromatography was done on a Waters AutoPurification System with 3100 Mass Detector. Mass spectra (MS) were determined on the Waters SQ Detector/3100 Mass Detector using electrospray techniques. Unless otherwise noted, the materials used in the examples were obtained from readily available commercial sources or synthesized by standard methods known to those skilled in the art of organic synthesis. Nuclear magnetic resonance spectra were recorded using a Jeol ECX 400 MHz spectrometer.
General scheme for synthesis of bridged phenanthridines of the invention. Final compounds of the invention can be isolated by filtration. Alternatively, the compounds can be isolated by normal, or reverse phase chromatography.
Certain groups of intermediates (1a and 1b) and groups of compounds of the present invention (1c, 1d, 1e and 1f) can be prepared according to the process outlined in the general synthetic schemes shown in Scheme I above.
A. Synthesis of compound 2b: To commercially available 2a (0.30 g, 1.98 mmol) in acetonitrile (1 mL) was added benzaldehyde (0.23 ml, 2.34 mmol), norbornylene (0.40 g, 4.25 mmol) and trifluoroacetic acid (0.24 ml, 2.14 mmol). The reaction was stirred at room temperature for 16 hours. The desired product 2b precipitated out of solution and was collected via filtration to provide 0.40 g of a white solid.
B. Synthesis of compound 2c: To 2b (0.52 g, 1.56 mmol) in 3 ml THF (tetrahydrofuran) and 1 ml water was added lithium hydroxide (0.37 g, 15.6 mmol). The reaction stirred at room temperature for five days. Reaction was not complete so one equivalent of potassium hydroxide was added and the reaction was heated to 60° C. for 16 hours. Ethyl acetate was added and the organic layer was washed with water. The organic layer was concentrated to give the desired acid 2c which was used without further purification.
C. Synthesis of compound 2d. To 2c (0.050 g, 0.15 mmol) in methylene chloride (1 ml) was added cyclopropylmethanamine (0.022 g, 0.31 mmol), and HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate: 0.037 g, 0.15 mmol). The reaction stirred at room temperature for 16 hours. The reaction mixture was diluted with water and extracted with methylene chloride. The organics were dried over sodium sulfate and concentrated. The crude mixture was purified using normal phase chromatography using a 10-60% ethyl acetate/hexanes gradient to provide 3.7 mgs of 2d as a white solid. Mass spectrometry (MS) of the molecule ion yielded the following: MS: m/z 373.2 (MH+).
To commercially available 4-(piperidin-1-ylsulfonyl)aniline 3a (0.06 g, 0.25 mmol) in 3 ml acetonitrile was added benzaldehyde (0.026 g, 0.25 mmol), norbornylene (0.035 g, 0.37 mmol), and TFA (Trifluoroacetic acid: 0.028 g, 0.25 mmol). The reaction stirred at room temperature for 18 hours. The desired compound 3b precipitated out of solution as the TFA salt and was collected by filtration to provide a white solid (64.9 mgs). MS: m/z 423.1 (MH+)
A. Synthesis of compound 4b. To commercially available 4a (0.2 g, 0.96 mmol) in methylene chloride (3 ml) was added commercially available valeric acid (0.096 g, 0.96 mmol), DIEA (N,N-Diisopropylethylamine: 0.25 g, 1.92 mmol), and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: 0.18 g, 0.96 mmol). The reaction stirred at room temperature for 18 hours. The organic layer was washed with 1N HCl and dried over sodium sulfate and concentrated. Crude 4b was used in the next step without further purification.
B. Synthesis of compound 4c. To 4b was added 1 ml of methylene chloride and 1 ml of TFA. The reaction stirred at room temperature for 2 hours. The reaction mixture was concentrated. Crude 4c was used in the next step.
C. Synthesis of compound 4d. To 4c (0.1 g, 0.52 mmol) in 3 ml of acetonitrile was added benzaldehyde (0.051 g, 0.48 mmol), norbornylene (0.067 g, 0.72 mmol), and TFA (0.055 g, 0.48 mmol). The reaction mixture stirred for 18 hours at room temperature and was concentrated. The crude mixture was purified using normal phase chromatography (ethyl acetate/hexanes, 10 to 80% gradient) to provide 65.4 mgs of 4d. MS: m/z 375.2 (MH+).
A. Synthesis of compound 5b: To commercially available 5a (0.3 g,. 1.44 mmol) in methylene chloride (4 ml) was added propyl isocyanate (0.11 g, 1.44 mmol). The reaction stirred at room temperature for 18 hours. A white solid precipitated out of solution. The white solid was collected by filtration and determined to be 5b.
B. Synthesis of compound 5c. To 5b was added 1 ml of TFA and 1 ml of methylene chloride. The reaction mixture stirred at room temperature for 2 hours. The mixture was concentrated and used in the next step without further purification.
C. Synthesis of compound 5d: To 5c (0.09 g, 0.46 mmol) in 3 ml of acetonitrile was added benzaldehyde (0.049 g, 0.46 mmol), norbornylene (0.066 g, 0.69 mmol), and TFA (0.052 g, 0.46 mmol). The reaction mixture stirred for 18 hours at room temperature. The desired product precipitated out of solution and was collected by filtration to yield 5d as a white solid and TFA salt (37.5 mgs). MS: m/z 376.2 (MH+).
Compound 6 was synthesized in the same manner as compound 5d except that propyl thioisocyanate was used instead of propyl isocyanate. The desired compound was purified by normal phase chromatography as described above. The desired compound was purified by normal phase chromatography as described above. MS: m/z 392.1 (MH+).
Compound 7 was synthesized in the same manner as compound 5d except that p-tolualdehyde was used instead of benzaldehyde. The desired compound was purified by normal phase chromatography as described above. MS: m/z 390.2 (MH+).
Compound 8 was synthesized in the same manner as compound 6 except that p-tolualdehyde was used instead of benzaldehyde. The desired compound was purified by normal phase chromatography as described above. MS: m/z 406.2 (MH+).
Compound 9 was synthesized in the same manner as compound 3b except that 2,6-dichlorobenzaldehyde was used instead of benzaldehyde. MS: m/z 491.1 (MH+).
Compound 10 was synthesized in the same manner as compound 3b except that p-tolualdehyde was used instead of benzaldehyde. The desired compound was purified by normal phase chromatography as described above. MS: m/z 437.2 (MH+).
Compound 11 was synthesized in the same manner as compound 3b except that 4-amino-N,N-dimethylbenzenesulfonamide was used instead of 3a. MS: m/z 383.1 (MH+). 1H NMR (CD2Cl2) δ 7.55 (s, 1H), 7.21-7.37 (m, 6H), 6.53-6.60 (d, J=8 Hz, 1H), 4.11-4.19 (br s, 1H), 3.52-3.60 (d, J=10 Hz, 1H), 2.61-2.70 (m, 2H), 2.56 (s, 6H), 2.11-2.20 (m,1H), 2.01-2.04 (m, 1H), 1.10-1.65 (m, 6H).
Compound 12 was synthesized in the same manner as compound 4d except that 3-methoxy propionic acid was used instead of valeric acid. MS: m/z 377.3 (MH+).
Compound 13 was synthesized in the same manner as compound 4d except that 4-oxazole carboxylic acid was used instead of valeric acid. MS: m/z 386.2 (MH+).
Compound 14 was synthesized in the same manner as compound 4d except that (tetrahydro-furan-2-yl)-acetic acid was used instead of valeric acid. MS: m/z 403.3 (MH+).
Compound 15 was synthesized in the same manner as compound 12 except that p-tolualdehyde was used instead of benzaldehyde. MS: m/z 391.3 (MH+).
Compound 16 was synthesized in the same manner as compound 14 except that p-tolualdehyde was used instead of benzaldehyde. MS: m/z 417.3 (MH+).
Compound 17 was synthesized in the same manner as compound 3b except that p-morpholinoaniline was used instead of 3a. The desired compound was purified by normal phase chromatography as described above. MS: m/z 361.2 (MH+).
Compound 18 was synthesized in the same manner as compound 17 except that p-tolualdehyde was used instead of benzaldehyde. The desired compound was purified by normal phase chromatography as described above. MS: m/z 375.2 (MH+).
Compound 19 was synthesized in the same manner as compound 4d except that cyclopropyl carboxylic acid was used instead of valeric acid. MS: m/z 359.3 (MH+).
Compound 20 was synthesized in the same manner as 19 except that 2,6-dichlorobenzaldehyde was used instead of benzaldehyde. MS: m/z 427.2 (MH+).
Compound 21 was synthesized in the same manner as compound 19 except that propionaldehyde was used instead of benzaldehyde. MS: m/z 311.3(MH+).
Compound 22 was synthesized in the same manner as compound 19 except that p-tolualdehyde was used instead of benzaldehyde. MS: m/z 373.3(MH+).
Compound 23 was synthesized in the same manner as 4d except that commercially available 4-amino acetanilide was used instead of 4c. MS: m/z 333.2(MH+).
Compound 24 was synthesized in the same manner as 23 except that 2,6-dichlorobenzaldehyde was used instead of benzaldehyde. MS: m/z 401.1(MH+).
Compounds 25a and 25b were synthesized in the same manner as 23 except the p-tolualdehyde was used instead of benzaldehyde. Upon purification, two compounds were isolated with the same molecular weight and assumed to be diastereomers. MS: m/z 347.3(MH+) and m/z 347.3(MH+).
Compound 26 was synthesized in the same manner as 5d except that 2,6-dichlorobenzaldehyde was used instead of benzaldehyde. MS: m/z 444.1 (MH+).
Compound 27 was synthesized in the same manner as compound 6 except that 2,6-dichlorobenzaldehyde was used instead of benzaldehyde. The desired compound was purified by normal phase chromatography as described above. MS: m/z 460.1 (MH+).
Compound 28 was synthesized in the same manner as compound 2d except that benzylamine was used in place of cyclopropylmethanamine. MS: m/z 409.2 (MH+).
Compound 29 was synthesized in the same manner as compound 4 except that p-tolualdehyde was used instead of benzaldehyde. MS: m/z 389.3 (MH+).
Compound 30 was synthesized in the same manner as compound 11 except that pyrimidine-5-carbaldehyde was used in place of benzaldehyde. MS: m/z 385.1(MH+).
Compound 31 was synthesized in the same manner as compound 11 except that thiazole-2-carbaldehyde was used in place of benzaldehyde. MS: m/z 390.1 (MH+).
Compound 32 was synthesized in the same manner as compound 11 except that N-(4-formylphenyl)acetamide was used in place of benzaldehyde. MS: m/z 440.2 (MH+).
Compound 33 was synthesized in the same manner as compound 11 except that 1-methyl-1H-pyrazole-5-carbaldehyde was used in place of benzaldehyde. MS: m/z 387.2 (MH+).
Compound 34 was synthesized in the same manner as compound 3b except that 4-(morpholinosulfonyl)aniline was used in place of 4-(piperidin-1-ylsulfonyl)aniline. MS: m/z 425.2(MH+). 1H NMR (CD2Cl2) δ 7.62 (s, 1H), 7.30-7.45 (m, 6H), 6.65 (d, J=8.8 Hz, 1H), 4.05 (br s, 1H), 3.68-3.77 (m, 4H), 3.62-3.67 (d, J=10 Hz, 1H), 2.91-2.99 (m, 4H), 2.65-2.78 (m, 2H), 2.19-2.29 (t, J=9 Hz, 1H), 2.11-2.15 (m, 1H), 1.10-1.75 (m, 6H).
Compound 35 was synthesized in the same manner as compound 3b except that (morpholinosulfonyl)aniline was used in place of 4-(piperidin-1-ylsulfonyl)aniline and pyrimidine-5-carbaldehyde was used in place of benzaldehyde. MS: m/z 427.3 (MH+).
Compound 36 was synthesized in the same manner as compound 3b except that (morpholinosulfonyl)aniline was used in place of 4-(piperidin-1-ylsulfonyl)aniline and thiazole-2-carbaldehyde was used in place of benzaldehyde. MS: m/z 432.2 (MH+). The resulting product was determined to be a 4:1 mixture of 2 diastereomers by 1H NMR. MS: m/z 432.2 (MH+). 1H NMR (CD2Cl2) δ 7.78-7.79 (d, J=3 Hz, 1H minor), 7.69-7.72 (d, J=3 Hz, 1H major), 7.53-7.58 (m, 1H), 7.41-7.43 (d, J=3 Hz, 1H minor), 7.33-7.38 (m, 1H), 7.31-7.33 (d, J=3 Hz, 1H major), 6.78-6.82 (d. J=8 Hz, 1H minor), 6.71-6.73 (d, J=8 Hz, 1H, major), 5.05 (s, 1H, minor), 4.75 (s, 1H, major), 4.62-4.65 (m, 1H, minor), 4.40-4.42 (m, 1H, major), 3.69-3.73 (m, 4H), 3.13-3.18 (m, 1H, minor), 2.91-2.99 (m, 4H), 2.81-2.85 (m, 1H major), 2.31-2.59 (m, 2H), 1.21-1.66 (m, 5H), 1.11-1.20 (m, 1H major), 0.88-0.94 (m, 1H, minor).
Compound 37 was synthesized in the same manner as compound 3b except that (morpholinosulfonyl)aniline was used in place of 4-(piperidin-1-ylsulfonyl)aniline and 1-methyl-1H-pyrazole-5-carbaldehyde was used in place of benzaldehyde. MS: m/z 429.2 (MH+). The resulting product was determined to be a 3:1 mixture of 2 diastereomers by 1H NMR. MS: m/z 429.2 (MH+).1H NMR (CD2Cl2) δ 7.61 (s, 1H, major), 7.55 (s, 1H, minor), 7.43 (s, 1H, minor), 7.31-7.36 (m, 2H), 6.67-6.70 (m, 1H), 6.36 (s, 1H, minor), 6.14 (s, 1H, major), 4.41-4.43 (d, 1H, minor), 4.22-4.24 (br s, 1H), 3.99-4.12 (d, J=8 Hz, 1H, major), 3.91 (s, 3H, major), 3.84 (s, 3H, minor), 3.69-3.73 (m, 4H), 3.08-3.11 (m, 1H, minor), 2.91-2.97 (m, 4H), 2.78-2.81 (m, 1H, major), 2.61-2.65 (m, 1H), 2.05-2.37 (m, 3H), 1.11-1.79 (m, 5H), 0.90-0.99 (m, 1H, minor).
Compound 38 was synthesized in the same manner as compound 3b except that 4-amino-N-cyclohexyl-N-methylbenzenesulfonamide was used in place of 4-(piperidin-1-ylsulfonyl)aniline. MS: m/z 451.2 (MH+).
Compound 39 was synthesized in the same manner as compound 3b except that 4-amino-N,N-dimethylbenzenesulfonamide was used in place of 4-(piperidin-1-ylsulfonyl)aniline. MS: m/z 355.1 (MH+).
Compound 40 was synthesized in the same manner as compound 3b except that 4-aminobenzenesulfonamide was used in place of 4-(piperidin-1-ylsulfonyl)aniline and 1-methyl-1H-pyrazole-5-carbaldehyde was used in place of benzaldehyde. MS: m/z 359.2 (MH+).
Compound 41 was synthesized in the same manner as compound 3b except that 4-(pyrrolidin-1-ylsulfonyl)aniline was used in place of 4-(piperidin-1-ylsulfonyl)aniline. MS: m/z 409.2 (MH+).
Compound 42 was synthesized in the same manner as compound 3b except that 4-(pyrrolidin-1-ylsulfonyl)aniline was used in place of 4-(piperidin-1-ylsulfonyl)aniline and 1-methyl-1H-pyrazole-5-carbaldehyde was used in place of benzaldehyde. MS: m/z 413.2 (MH+).
Compound 43 was synthesized in the same manner as compound 3b except that 4-(2-methylthiazol-4-yl)aniline was used in place of 4-(piperidin-1-ylsulfonyl)aniline. MS: m/z 373.3(MH+).
Compound 44 was synthesized in the same manner as compound 3b except that 4-(2-methylthiazol-4-yl)aniline was used in place of 4-(piperidin-1-ylsulfonyl)aniline and 1-methyl-1H-pyrazole-5-carbaldehyde was used in place of benzaldehyde. MS: M/z 377.3 (MH+).
Compound 45 was synthesized in the same manner as compound 3b except that 4-(5-butyl-1,3,4-oxadiazol-2-yl)aniline was used in place of 4-(piperidin-1-ylsulfonyl)aniline. MS: m/z 400.4(MH+).
Compound 46 was synthesized in the same manner as compound 3b except that N-(4-aminophenylsulfonyl)acetamide was used in place of 4-(piperidin-1-ylsulfonyl)aniline. MS: m/z 397.3 (MH+).
Compound 47 was synthesized in the same manner as compound 3b except that N-(4-aminophenylsulfonyl)acetamide was used in place of 4-(piperidin-1-ylsulfonyl)aniline and 1-methyl-1H-pyrazole-5-carbaldehyde was used in place of benzaldehyde. MS: m/z 401.3(MH+).
Compound 48 was synthesized in the same manner as 3b except that cyclopentadiene was used in place of norbornylene. MS: m/z 395.2(MH+).
Compound 49 was synthesized in the same manner as 3b except that cyclopentadiene was used in place of norbornylene and 4-amino-N,N-dimethylbenzenesulfonamide was used in place of 4-(piperidin-1-ylsulfonyl)aniline. MS: m/z 355.1 (MH+).
To compound 11 (0.05 g, 0.131 mmol) in methylene chloride (2 mL) was added DIEA (0.034 g, 0.261 mmol) and propane-2-sulfonyl chloride (0.018 g, 0.131 mmol). The reaction mixture stirred at room temperature for 16 hours. The organics were washed with water, dried over sodium sulfate and concentrated. The crude mixture was purified using reverse chromatography to yield 1.8 mgs of 50. MS: m/z 489.1 (MH+).
A. Synthesis of 51a. To tert-butyl 4-aminophenylcarbamate 5a (0.1 g, 0.48 mmol) in methylene chloride was added triethylamine (0.13 mL, 0.96 mmol) and propane-2-sulfonyl chloride (0.068 g, 0.48 mmol). The reaction mixture stirred for 20 hours at room temperature. The organics were washed with ammonium chloride solution, extracted with methylene chloride, dried over sodium sulfate and concentrated. The crude material was used in the next step without further purification.
B. Synthesis of 51b. To 51a was added 1 mL of methylene chloride and 1 mL of TFA. The reaction mixture stirred for 16 hours at room temperature. The reaction mixture was concentrated and the crude material was used in the next step without further purification.
C. Synthesis of 51. To 51b (0.1 g, 0.48 mmol) in 3 ml of acetonitrile was added benzaldehyde (0.051 g, 0.48 mmol), norbornylene (0.067 g, 0.72 mmol), and TFA (0.055 g, 0.48 mmol). The reaction mixture stirred for 18 hours at room temperature and was concentrated. The crude mixture was purified using normal phase chromatography using an ISCO and 10 to 80% gradient (ethyl acetate/hexanes) to provide 65.4 mgs of 51. MS: m/z 397.2(MH+).
Compound 52 was synthesized in the same manner as compound 51 except that methanesulfonyl chloride was used in place of propane-2-sulfonyl chloride. The reaction with methanesulfonyl chloride yielded the di-alkylated amine rather than the mono-alkylated amine. MS: m/z 447.1(MH+).
The ability of compounds to act as agonists or inverse agonists at human CB2 and CB1 receptors (hCB2, hCB1, respectively) was determined by measuring changes in intracellular cAMP levels. Chinese Hamster Ovary (CHO-K1) cell lines stably expressing hCB2 (Genebank: X74328) or hCB1 (Genebank: X54937) were purchased from Euroscreen (Gosselies, Belgium).
Cell lines were grown in suspension in EX-CELL 302 CHO Serum-free medium (Sigma, catalog #14324C) supplemented with 1% Fetal Bovine Serum, glutamine and with or without non-essential amino-acids under 0.4 mg/mL G418 selection.
Receptor mediated responses were determined by measuring changes in intracellular cAMP using LANCE cAMP detection kit (catalog #AD0264, PerkinElmer, Wellesley, Mass.) based on time-resolved fluorescence resonance energy transfer (TR-FRET). Changes in cAMP were determined in cells pre-incubated with IBMX (isobutyl methylxanthine) and prestimulated with NKH-477 (a water soluble forskolin derivative, catalog #1603, Tocris, Ellisville, Mo.) to increase basal cAMP levels as detailed below.
On the day of the experiment, cells were spun at low speed for 5 min at room temperature. The supernatant was removed and cells were resuspended in stimulation buffer (Hanks Buffered Salt Solution/5 mM HEPES, containing 0.5 mM IBMX (catalog #17018, Sigma) and 0.02% BSA (PerkinElmer, catalog #CR84-100)). Cell clumps were removed by filtering through cell strainer 40 μm (BD Falcon, Discovery Labware, Bedford, Mass.) and diluted to 2×105 cells/mL. Antibody supplied with the LANCE cAMP immunoassay kit was then added according to the manufacturer's instructions. An aliquot of cells was taken for un-induced controls. To the remaining cells was added NKH-477 (a water soluble forskolin derivative, Tocris catalog #1603) to a final concentration of 2-8 μM. Cells were then incubated for either 25 or 30 min at room temperature prior to adding to Proxiplates containing test compounds (final DMSO concentration was less than 0.5%) with a Multidrop bulk dispenser, followed by a sixty minute incubation at room temperature. The response was stopped by addition of the detection mix supplied with the LANCE kit.
The reagents were allowed to equilibrate for three hours prior to reading on an Envision multi-mode detector (PerkinElmer). TR-FRET was measured using a 330-380 nm excitation filter, 615 and 665 nm emission filters, dichroic mirror 380 nm and Z=1 mm.
Cyclic AMP concentrations in each well were back-calculated from a cAMP standard curve run concurrently during each assay. Each plate contained 16 wells of forskolin stimulated cells and 16 wells of forskolin plus CP55,940-treated. CP55,940-treated cells were treated with CP55,940 (Tocris catalog #0949) at 1 μM and the maximal response was used as the full range (100%) standard. WIN55,212 (Tocris catalog #1038) was used as an internal standard. Concentrations of cAMP were expressed as a percent of the difference of these two groups of wells. Concentration-response data including EC50 (the concentration of compound producing 50% of the maximal response) and intrinsic activity (the percent maximal activation compared to full activation by CP55,940) were determined using a four-parameter non-linear regression algorithm (Xlfit equation 251, IDBS). Assays were performed with at least three replicates per compound and averaged results for the several compounds of the invention are shown in Table I.
This test identifies compounds which exhibit analgesic activity against visceral pain or pain associated with activation of low pH-sensitive nociceptors [see Barber and Gottschlich (1986) Med. Res. Rev. 12: 525-562; Ramabadran and Bansinath (1986) Pharm. Res. 3: 263-270]. Intraperitoneal administration of dilute acetic acid solution causes a writhing behavior in mice. A writhe is defined as a contraction of the abdominal muscles accompanied by an extension of the forelimbs and elongation of the body. The number of writhes observed in the presence and absence of test compounds is counted to determine the analgesic activity of the compounds.
Male ICR mice, 20-40 grams in weight, served as experimental subjects. To determine the activity and potency of test compounds, mice were weighed and then injected with different doses of the compound solution or vehicle subcutaneously in the back of the neck and then returned to their home cages. Thirty minutes later, body temperatures were obtained using a digital rectal probe and then mice were immediately injected with 10 ml/kg of a 0.6% (v/v) acetic acid solution into the right lower quadrant of the abdomen. Mice were then placed in individual observation chambers (usually a 4000 ml beaker) with a fine layer of bedding at the bottom and the recording of the number of writhes begun immediately. The number of writhes was counted over a 15-min period starting from the time of the acetic acid injection. Raw data were analyzed using a one-way ANOVA followed by Dunnett's post-tests. For dose-response analysis, raw data were converted to % maximum possible effect (% MPE) using the formula:
% MPE=((Wc−Wv)/(0−Wv))*100
where Wc is the number of writhes in compound-treated mice and Wv is the mean number of writhes in vehicle-treated mice. The dose which elicited 50% attenuation of hypersensitivity (ED50) was determined using linear regression analysis. (Tallarida & Murray, 1987).
Results in the mouse acetic acid-induced writhing assay for compounds 3b, 34 and 46 are shown in Table II.
This application claims the benefit of U.S. provisional patent application Ser. No. 60/904,644 filed Mar. 2nd, 2007 the specification of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60904644 | Mar 2007 | US |
