专利摘要:

公开号:AU2003284342A1
申请号:U2003284342
申请日:2003-10-22
公开日:2004-05-25
发明作者:Stephen L. Buchwald;Michael Heffernan;Roman V. Rariy;Timothy M. Swager
申请人:Collegium Pharmaceutical Inc;
IPC主号:A61K
专利说明:
WO 2004/039320 PCT/US2003/033681 STEREOISOMERS OF p-HYDROXY-MILNACIPRAN, AND METHODS OF USE THEREOF Background of the Invention 5 Efficacy and tolerability are important factors determining the choice of a medication for treatment of mental depression and other mental disorders including Functional Somatic Disorders. The move from tricyclic antidepressants (TCAs) to selective serotonin reuptake inhibitors (SSRIs) involved not only the loss of the direct receptor interactions responsible for the adverse side effects of TCAs, but also the ability to inhibit 10 the reuptake of norepinephrine. Selectivity for the single neurotransmitter, serotonin, may explain why SSRIs tend to be less efficacious than the TCAs, especially in more serious forms of depression (Lopez-Ibor J. et al., 1996, Int. Clin. Psychophann., 11:41-46). Older TCAs are associated with significant behavioral toxicity, notably psychomotor and cognitive impairment and sedation. SSRIs are largely devoid of these effects, but 15 gastrointestinal disturbances such as nausea and dyspepsia are common with these agents (Hindmarch ., 1997, Human Psychopharmacology, 12:115-119). For example, for widely prescribed SSRI sertraline (Zoloft@, Pfizer) the top three adverse events associated with discontinuation of treatment were nausea, insomnia, and diarrhea (Physician's Desk Reference, 57th Edition, 2003, Thomson Medical). 20 Efforts toward improving antidepressant medications are guided by cumulative evidence from neurochemical and clinical studies supporting the therapeutic potential of enhancing monoamine function in depression. A number of antidepressant drugs, serotonin and norepinephrine reuptake inhibitors (SNRIs), including duloxetine, venlafaxine, and milnacipran, have been developed based on their interaction with both serotonin (5-HT) and 25 norepinephrine (NE) receptors. Milnacipran is also often referred to as norepinephrine and serotonin reuptake inhibitor (NSRI) since its norepinephrine ("NE") to serotonin ("5-HT") ratio is 2:1 (Moret et al., 1985, Neuropharmacology, 24:1211-1219; Palmier et al., 1989, Eur. J. Clin. Pharmacol., 37:235-238). Current clinical evidence suggests that these new agents may offer improved efficacy and/or faster onset of action compared with SSRIs 30 (Tran P.V. et al., 2003, J. Clin. Psychopharmacol., 23:78-86). Recent trials with milnacipran suggest that this compound is effective in relieving pain both associated with, and independent of, depression (Briley M., 2003, Curr. Opin. Investig. Drugs, 4:42-45; Cypress Bioscience Inc., Cypress Bioscience Inc. Announces Final Results of Milnacipran -1- WO 2004/039320 PCT/US2003/033681 Phase II Clinical Trial in Fibromyalgia, Media Release, March 21, 2003, Available from: URL: http://www.cypressbio.com). Milnacipran and methods for its synthesis are described in U.S. Patent No. 4,478,836. Milnacipran (midalcipran, midacipran, F 2207) inhibits the uptake of both, 5 norepinephrine (NE) and serotonin (5-HT), with an NE to 5-HT ratio of 2:1 (Moret et al., 1985, Neuropharmacology, 24:1211-1219; Palmier et al., 1989, Eur. J. Clin. Pharmacol., 37:235-238) but does not affect the uptake of dopamine. Milnacipran has no affinity for alpha or beta adrenergic, muscarinic, histaminergic, and dopaminergic receptors. This suggests that milnacipran has a low potential to produce anticholinergic, sedative, and 10 stimulant effects. Milnacipran does not affect the number of beta adrenoceptors in rat cortex after chronic administration (Briley M. et al., Int. Clin. Psychopharmac., 1996, 11:10-14). Additional information regarding milnacipran may be found in the Merck Index, 12th Edition, at entry 6281. Milnacipran (Ixel@, Pierre Fabre), has demonstrated numerous adverse reactions in 15 human clinical trials with tolerability decreasing with increasing dose (Puech A. et al., 1997, Int. Clin. Psychopharm., 12:99-108). In the double-blind, randomized, multicenter clinical study the most frequent spontaneously reported adverse events for 100 mg/day milnacipran twice daily were as follows: abdominal pain (13%), constipation (10%), and headache (9%). Interestingly, when in the same study milnacipran was given 200 mg/day 20 twice daily, pain related adverse reactions decreased (headache to 8% and abdominal pain to 7%) but nausea and vomiting were more pronounced side effects and were reported by 7% of the patients (Guelfi J.D., 1998, Int. Clin. Psychopharm., 13:121-128). In a double blind comparative study involving 219 elderly patients with depression the only adverse event reported more frequently for milnacipran recipients than for TCA imipramine 25 recipients was nausea. Patients received either milnacipran or imipramine 75-100 mg/day twice daily for 8 weeks (Tignol J. et al., 1998, Acta Psychiatrica Scandinavica, 97:157 165). It was also observed that when milnacipran was administered intravenously to 10 patients, five of them reported transient nausea. Nausea was primarily reported at the moment of peak of milnacipran plasma level (Caron J. et al., 1993, Eur. 30 Neuropsychopharmacol., 3:493-500). This study clearly demonstrates that nausea is directly correlated with the milnacipran blood plasma concentration. In addition, it strongly suggests that the nausea can be a centrally mediated side effect since the drug was given intravenously in this study. Data from other studies suggest that milnacipran may also -2- WO 2004/039320 PCT/US2003/033681 induce a locally mediated nausea via gastric irritation (the rapid onset of the nausea was observed even prior to achieving peak plasma levels). The incidence of spontaneously reported milnacipran adverse experiences in placebo-controlled clinical trials is given in Figure 63 (adverse effect is listed if frequency 5 was more than 2% in milnacipran 100 mg/day group). As it can be clearly seen from data presented in Figure 63, the incidence of certain adverse events increases with dosage, including nausea, vomiting, sweating, hot flashes, palpitations, tremor, anxiety, dysuria, and insomnia. It is important to note that in one of the early depression trials, even after one week 10 of milnacipran dose escalation employed to reduce side effects, the most commonly reported reason for discontinuation of treatment because of adverse effects was nausea and vomiting (Leinonen E., 1997, Acta Psychiatr. Scand., 96:497-504). In the recent fibromyalgia clinical trial with the long dose escalation period (four weeks) which was implemented in order to reduce milnacipran side effects and increase patient's tolerance, 15 the most common dose-related side effect reported by patients was nausea (Cypress Bioscience Inc., Cypress Bioscience Inc. Announces Final Results of Milnacipran Phase II Clinical Trial in Fibromyalgia, Media Release, March 21, 2003). The data presented in Figure 63 demonstrates that the currently immediate available release formulation of milnacipran is not suitable for the treatment of health conditions that 20 require milnacipran doses equal or above 100 mg/day given either as once a day or twice a day due to high incidence of treatment-emergent side effects that leads to poor patient's tolerance. Higher doses are required in the treatment of severe depression and other associated disorders. As shown in one of the early antidepressant clinical trials, milnacipran dosage of 200 mg/day was superior to the lower doses (Von Frenckell R et al., 25 1990, Int. Clin. Psychopharmacology., 5:49-56). Milnacipran dosing regime of 100-250 mg daily was recently reported for the treatment of fibromyalgia (US Patent No. 6,602,911). It would be very difficult to reach the upper limits of the dose range using the currently available formulation due to the dose related treatment emergent side effects and the need to titrate over a long period to reach the required dose. 30 The (+)-dextro enantiomer of milnacipran (F2695, (+)-1S,2R-milnacipran) is roughly twice as active in inhibiting norepinephrine and serotonin reuptake as the racemic mixture. See Viazzo et al. Tetrahedron Lett. 1996, 37, 4519-4522; Deprez et al. Eur. J. -3- WO 2004/039320 PCT/US2003/033681 Drug Metab. Pharmacokinet. 1998, 23, 166-171. Moreover, the (-)-levro enantiomer of milnacipran (F2696, (-)-lR,2S-milnacipran) is much less potent. See id. In sum, although milnacipran is reasonably effective in treating major depressive episodes, more efficacious methods are needed to treat effectively major depressive 5 episodes and other mental disorders including Functional Somatic Disorders. Summary of the Invention One aspect of the present invention relates to the enantiomers of para-hydroxy milnacipran or congeners thereof Biological assay studies revealed that (+)-para-hydroxy milnacipran is an approximately two-fold more potent inhibitor of norepinephrine uptake 10 compared to inhibition of serotonin uptake. In contrast, (-)-para-hydroxy-milnacipran is an approximately two-fold more potent inhibitor of serotonin uptake compared to inhibition of norepinephrine uptake. The inhibition properties of each enantiomer ofpara-hydroxy milnacipran stand in contrast to that of the racemic mixture which inhibits serotonin uptake and norepinephrine uptake with approximately equal potency. Another aspect of the 15 present invention relates to salts and prodrug forms of the aforementioned compounds. A third aspect of the present invention relates to methods of treating mammals suffering from various mental disorders including Functional Somatic Disorders, e.g. depression, chronic pain, or fibromyalgia, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of the present invention. Yet another 20 aspect of the present invention relates to formulations comprising a compound of the present invention, and a pharmaceutically acceptable excipient. Brief Description of the Figures Figure 1 depicts the synthetic route used to prepare the individual enantiomers of p hydroxy-milnacipran. 25 Figure 2 depicts a 1H NMR spectrum of lactone CS1590. Figure 3 depicts a 1 3 C NMR spectrum of lactone CS 1590. Figure 4 depicts a 'H NMR spectrum of amide CS1608. Figure 5 depicts a 1 3 C NMR spectrum of amide CS 1608. Figure 6 depicts a 1H NMR spectrum of CS1628. 30 Figure 7 depicts a 1 3 C NMR spectrum of CS1628. Figure 8 depicts a 1H NMR spectrum of CS 1649. Figure 9 depicts a 13 C NMR spectrum of CS1649. -4- WO 2004/039320 PCT/US2003/033681 Figure 10 depicts a HPLC chromatogram of CS 1665 (HPLC Conditions: 10% to 95% acetonitrile within 8 min; 2 min at 95%, LM with 0.1% TFA, Flow: 2.OmL/min, Saule: Zorbax XDB-C8). Figure 11 depicts a mass spectrum of CS 1665. 5 Figure 12 depicts a IH NMR spectrum of CS1665. Figure 13 depicts a 1 3 C NMR spectrum of CS 1665. Figure 14 depicts a HPLC chromatogram of CS1710 (HPLC Conditions: 10% to 95% acetonitrile within 8 min; 2 min at 95%, LM with 0.1% TFA, Flow: 2.0mL/min, Saule: Zorbax XDB-C8). 10 Figure 15 depicts a mass spectrum of CS1710. Figure 16 depicts a 'H NMR spectrum of CS 1710. Figure 17 depicts a 13 C NMR spectrum of CS1710. Figure 18 depicts a HPLC chromatogram of CS1713 (HPLC Conditions: 10% to 95% acetonitrile within 8 min; 2 min at 95%, LM with 0.1% TFA, Flow: 2.OmL/min, Saule: 15 Zorbax XDB-C8). Figure 19 depicts a mass spectrum of CS1713. Figure 20 depicts a 1H NMR spectrum of CS1713. Figure 21 depicts a 1 3 C NMR spectrum of CS1713. Figure 22 depicts a HPLC chromatogram of CS1714 (HPLC Conditions: 10% to 20 95% acetonitrile within 8 min; 2 min at 95%, LM with 0.1% TFA, Flow: 2.OmL/min, Saule: Zorbax XDB-C8). Figure 23 depicts a mass spectrum of CS 1714. Figure 24 depicts a 'H NMR spectrum of CS 1714. Figure 25 depicts a 1 3 C NMR spectrum of CS1714. 25 Figure 26 depicts a HPLC chromatogram of CS1814 (racemic p-Hydroxy Milnacipran Hydrochloride; HPLC Conditions: 10% to 95% acetonitrile within 8 min; 2 min at 95%, LM with 0.1% TFA, Flow: 2.OmL/min, Saule: Zorbax XDB-C8). Figure 27 depicts a LC/MS chromatogram of CS1814. Figure 28 depicts a mass spectrum of selected peaks from the LC/MS 30 chromatogram of CS1814. Figure 29 depicts a mass spectrum of a peak from the LC/MS chromatogram of CS1814. Figure 30 depicts a 'H NMR spectrum of CS 1814. -5- WO 2004/039320 PCT/US2003/033681 Figure 31 depicts a 1 3 C NMR spectrum of CS 1814. Figure 32 depicts biological activity data for CS1814 in assays using receptors from human (hum) and rat. Figure 33 depicts biological activity data for CS1814 in assays using receptors from 5 human (hum), mouse, guinea pig (gp), syrian hamster (syh), and rat. Figure 34 depicts biological activity data for CS1814 in assays using receptors from human (hum) and rat. Figure 35 depicts biological activity data for CS1814 in assays using receptors from human (hum). 10 Figure 36 depicts biological activity data for various reference compounds. Figure 37 depicts biological activity data for various reference compounds. Figure 38 depicts biological activity data for various reference compounds. Figure 39 depicts a graph of % inhibition of Norephinephrine Transporter (NET) by CS1814 (Vial #1). 15 Figure 40 depicts a graph of% inhibition of Serotonin Transporter (SERT) by CS1814 (Vial #1). Figure 41 depicts biological activity data for CS1713 (Vial #2) and CS1714 (Vial #3). Figure 42 depicts biological activity data for CS1713 (Vial #2) and CS1714 (Vial 20 #3). Figure 43 depicts biological activity data for CS1713 (Vial #2) and CS1714 (Vial #3). Figure 44 depicts biological activity data for CS1713 (Vial #2), CS1714 (Vial #3), and CS1814 (Vial #1). 25 Figure 45 depicts biological activity data for CS1713 (Vial #2), CS1714 (Vial #3), and CS1814 (Vial #1). Figure 46 depicts biological activity data for CS1714 (Vial #3). Figure 47 depicts a graph of% inhibition of Norepinephrine Uptake by CS1814 (CEL-1) and Desipramine. 30 Figure 48 depicts a graph of % inhibition of Serotonin Uptake by CS1814 (CEL-1) and Fluoxetine. Figure 49 depicts a graph of% inhibition of Norepinephrine Transporter by CS 1713 (CEL-3) and Desipramine. -6- WO 2004/039320 PCT/US2003/033681 Figure 50 depicts a graph of % inhibition of Serotonin Transporter by CS 1713
(CEL
3) and GBR-12909. Figure 51 depicts a graph of % inhibition of Norepinephrine Uptake by CS1713 5 (CEL 3) and Desipramine. Figure 52 depicts a graph of % inhibition of Serotonin Uptake by CS1713 (CEL 3) and Fluoxetine. Figure 53 depicts a graph of % inhibition of Norepinephrine Transporter by 10 CS1714 (CEL-5) and Desipramine. Figure 54 depicts a graph of % inhibition of Serotonin Transporter by CS 1714 (CEL-5) and GBR-12909. Figure 55 depicts a graph of % inhibition of Norepinephrine Uptake by CS1714 (CEL-5) and Desipramine. 15 Figure 56 depicts a graph of % inhibition of Serotonin Uptake by CS 1714 (CEL-5) and Fluoxetine. Figure 57 depicts cellular assay data for reference compounds. Figure 58 depicts cellular assay data for reference compounds. Figure 59 depicts a summary of significant primary results for CS1814. 20 Figure 60 depicts a summary of significant primary results for CS1713 (Vial #2) and CS1714 (Vial #3). Figure 61 depicts a summary of significant primary results for CS1814 (Vial #1), CS1713 (Vial #2) and CS1714 (Vial #3). Figure 62 depicts a summary of secondary results for CS1814 (Vial #1), CS1713 25 (Vial #2) and CS1714 (Vial #3). Figure 63 depicts incidences of spontaneously reported milnacipran adverse experiences in placebo-controlled clinical trials. Detailed Description of the Invention Definitions 30 For convenience, certain terms employed in the specification, examples, and appended claims are collected here. The term "milnacipran" refers to the racemic mixture of the tri-substituted cyclopropane depicted below. -7- WO 2004/039320 PCT/US2003/033681 H Et 2 N
NH
2 0 Racemic milnacipran The term "ED 50 " means the dose of a drug which produces 50% of its maximum response or effect. Alternatively, the dose which produces a pre-determined response in 50% of test subjects or preparations. 5 Compounds refered to in the specification and figures are identified using a six character alpha-numeric code. For example, racemic p-hydroxy-milnacipran is CS1814. In certain instances, the six-character alpha-numeric code is followed by forward slash and a number. The forward slash followed by a number indicates the batch from which the data was taken. For example, CS 1814/1 indicates that the compound is p-hydroxy-milnacipran 10 and the data was taken from batch 1. The term "LD 5 0 " means the dose of a drug which is lethal in 50% of test subjects. The term "therapeutic index" refers to the therapeutic index of a drug defined as LD5 0
/ED
5 0 The term "structure-activity relationship (SAR)" refers to the way in which altering 15 the molecular structure of drugs alters their interaction with a receptor, enzyme, etc. The term "agonist" refers to a compound that mimics the action of natural transmitter or, when the natural transmitter is not known, causes changes at the receptor complex in the absence of other receptor ligands. The term "antagonist" refers to a compound that binds to a receptor site, but does 20 not cause any physiological changes unless another receptor ligand is present. The term "competitive antagonist" refers to a compound that binds to a receptor site; its effects can be overcome by increased concentration of the agonist. The term "partial agonist" refers to a compound that binds to a receptor site but does not produce the maximal effect regardless of its concentration. 25 The term "inverse agonist" refers to a compound that binds to a constitutively active receptor site and reduces its physiological function. The term "ligand" refers to a compound that binds at the receptor site. -8- WO 2004/039320 PCT/US2003/033681 The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium. The term "electron-withdrawing group" is recognized in the art, and denotes the 5 tendency of a substituent to attract valence electrons from neighboring atoms, i.e., the substituent is electronegative with respect to neighboring atoms. A quantification of the level of electron-withdrawing capability is given by the Hammett sigma (a) constant. This well known constant is described in many references, for instance, J. March, Advanced Organic Chemistry, McGraw Hill Book Company, New York, (1977 edition) pp. 251-259. 10 The Hammett constant values are generally negative for electron donating groups (c[P] = 0.66 for NH 2 ) and positive for electron withdrawing groups (a[P] = 0.78 for a nitro group), a[P] indicating para substitution. Exemplary electron-withdrawing groups include nitro, acyl, formyl, alkylsulfonyl, arylsulfonyl, trifluoromethyl, cyano, chloride, and the like. Exemplary electron-donating groups include amino, methoxy, and the like. 15 The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Cl-C 30 for straight chain, C 3
-C
30 for branched chain), and more 20 preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group). The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in 25 length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower 30 alkenyl" and "lower alkynyl" have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl. The term "aryl" as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, -9- WO 2004/039320 PCT/US2003/033681 furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described 5 above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, alkylsulfonyl, arylsulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF 3 , -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two 10 or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls. The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ortho-dimethylbenzene are 15 synonymous. The terms "heterocyclyl" or "heterocyclic group" refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, 20 phcnoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, 25 thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, 30 alkylthio, alkylsulfonyl, arylsulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF 3 , -CN, or the like. The terms "polycyclyl" or "polycyclic group" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more -10- WO 2004/039320 PCT/US2003/033681 carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, 5 imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, alkylsulfonyl, arylsulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF 3 , -CN, or the like. As used herein, the term "nitro" means -N02; the term "halogen" designates -F, -Cl, -Br or -I; the term "sulfhydryl" means -SH; the terms "hydroxy" and "hydroxyl" mean -OH; 10 and the term "sulfonyl" means -SO 2 -. The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: R R '10 10 1+ -N -N- Ric R or 9 R9 wherein R9, R 10 and R' 10 each independently represent a group permitted by the rules of 15 valence. The term "acylamino" is art-recognized and refers to a moiety that can be represented by the general formula: 0
-N
1 1 R',I R, wherein R9 is as defined above, and R'l I represents a hydrogen, an alkyl, an alkenyl or 20 -(CH2)m-R8, where m and R8 are as defined above. The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula: 0 N- R9 Rio wherein R9, R 10 are as defined above. Preferred embodiments of the amide will not 25 include imides which may be unstable. -11- WO 2004/039320 PCT/US2003/033681 The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2)m-R8, wherein m and R8 are defined above. Representative alkylthio groups include methylthio, ethyl thio, and the like. 5 The term "carbonyl" is art recognized and includes such moieties as can be represented by the general formula: 0 0 1L X-R.R , or -X R'11 wherein X is a bond or represents an oxygen or a sulfur, and RI I represents a hydrogen, an alkyl, an alkenyl, -(CH2)m-R8 or a pharmaceutically acceptable salt, R'I i represents a 10 hydrogen, an alkyl, an alkenyl or -(CH 2 )m-R8, where m and R8 are as defined above. Where X is an oxygen and RI I or R'l I is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R 1 I is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when RI I is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen, and R'l I is hydrogen, the formula represents a 15 "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where X is a sulfur and RI I or R'l I is not hydrogen, the formula represents a "thiolester." Where X is a sulfur and RI I is hydrogen, the formula represents a "thiolcarboxylic acid." Where X is a sulfur and Rl l' is hydrogen, the formula represents a "thiolformate." On the other hand, where X is a bond, and R 1 1 is 20 not hydrogen, the above formula represents a "ketone" group. Where X is a bond, and RI I is hydrogen, the above formula represents an "aldehyde" group. The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons 25 covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -0-alkyl, -0 alkenyl, -0-alkynyl, -O-(CH2)m-R 8 , where m and R 8 are described above. The term "sulfonate" is art recognized and includes a moiety that can be represented by the general formula: -12- WO 2004/039320 PCT/US2003/033681 0 II
-S-R
4 1 || in which R 4 1 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl. The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and 5 nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively. The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, 10 trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations 15 utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference. The term "sulfate" is art recognized and includes a moiety that can be represented by the general formula: 0 II -0-S-OR 41 20 in which R 4 1 is as defined above. The term "sulfonylamino" is art recognized and includes a moiety that can be represented by the general formula: 0 -N-S-R R The term "sulfamoyl" is art-recognized and includes a moiety that can be 25 represented by the general formula: -13- WO 2004/039320 PCT/US2003/033681 0 11 -S-N o Ra The term "sulfonyl", as used herein, refers to a moiety that can be represented by the general formula: 0 11 -S-R44 5 in which R 4 4 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. The term "sulfoxido" as used herein, refers to a moiety that can be represented by the general formula: 0 I I
-
S-R 4 10 in which R 4 4 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl. A "selenoalkyl" refers to an alkyl group having a substituted seleno group attached thereto. Exemplary "selenoethers" which may be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and -Se-(CH2)m-R7, m and R 7 being defined 15 above. Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls. As used herein, the definition of each expression, e.g. alkyl, m, n, etc., when it 20 occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which 25 does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include -14- WO 2004/039320 PCT/US2003/033681 acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this 5 invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds. The phrase "protecting group" as used herein means temporary substituents which 10 protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2 "d ed.; Wiley: New York, 1991). 15 Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an 20 alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention. If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group 25 cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure 30 enantiomers. Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof (e.g., functioning as analgesics), wherein one or more simple variations of substituents are -15- WO 2004/039320 PCT/US2003/033681 made which do not adversely affect the efficacy of the compound in binding to sigma receptors. In general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional 5 synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. 10 Synthesis of Individual Enanteriomers of p-Hydroxy-Milnacipran The condensation reaction of 4 -methoxybenzylcyanide and enantiomerically pure epichlorhydrin (Figure 1), which are both commercially available, gave access to the corresponding lactones CS1590 and CS1591 in satisfactory yield. Subsequent opening of the lactone in the presence of lithiumdiethylamide, generated from n-butyllithium and 15 diethylamine, furnished CS1608 and the corresponding enantiomer, respectively. Conversion of the primary alcohols CS1608 and CS1609 to the azides CS1628 and C1648 was accomplished in a pot procedure by in situ generation of the corresponding mesylates followed by nucleophilic displacement with sodium azide. Following this protocol the desired azides were obtained in 36 - 40% yield. Subsequent removal of the protecting group 20 was carried out in the presence of borontribromide at -30 *C for 48 h and produced the deprotected phenols CS1649 and CS1658 in 66% yield. Final reduction of the azide moiety in CS1649 and CS1658 under standard reaction conditions furnished the desired target compounds CSI 665 and CS171 0. Preparation of the corresponding hydrochloric acid salts was accomplished by using hydrochloric acid in dioxane and subsequent removal of the 25 solvent. Methods for the Resolution of Enantiomers One alternative procedure for the isolation of an individual enantiomer is by resolution of an enantiomer from a racemic mixture. Today, chiral separations of cationic drugs by capillary electrophoresis are generally carried out by adding negatively charged 30 cyclodextrins (CDs) to the running buffer, while anionic or neutral drug separations require the use of dual-CD systems (mixtures of neutral and charged CDs). Chiral separation of some basic drugs (idazoxan, efaroxan, milnacipran) has been studied by mixtures of sulfated-p--CD (S-PCD) and hydroxypropl-y-CD (HP-y-CD). The influence of the -16- WO 2004/039320 PCT/US2003/033681 following parameters (nature and concentration of neutral CD, concentration of S-p-CD) on many separation factors (electrophoretic mobility, selectivity, efficiency, asymmetry factor, resolution) demonstrated that dual-CD systems are useful for chiral separation of basic drugs in order to improve the symmetry of the second-migrating enantiomer. Indeed, the 5 neutral CD reduces the extent of electromigration dispersion by mobility tuning. Finally, the 0.5 mg/mL S-p-CD/5 mg/mL HP-y-CD dual system has allowed the chiral separation of idazoxan, efaroxan and milnacipran enantiomers in less than 9 min. See generally Grard, S. et al. Electrophoresis 2000, 21, 3028-3034. Biological Activity Analysis 10 The results from the biological testing of CS1814, CS1713, CS1714, and various reference compounds are presented in Figures 32-62. CS1814 (Vial #1), CS1713 (Vial #2), and CS1714 (Vial #3) were evaluated in various radioligand binding assays, and for inhibition of CYP450 3A4 at initial concentrations of 10 pM. As depicted in Figures 59 and 60, significant activity (250%) was observed 15 for displacement of radioligand from Serotonin Transporter binding sites (Vial #1 Ki = 6.73 nM, Vial #2 Ki = 3.88 nM, Vial #3 Ki = 8.15 nM) and Norepinephrine Transporter binding sites (Vial #1 Ki = 0.218 pM, Vial #2 Ki = 0.112 pM, Vial #3 Ki = 1.68 pM). In addition, CS1713 (Vial #2), CS1714 (Vial #3), and CS1814 (Vial #1) 20 were evaluated for inhibition of cellular Serotonin and Norepinephrine Uptake. As depicted in Figure 61, CS 1814 (Vial #1) is approximately equipotent in inhibiting serotonin and norepinephrine uptake (IC 5 0 = 28.6 nM for norepinephrine, IC 50 = 21.7 nM for serotonin). Interestingly, CS 1713 (Vial #2) is a more potent inhibitor of norepinephrine uptake than serotonin uptake (IC 50 = 25 10.3 nM for norepinephrine, IC 50 = 22 nM for serotonin). In contrast, CS1714 (Vial #3) is a more potent inhibitor of serotonin uptake compared to norepinephrin uptake (IC 50 = 88.5 nM for norepinephrine, IC 50 = 40.3 nM for serotonin). The fact that CS1713 (Vial #2) is a more potent inhibitor of norepinephrine uptake would render it a superior therapeutic agent for treating 30 diseases linked to norepinephrine uptake. In addition, the CS1714 (Vial #3) would useful for treating conditions requiring selective inhibition of serotonin uptake. -17- WO 2004/039320 PCT/US2003/033681 Importantly, no cytotoxicity was observed for CS1713 (Vial #2), CS1714 (Vial #3), or CS1814 (Vial #1) at 10 pM. In addition, CS1814 (Vial #1) is a selective inhibitor of norepinephrine and serotonin receptors. The fact that CS1814 generally does not bind well to other receptors, as depicted in 5 Figures 32 and 33, substantially reduces the risk of negative side effects associated with administering the compound to a patient. Therefore, it is likely that CS1713 and CS1714 will not have detrimental side effects. Compounds & Methods of the Invention In certain embodiments, a compound of the present invention is an isolated 10 compound represented by A: R R
R
1
R
1 RR
R
3 -N R1N/ R3 X R1 R A wherein X represents independently for each occurrence 0, S, or NR; R represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, 15 heteroaryl, arylalkyl, formyl, acyl, silyl, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m-Rso; RI represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, 20 alkylamino, arylamino, arylakylamino, sulfhydryl, alkylthio, arylthio, arylakylthio, nitro, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m-Ro;
R
2 represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, 25 heteroaryl, arylalkyl, or -(CH2)m-Ro;
R
3 represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, or -(CH2)m-Ro; -18- WO 2004/039320 PCT/US2003/033681 R4 is absent or present between one and four times inclusive;
R
4 , if present, represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, arylakylamino, sulfhydryl, alkylthio, arylthio, 5 arylakylthio, nitro, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m R8o;
R
80 represents independently for each occurrence an aryl, cycloalkyl, cycloalkenyl, 10 heterocyclyl, or polycyclyl moiety; m is independently for each occurrence an integer in the range 0 to 8 inclusive; and the compound is a single enantiomer; or a pharmaceutically acceptable salt or prodrug thereof. 15 In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents 0. In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein R represents H. In certain embodiments, the compounds of the present invention are represented by 20 A and the attendant definitions, wherein R 1 represents H. In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein R 2 represents H. In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein R 3 represents alkyl. 25 In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein R4 is absent. In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents 0; and R represents H. In certain embodiments, the compounds of the present invention are represented by 30 A and the attendant definitions, wherein X represents 0; R represents H; and R' represents H. -19- WO 2004/039320 PCT/US2003/033681 In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents 0; R represents H; R, represents H; and R2 represents H. In certain embodiments, the compounds of the present invention are represented by 5 A and the attendant definitions, wherein X represents 0; R represents H; RI represents H;
R
2 represents H; and R 3 represents alkyl. In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents 0; R represents H; R, represents H; R2 represents H; R 3 represents alkyl; and R 4 is absent. 10 In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents 0; R represents H; R1 represents H; R2 represents H; R 3 represents ethyl; and R 4 is absent. In an assay based on a mammalian GPCR, certain compounds according to structure 15 A have IC 50 values less than 10 pM, more preferably less than 1 gM, even more preferably less than 100 nM, and most preferably less than 10 nM. In an assay based on a mammalian GPCR, certain compounds according to structure A have EC 5 0 values less than 10 pM, more preferably less than 1 PM, even more preferably less than 100 nM, and most preferably less than 10 nM. 20 In certain embodiments, compounds according to structure A are effective in the treatment of a mammal suffering from depression. In certain embodiments, compounds according to structure A are effective in the treatment of a mammal suffering from fibromyalgia syndrome. In certain embodiments, compounds according to structure A are effective in the 25 treatment of a mammal suffering from mental disorders including Functional Somatic Disorders, for example, depression, fibromyalgia syndrome, chronic fatigue syndrome, pain, attention deficit/hyperactivity disorder, and visceral pain syndromes (VPS), such as irritable bowel syndrome (IBS), noncardiac chest pain (NCCP), functional dyspepsia, interstitial cystitis, essential vulvodynia, urethral syndrome, orchialgia, and affective 30 disorders, including depressive disorders (major depressive disorder, dysthymia, atypical depression) and anxiety disorders (generalized anxiety disorder, phobias, obsessive compulsive disorder, panic disorder, post-traumatic stress disorder), premenstrual dysphoric -20- WO 2004/039320 PCT/US2003/033681 disorder, temperomandibular disorder, atypical face pain, migraine headache, and tension headache. In certain embodiments, a compound of the present invention is an isolated 5 compound represented by B: xR 1
R
1 R R R R 2R R
R
3 -N N R Ri R 3 X R1 R B wherein X represents independently for each occurrence 0, S, or NR; R represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, 10 heteroaryl, arylalkyl, formyl, acyl, silyl, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m-Ro; R I represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, 15 alkylamino, arylamino, arylakylamino, sulfhydryl, alkylthio, arylthio, arylakylthio, nitro, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m-Ro;
R
2 represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, 20 heteroaryl, arylalkyl, or -(CH2)m-R8o;
R
3 represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, or -(CH2)m-R8o;
R
4 is absent or present between one and four times inclusive;
R
4 , if present, represents independently for each occurrence H, alkyl, cycloalkyl, 25 alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, arylakylamino, sulfhydryl, alkylthio, arylthio, -21- WO 2004/039320 PCT/US2003/033681 arylakylthio, nitro, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m Rao; 5 R 8 0 represents independently for each occurrence an aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl moiety; m is independently for each occurrence an integer in the range 0 to 8 inclusive; and the compound is a single enantiomer; or a pharmaceutically acceptable salt or prodrug thereof. 10 In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents 0. In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein R represents H. 15 In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein R' represents H. In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein R 2 represents H. In certain embodiments, the compounds of the present invention are represented by 20 B and the attendant definitions, wherein R represents alkyl. In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein R 4 is absent. In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents 0; and R represents H. 25 In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents 0; R represents H; and RI represents H. In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents 0; R represents H; R' represents H; 30 and R 2 represents H. In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents 0; R represents H; R' represents H;
R
2 represents H; and R 3 represents alkyl. -22- WO 2004/039320 PCT/US2003/033681 In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents 0; R represents H; R' represents H; R2 represents H; R3 represents alkyl; and R 4 is absent. In certain embodiments, the compounds of the present invention are represented by 5 B and the attendant definitions, wherein X represents 0; R represents H; R1 represents H;
R
2 represents H; R 3 represents ethyl; and R 4 is absent. In an assay based on a mammalian GPCR, certain compounds according to structure B have IC 50 values less than 10 pM, more preferably less than 1 pM, even more preferably 10 less than 100 nM, and most preferably less than 10 nM. In an assay based on a mammalian GPCR, certain compounds according to structure B have EC 5 o values less than 10 M, more preferably less than 1 gM, even more preferably less than 100 nM, and most preferably less than 10 nM. In certain embodiments, compounds according to structure B are effective in the 15 treatment of a mammal suffering from depression. In certain embodiments, compounds according to structure B are effective in the treatment of a mammal suffering from fibromyalgia syndrome. In certain embodiments, compounds according to structure B are effective in the treatment of a mammal suffering from mental disorders including Functional Somatic 20 Disorders, for example, depression, fibromyalgia syndrome, chronic fatigue syndrome, pain, attention deficit/hyperactivity disorder, and visceral pain syndromes (VPS), such as irritable bowel syndrome (IBS), noncardiac chest pain (NCCP), functional dyspepsia, interstitial cystitis, essential vulvodynia, urethral syndrome, orchialgia, and affective disorders, including depressive disorders (major depressive disorder, dysthymia, atypical 25 depression) and anxiety disorders (generalized anxiety disorder, phobias, obsessive compulsive disorder, panic disorder, post-traumatic stress disorder), premenstrual dysphoric disorder, temperomandibular disorder, atypical face pain, migraine headache, and tension headache. In certain embodiments, the compound of the invention is selected from the group 30 1S, 2R 1-(4-Methoxy-phenyl9-3-oxa-bicyclo[3.1.0]hexan-2-one (CS1590), 1R, 2S 1-(4 Methoxy-phenyl9-3-oxa-bicyclo[3.1.0]hexan-2-one (CS1591),IS, 2R 2-Hydroxymethyl-1 (4-methoxy-phenyl)-cyclopropanecarboxylic acid diethylamide (CS1608), 1R, 2S 2 Hydroxymethyl- 1 -(4-methoxy-phenyl)-cyclopropanecarboxylic acid diethylamide -23- WO 2004/039320 PCT/US2003/033681 (CS 1609), iS, 2R 2-Azidomethyl- 1 -(4-methoxy-phenyl)-cyclopropanecarboxylic acid diethylamide (CS 1628), 1 R, 2S 2-Azidomethyl- I -(4-methoxy-phenyl) cyclopropanecarboxylic acid diethylamide (CS1648), 1S, 2R 2-Azidomethyl-1-(4 hydroxy-phenyl)-cyclopropanecarboxylic acid diethylamide (CS 1649), 1 R, 2S 2 5 Azidomethyl- 1 -(4-hydroxy-phenyl)-cyclopropanecarboxylic acid diethylamide (CS 1658), iS, 2R 2-Aminomethyl-1-(4-hydroxy-phenyl)-cyclopropanecarboxylic acid diethyl amide (CS1665), 1R, 2S 2-Aminomethyl-1-(4-hydroxy-phenyl)-cyclopropanecarboxylic acid diethyl amide (CS1710), and racemic 2-Aminomethyl-1-(4-hydroxy-phenyl) cyclopropanecarboxylic acid diethyl amide (CS1814). 10 In certain embodiments, the present invention relates to a formulation, comprising a compound represented by any of the structures outlined above; and a pharmaceutically acceptable excipient. In certain embodiments, the compounds of this invention can be administered adjunctively with other active compounds such as analgesics, anti-inflammatory drugs, 15 antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators, anti asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastro intestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, antinarcoleptic, and anorectics. 20 Specific examples of compounds that can be adjunctively administered with the compounds of this invention include, but are not limited to, aceclofenac, acetaminophen, adomexetine, almotriptan, alprazolam, amantadine, amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine, amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, azatadine, beclomethasone, benactyzine, benoxaprofen, bermoprofen, 25 betamethasone, bicifadine, bromocriptine, budesonide, buprenorphine, bupropion, buspirone, butorphanol, butriptyline, caffeine, carbamazepine, carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine, choline salicylate, citalopram, clomipramine, clonazepam, clonidine, clonitazene, clorazepate, clotiazepam, cloxazolam, clozapine, codeine, corticosterone, cortisone, cyclobenzaprine, cyproheptadine, demexiptiline, 30 desipramine, desomorphine, dexamethasone, dexanabinol, dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, dezocine, diazepam, dibenzepin, diclofenac sodium, diflunisal, dihydrocodeine, dihydroergotamine, dihydromorphine, dimetacrine, divalproxex, dizatriptan, dolasetron, donepezil, dothiepin, doxepin, duloxetine, ergotamine, -24- WO 2004/039320 PCT/US2003/033681 escitalopram, estazolam, ethosuximide, etodolac, femoxetine, fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine, flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan, gabapentin, galantamine, gepirone, ginko bilboa, granisetron, haloperidol, huperzine A, hydrocodone, hydrocortisone, hydromorphone, hydroxyzine, 5 ibuprofen, imipramine, indiplon, indomethacin, indoprofen, iprindole, ipsapirone, ketaserin, ketoprofen, ketorolac, lesopitron, levodopa, lipase, lofepramine, lorazepam, loxapine, maprotiline, mazindol, mefenamic acid, melatonin, melitracen, memantine, meperidine, meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone, methamphetamine, methocarbamol, methyldopa, methylphenidate, methylsalicylate, 10 methysergid(e), metoclopramide, mianserin, mifepristone, milnacipran, minaprine, mirtazapine, moclobemide, modafinil, molindone, morphine, morphine hydrochloride, nabumetone, nadolol, naproxen, naratriptan, nefazodone, neurontin, nomifensine, nortriptyline, olanzapine, olsalazine, ondansetron, opipramol, orphenadrine, oxaflozane, oxaprazin, oxazepam, oxitriptan, oxycodone, oxymorphone, pancrelipase, parecoxib, 15 paroxetine, pemoline, pentazocine, pepsin, perphenazine, phenacetin, phendimetrazine, phenmetrazine, phenylbutazone, phenytoin, phosphatidylserine, pimozide, pirlindole, piroxicam, pizotifen, pizotyline, pramipexole, prednisolone, prednisone, pregabalin, propanolol, propizepine, propoxyphene, protriptyline, quazepam, quinupramine, reboxitine, reserpine, risperidone, ritanserin, rivastigmine, rizatriptan, rofecoxib, ropinirole, rotigotine, 20 salsalate, sertraline, sibutramine, sildenafil, sulfasalazine, sulindac, sumatriptan, tacrine, temazepam, tetrabenozine, thiazides, thioridazine, thiothixene, tiapride, tiasipirone, tizanidine, tofenacin, tolmetin, toloxatone, topiramate, tramadol, trazodone, triazolam, trifluoperazine, trimethobenzamide, trimipramine, tropisetron, valdecoxib, valproic acid, venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone, zolmitriptan, zolpidem, 25 zopiclone and isomers, salts, and combinations thereof. By adjunctive administration is meant simultaneous administration of the compounds, in the same dosage form, simultaneous administration in separate dosage forms, and separate administration of the compounds. In certain embodiments, the present invention relates to ligands for a GPCR, e.g., a 30 receptor for a neurotransmitter, wherein the ligands are represented by a structure outlined above, and any of the sets of definitions associated with a structure. In certain embodiments, the ligands of the present invention are antagonists, agonists, partial agonists or inverse agonists of a GPCR. In certain preferred embodiments, the ligands of the present -25- WO 2004/039320 PCT/US2003/033681 invention are antagonists of the reuptake of serotonin or norepinephrine or both. In any event, the ligands of the present invention preferably exert their effect on a GPCR at a concentration less than about 10 micromolar, more preferably less than about 1 micromolar, even more preferably at a concentration less than about 100 nanomolar, and most preferably 5 at a concentration less than 10 nanomolar. In certain preferred embodiments, the ligands of the present invention are antagonists of a the reuptake of serotonin or norepinephrine or both at a concentration less than about 10 micromolar, more preferably less than about 1 micromolar, even more preferably at a concentration less than about 100 nanomolar, and most preferably at a concentration less than 10 nanomolar. 10 The compounds of the invention are indicated for use in the treatment of inflammatory, immunological, bronchopulmonary, cardiovascular, oncological or CNS degenerative disorders; preferably for oral or topical treatment of inflammatory and/or immunological disorders, such as the oral or topical treatment of airway diseases involving inflammatory conditions, e.g. asthma, bronchitis; or atopic diseases, e.g. rhinitis or atopic 15 dermatitis; inflammatory bowel diseases, e.g. Crohn's disease or colitis; autoimmune diseases e.g. multiple sclerosis, diabetes, atherosclerosis, psoriasis, systemic lupus erythematosus or rheumatoid arthritis; malignant diseases, e.g. skin or lung cancer; HIV infections or AIDS; or for inhibiting rejection of organs/transplants. The compounds of the invention are also indicated for use in treatment of heart failure, and in treatment of diabetic 20 patients with macular edema or diabetic retinopathy. One embodiment of the invention is the treatment of a patient having inflammatory pain. For example, administration of certain kinase inhibitors significantly diminishes both acute and chronic hyperalgesia resulting from exposure to the inflammatory agent carrageenan; moreover, administration of certain kinase inhibitors diminishes hyperalgesia 25 due to diabetes, chemotherapy or traumatic nerve injury. Such inflammatory pain may be acute or chronic and can be due to any number of conditions characterized by inflammation including, without limitation, sunburn, rheumatoid arthritis, osteoarthritis, colitis, carditis, dermatitis, myositis, neuritis and collagen vascular diseases. In addition, administration of a compound of the present invention to a subject immediately prior to, during or after an 30 inflammatory event can ameliorate both the acute pain and the chronic hyperalgesia that the subject would otherwise experience. Another preferred embodiment of the invention is the treatment of a patient having neuropathic pain. Such patients can have a neuropathy classified as a radiculopathy, -26- WO 2004/039320 PCT/US2003/033681 mononeuropathy, mononeuropathy multiplex, polyneuropathy or plexopathy. Diseases in these classes can be caused by a variety of nerve-damaging conditions or procedures, including, without limitation, trauma, stroke, demyelinating diseases, abscess, surgery, amputation, inflammatory diseases of the nerves, causalgia, diabetes, collagen vascular 5 diseases, trigeminal neuralgia, rheumatoid arthritis, toxins, cancer (which can cause direct or remote (e.g. paraneoplastic) nerve damage), chronic alcoholism, herpes infection, AIDS, and chemotherapy. Nerve damage causing hyperalgesia can be in peripheral or CNS nerves. This embodiment of the invention is based on the fact that administration of certain kinase inhibitors significantly diminishes hyperalgesia due to diabetes, chemotherapy or traumatic 10 nerve injury. Preferred embodiments of the present invention include a composition combining a compound of the present invention with one or more additional pain-reducing agents and a method of administering such a composition. An individual pain medication often provides only partially effective pain alleviation because it interferes with just one pain-transducing 15 pathway out of many. Alternatively, a compound of the present invention can be administered in combination with a pain-reducing (analgesic) agent that acts at a different point in the pain perception process. Fibromyalgia syndrome is a chronic and debilitating condition characterized by widespread pain and stiffness throughout the body, accompanied by severe fatigue and 20 headache. It affects an estimated 2%-4% of the population worldwide and is the second most common diagnosis by rheumatologists in the United States, after osteoarthritis. Despite the high prevalence and severity of this syndrome, there are no approved treatments specifically for FMS in the United States or elsewhere. Another preferred embodiment of the present invention relates to treating fibromyalgia by adminstering a therapeutically 25 effective amount of a compound of the present invention to a mammal in need thereof. Prodrugs and Intermediates It will be appreciated by those skilled in the art that, although certain protected derivatives of the compounds of the present invention, which may be made prior to a final deprotection stage, may not possess pharmacological activity as such, they may be 30 administered parenterally or orally and thereafter metabolized in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as "prodrugs". Moreover, certain compounds of the present invention may act as prodrugs of other compounds of the present invention. Critically, all prodrugs -27- WO 2004/039320 PCT/US2003/033681 of compounds of the present invention are included within the scope of the present invention. Novel intermediates as described hereinbefore and their use in the manufacture of other compounds of the present invention also form part of the invention. Pharmaceutical Compositions 5 In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for 10 administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, 15 a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. The phrase "therapeutically-effective amount" as used herein means that amount of 20 a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of 25 sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid 30 filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some -28- WO 2004/039320 PCT/US2003/033681 examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) 5 excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; 10 (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. As set out above, certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming 15 pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term "pharmaceutically-acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form 20 with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. 25 (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19) The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, 30 sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2 -29- WO 2004/039320 PCT/US2003/033681 acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts 5 with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, 10 carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, 15 ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra) Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the 20 compositions. Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, 25 propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The 30 formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount -30- WO 2004/039320 PCT/US2003/033681 of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 5 70 per cent, most preferably from about 10 per cent to about 30 per cent. In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned 10 formulation renders orally bioavailable a compound of the present invention. Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention 15 with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non 20 aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste. 25 In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, 30 carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary -31- WO 2004/039320 PCT/US2003/033681 ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of 5 capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more 10 accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid 15 diluent. The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or 20 controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which 25 can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The 30 active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. Extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in "Remington - The science and practice of pharmacy" -32- WO 2004/039320 PCT/US2003/033681 (20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). A diffusion system typically consists of two types of devices, reservoir and matrix, and is well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of 5 materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethyleellulose, and carbopol 934, 10 polyethylene oxides. Fatty compounds include, but are not limited to, various waxes such as camauba wax and glyceryl tristearate. Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable 15 coating materials in suitable proportion. The devices with different drug release mechanisms described above could be combined in a final dosage form comprising single or multiple units. Examples of multiple units include multilayer tablets, capsules containing tablets, beads, granules, etc. An immediate release portion can be added to the extended release system by means 20 of either applying an immediate release layer on top of the extended release core using coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads. Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry 25 granulation processes. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as any of many different kinds of starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for 30 example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including -33- WO 2004/039320 PCT/US2003/033681 acacia, alginates, methylcellulose, and polyvinylpyrrolidine can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium 5 stearate, stearic acid and hydrogenated vegetable oils. Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In a congealing method, the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed. 10 Delayed release formulations are created by coating a solid dosage form with a film of a polymer which is insoluble in the acid environment of the stomach, and soluble in the neutral environment of small intestines. The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing 15 composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and/or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as 20 will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic 25 polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed 30 from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename Eudragit*. (Rohm Pharma; Westerstadt, Germany), including Eudragit*. L30D-55 and L1OO-55 (soluble at pH 5.5 and above), Eudragit. L-100 (soluble -34- WO 2004/039320 PCT/US2003/033681 at pH 6.0 and above), Eudragit*. S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and Eudragits*. NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability); vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid 5 copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied. The preferred coating weights for particular coating materials may be readily 10 determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies. The coating composition may include conventional additives, such as plasticizers, 15 pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. % to 50 wt. % relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, 20 castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution. One 25 effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone), may also be added to the coating composition. Alternatively, a delayed release tablet may be formulated by dispersing the drug 30 within a matrix of a suitable material such as a hydrophilic polymer or a fatty compound. The hydrophilic polymers may be comprised of polymers or copolymers of cellulose, cellulose ester, acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, and vinyl or enzymatically degradable polymers or copolymers as described above. These hydrophilic -35- WO 2004/039320 PCT/US2003/033681 polymers are particularly useful for providing a delayed release matrix. Fatty compounds for use as a matrix material include, but are not limited to, waxes (e.g. camauba wax) and glycerol tristearate. Once the active ingredient is mixed with the matrix material, the mixture can be compressed into tablets. 5 Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl 10 carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as 15 wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and 20 tragacanth, and mixtures thereof. Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a 25 suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations 30 containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a -36- WO 2004/039320 PCT/US2003/033681 pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, 5 paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain 10 customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can 15 also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel. Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. 20 Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, 25 antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, 30 vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. -37- WO 2004/039320 PCT/US2003/033681 These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, 5 and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the 10 absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished 15 by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include 20 poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in 25 combination with a pharmaceutically acceptable carrier. The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or 30 inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually -38- WO 2004/039320 PCT/US2003/033681 by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. 5 The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. 10 These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. Regardless of the route of administration selected, the compounds of the present 15 invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention niay be varied so as to obtain an amount of the active ingredient which is 20 effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion 25 or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine 30 and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to -39- WO 2004/039320 PCT/US2003/033681 achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such 5 an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.000 1 to about 100 mg per kilogram of body weight per day. If desired, the effective daily dose of the active compound may be administered as 10 two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). 15 In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the subject compounds, as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially 20 formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical 25 application, for example, as a cream, ointment or spray applied to the skin, lungs, or oral cavity; or (4) intravaginally or intravectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other 30 pharmaceuticals. The term "treatment" is intended to encompass also prophylaxis, therapy and cure. -40- WO 2004/039320 PCT/US2003/033681 The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general. The compound of the invention can be administered as such or in admixtures with 5 pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered. 10 The addition of the active compound of the invention to animal feed is preferably accomplished by preparing an appropriate feed premix containing the active compound in an effective amount and incorporating the premix into the complete ration. Alternatively, an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed. The way in which such feed premixes and 15 complete rations can be prepared and administered are described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A., 1969 or "Livestock Feeds and Feeding" 0 and B books, Corvallis, Ore., U.S.A., 1977). Combination Therapy The compounds of the invention may be administered to a patient in combination 20 with one or more therapeutic agents. The complementary drug or drugs may be mixed with the primary drug and formulated into a single tablet, pill, capsule, or solution for parenteral administration, and the like. Alternatively, the primary drug and complimentary drug may be administered via separate compositions, e.g. separate tablets or solutions. The primary drug may be administered at the same time as the complementary drug or the primary drug 25 may be administered intermittently with the complementary drug. The dosage of the complementary drug will generally be dependent upon a number of factors including the health of the patient being treated, the extent of treatment desired, the nature and kind of concurrent therapy, if any, and the frequency of treatment and the nature of the effect desired. In general, dosage ranges of the complementary drugs often range from about 30 0.001 to about 250 mg/kg body weight per day. For a normal adult having a body weight of about 70 kg, a dosage in the range of from about 0.1 to about 25 mg/kg body weight is typically preferred. However, some variability in this general dosage range may be required depending upon the age and weight of the subject being treated, the intended route of -41- WO 2004/039320 PCT/US2003/033681 administration, the particular agent being administered and the like. Since two or more different active agents are being used together in a combination therapy, the potency of each agent and the interactive effects achieved using them together must be considered. However, the determination of dosage ranges and optimal dosages for a particular mammal 5 is also well within the ability of one of ordinary skill in the art having the benefit of the instant disclosure. In certain embodiments, the compounds of this invention can be administered adjunctively with other active compounds such as analgesics, anti inflammatory drugs, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxioltyics, sedatives, hypnotics, antipsychotics, 10 bronchodilators, anti asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastro-intestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, antinarcoleptic, and anorectics. Another embodiment of the present invention relates to a combination therapy comprising the compounds of the invention and one or more compounds that inhibit the 15 serotonin transporter, norepinephrine transporter, or both. The relative proportion of the therapeutic agents in the combination therapy is selected to achieve a specific level of inhibition for the serotonin transporter and the norepinephrine transporter. For example, in certain embodiments it may be beneficial to treat a patient using a combination therapy that inhibits the serotonin transporter and the norepinephrine transporter with equal potency. 20 However, in certain embodiments, it may be beneficial to treat a patient using a combination therapy that inhibits the serotonin transporter to a greater extent than the norepinephrine transporter. For example, in certain embodiments, the ratio of inhibition of the serotonin transporter relative to the norepinephrine transporter is two, four, six, or ten. Alternatively, in certain embodiments, the ratio of inhibition of the norepinephrine 25 transporter relative to the serotonin transporter is two, four, six, or ten. In certain embodiments, the combination therapy comprises two therapeutic agents. However, the combination therapy could include more than two therapeutic agents, e.g. three, four, five, etc. In certain embodiments, the combination therapy comprises a selective serotonin 30 reuptake inhibitor (SSRI) and compound of formula A or B. In certain embodiments, the combination therapy comprises a selective norepinephrine reuptake inhibitor (SNRI) and compound of formula A or B. In certain embodiments, the combination therapy comprises a SSRI, a SNRI, and a compound of formula A or B. In certain embodiments, the -42- WO 2004/039320 PCT/US2003/033681 combination therapy comprises at least one of the compounds of the invention, a SSRI, and a SNRI. In certain embodiments, a SNRI is milnacipran. In certain embodiments, the combination therapy comprises CS1713 and a SSRI. In certain embodiments, the combination therapy comprises CS1713 and a SNRI. In certain 5 embodiments, the combination therapy comprises CS1713, a SSRI, and a SNRI. In certain embodiments, the invention relates to the above-mentioned combination therapies of CS1713 which further comprise CS1814. In certain embodiments, the combination therapy comprises CS1714 and a SSRI. In certain embodiments, the combination therapy comprises CS1714 and a SNRI. In certain 10 embodiments, the combination therapy comprises CS1714, a SSRI, and a SNRI. In certain embodiments, the invention relates to the above-mentioned combination therapies of CS1714 which further comprise CS1814. In certain embodiments, the combination therapy comprises CS1814 and a SSRI. In certain embodiments, the combination therapy comprises CS1814 and a SNRI. In certain 15 embodiments, the combination therapy comprises CS1814, a SSRI, and a SNRI. Combinatorial Libraries The subject compounds readily lend themselves to preparation using the methods of combinatorial chemistry, providing access to combinatorial libraries of compounds for the screening of pharmaceutical, agrochemical or other biological or medically-related activity 20 or material-related qualities. A combinatorial library for the purposes of the present invention is a mixture of chemically related compounds which may be screened together for a desired property; said libraries may be in solution or covalently linked to a solid support. The preparation of many related compounds in a single reaction greatly reduces and simplifies the number of screening processes which need to be carried out. Screening for 25 the appropriate biological, pharmaceutical, agrochemical or physical property may be done by conventional methods. Diversity in a library can be created at a variety of different levels. For instance, the substrate aryl groups used in a combinatorial approach can be diverse in terms of the core aryl moiety, e.g., a variegation in terms of the ring structure, and/or can be varied with 30 respect to the other substituents. A variety of techniques are available in the art for generating combinatorial libraries of small organic molecules. See, for example, Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Patents 5,359,115 and 5,362,899: the Ellman U.S. Patent -43- WO 2004/039320 PCT/US2003/033681 5,288,514: the Still et al. PCT publication WO 94/0805 1; Chen et al. (1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publications W092/10092, W093/09668 and W091/07087; and the Lerner et al. PCT publication W093/20242). Accordingly, a variety of libraries on the order of about 16 to 1,000,000 or more diversomers can be 5 synthesized and screened for a particular activity or property. In an exemplary embodiment, a library of substituted diversomers can be synthesized using the subject reactions adapted to the techniques described in the Still et al. PCT publication WO 94/0805 1, e.g., being linked to a polymer bead by a hydrolyzable or photolyzable group, e.g., located at one of the positions of substrate. According to the Still 10 et al. technique, the library is synthesized on a set of beads, each bead including a set of tags identifying the particular diversomer on that bead. In one embodiment, which is particularly suitable for discovering enzyme inhibitors, the beads can be dispersed on the surface of a permeable membrane, and the diversomers released from the beads by lysis of the bead linker. The diversomer from each bead will diffuse across the membrane to an 15 assay zone, where it will interact with an enzyme assay. Detailed descriptions of a number of combinatorial methodologies are provided below. A. Direct Characterization A growing trend in the field of combinatorial chemistry is to exploit the sensitivity of techniques such as mass spectrometry (MS), e.g., which can be used to characterize sub 20 femtomolar amounts of a compound, and to directly determine the chemical constitution of a compound selected from a combinatorial library. For instance, where the library is provided on an insoluble support matrix, discrete populations of compounds can be first released from the support and characterized by MS. In other embodiments, as part of the MS sample preparation technique, such MS techniques as MALDI can be used to release a 25 compound from the matrix, particularly where a labile bond is used originally to tether the compound to the matrix. For instance, a bead selected from a library can be irradiated in a MALDI step in order to release the diversomer from the matrix, and ionize the diversomer for MS analysis. B) Multipin Synthesis 30 The libraries of the subject method can take the multipin library format. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS 81:3998-4002) introduced a method for generating compound libraries by a parallel synthesis on polyacrylic acid-grated polyethylene pins arrayed in the microtitre plate format. The Geysen technique can be used -44- WO 2004/039320 PCT/US2003/033681 to synthesize and screen thousands of compounds per week using the multipin method, and the tethered compounds may be reused in many assays. Appropriate linker moieties can also been appended to the pins so that the compounds may be cleaved from the supports after synthesis for assessment of purity and further evaluation (c.f., Bray et al. (1990) 5 Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem 197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166). C) Divide-Couple-Recombine In yet another embodiment, a variegated library of compounds can be provided on a set of beads utilizing the strategy of divide-couple-recombine (see, e.g., Houghten (1985) i PNAS 82:5131-5135; and U.S. Patents 4,631,211; 5,440,016; 5,480,971). Briefly, as the name implies, at each synthesis step where degeneracy is introduced into the library, the beads are divided into separate groups equal to the number of different substituents to be added at a particular position in the library, the different substituents coupled in separate reactions, and the beads recombined into one pool for the next iteration. 15 In one embodiment, the divide-couple-recombine strategy can be carried out using an analogous approach to the so-called "tea bag" method first developed by Houghten, where compound synthesis occurs on resin sealed inside porous polypropylene bags (Houghten et al. (1986) PNAS 82:5131-5135). Substituents are coupled to the compound bearing resins by placing the bags in appropriate reaction solutions, while all common steps 20 such as resin washing and deprotection are performed simultaneously in one reaction vessel. At the end of the synthesis, each bag contains a single compound. D) Combinatorial Libraries by Light-Directed, Spatially Addressable Parallel Chemical Synthesis A scheme of combinatorial synthesis in which the identity of a compound is given 25 by its locations on a synthesis substrate is termed a spatially-addressable synthesis. In one embodiment, the combinatorial process is carried out by controlling the addition of a chemical reagent to specific locations on a solid support (Dower et al. (1991) Annu Rep Med Chem 26:271-280; Fodor, S.P.A. (1991) Science 251:767; Pirrung et al. (1992) U.S. Patent No. 5,143,854; Jacobs et al. (1994) Trends Biotechnol 12:19-26). The spatial 30 resolution of photolithography affords miniaturization. This technique can be carried out through the use protection/deprotection reactions with photolabile protecting groups. The key points of this technology are illustrated in Gallop et al. (1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared for coupling through the covalent -45- WO 2004/039320 PCT/US2003/033681 attachment of photolabile nitroveratryloxycarbonyl (NVOC) protected amino linkers or other photolabile linkers. Light is used to selectively activate a specified region of the synthesis support for coupling. Removal of the photolabile protecting groups by light (deprotection) results in activation of selected areas. After activation, the first of a set of 5 amino acid analogs, each bearing a photolabile protecting group on the amino terminus, is exposed to the entire surface. Coupling only occurs in regions that were addressed by light in the preceding step. The reaction is stopped, the plates washed, and the substrate is again illuminated through a second mask, activating a different region for reaction with a second protected building block. The pattern of masks and the sequence of reactants define the 10 products and their locations. Since this process utilizes photolithography techniques, the number of compounds that can be synthesized is limited only by the number of synthesis sites that can be addressed with appropriate resolution. The position of each compound is precisely known; hence, its interactions with other molecules can be directly assessed. In a light-directed chemical synthesis, the products depend on the pattern of 15 illumination and on the order of addition of reactants. By varying the lithographic patterns, many different sets of test compounds can be synthesized simultaneously; this characteristic leads to the generation of many different masking strategies. E) Encoded Combinatorial Libraries In yet another embodiment, the subject method utilizes a compound library provided 20 with an encoded tagging system. A recent improvement in the identification of active compounds from combinatorial libraries employs chemical indexing systems using tags that uniquely encode the reaction steps a given bead has undergone and, by inference, the structure it carries. Conceptually, this approach mimics phage display libraries, where activity derives from expressed peptides, but the structures of the active peptides are 25 deduced from the corresponding genomic DNA sequence. The first encoding of synthetic combinatorial libraries employed DNA as the code: A variety of other forms of encoding have been reported, including encoding with sequenceable bio-oligomers (e.g., oligonucleotides and peptides), and binary encoding with additional non-sequenceable tags. 1) Tagging with sequenceable bio-oligomers 30 The principle of using oligonucleotides to encode combinatorial synthetic libraries was described in 1992 (Brenner et al. (1992) PNAS 89:5381-5383), and an example of such a library appeared the following year (Needles et al. (1993) PNAS 90:10700-10704). A combinatorial library of nominally 77 (= 823,543) peptides composed -46- WO 2004/039320 PCT/US2003/033681 of all combinations of Arg, Gln, Phe, Lys, Val, D-Val and Thr (three-letter amino acid code), each of which was encoded by a specific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively), was prepared by a series of alternating rounds of peptide and oligonucleotide synthesis on solid support. In this work, the amine linking functionality on 5 the bead was specifically differentiated toward peptide or oligonucleotide synthesis by simultaneously preincubating the beads with reagents that generate protected OH groups for oligonucleotide synthesis and protected NH 2 groups for peptide synthesis (here, in a ratio of 1:20). When complete, the tags each consisted of 69-mers, 14 units of which carried the code. The bead-bound library was incubated with a fluorescently labeled antibody, and 10 beads containing bound antibody that fluoresced strongly were harvested by fluorescence activated cell sorting (FACS). The DNA tags were amplified by PCR and sequenced, and the predicted peptides were synthesized. Following such techniques, compound libraries can be derived for use in the subject method, where the oligonucleotide sequence of the tag identifies the sequential combinatorial reactions that a particular bead underwent, and 15 therefore provides the identity of the compound on the bead. The use of oligonucleotide tags permits exquisitely sensitive tag analysis. Even so, the method requires careful choice of orthogonal sets of protecting groups required for alternating co-synthesis of the tag and the library member. Furthermore, the chemical lability of the tag, particularly the phosphate and sugar anomeric linkages, may limit the 20 choice of reagents and conditions that can be employed for the synthesis of non-oligomeric libraries. In preferred embodiments, the libraries employ linkers permitting selective detachment of the test compound library member for assay. Peptides have also been employed as tagging molecules for combinatorial libraries. Two exemplary approaches are described in the art, both of which employ 25 branched linkers to solid phase upon which coding and ligand strands are alternately elaborated. In the first approach (Kerr JM et al. (1993) J Am Chem Soc 115:2529-2531), orthogonality in synthesis is achieved by employing acid-labile protection for the coding strand and base-labile protection for the compound strand. In an alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170), 30 branched linkers are employed so that the coding unit and the test compound can both be attached to the same functional group on the resin. In one embodiment, a cleavable linker can be placed between the branch point and the bead so that cleavage releases a molecule containing both code and the compound (Ptek et al. (1991) Tetrahedron Lett 32:3891 -47- WO 2004/039320 PCT/US2003/033681 3894). In another embodiment, the cleavable linker can be placed so that the test compound can be selectively separated from the bead, leaving the code behind. This last construct is particularly valuable because it permits screening of the test compound without potential interference of the coding groups. Examples in the art of independent cleavage 5 and sequencing of peptide library members and their corresponding tags has confirmed that the tags can accurately predict the peptide structure. 2) Non-sequenceable Tagging: Binary Encoding An alternative form of encoding the test compound library employs a set of non sequencable electrophoric tagging molecules that are used as a binary code (Ohlmeyer et al. 10 (1993) PNAS 90:10922-10926). Exemplary tags are haloaromatic alkyl ethers that are detectable as their trimethylsilyl ethers at less than femtomolar levels by electron capture gas chromatography (ECGC). Variations in the length of the alkyl chain, as well as the nature and position of the aromatic halide substituents, permit the synthesis of at least 40 such tags, which in principle can encode 240 (e.g., upwards of 1012) different molecules. 15 In the original report (Ohlmeyer et al., supra) the tags were bound to about 1% of the available amine groups of a peptide library via a photocleavable o-nitrobenzyl linker. This approach is convenient when preparing combinatorial libraries of peptide-like or other amine-containing molecules. A more versatile system has, however, been developed that permits encoding of essentially any combinatorial library. Here, the compound would be 20 attached to the solid support via the photocleavable linker and the tag is attached through a catechol ether linker via carbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem 59:4723-4724). This orthogonal attachment strategy permits the selective detachment of library members for assay in solution and subsequent decoding by ECGC after oxidative detachment of the tag sets. 25 Although several amide-linked libraries in the art employ binary encoding with the electrophoric tags attached to amine groups, attaching these tags directly to the bead matrix provides far greater versatility in the structures that can be prepared in encoded combinatorial libraries. Attached in this way, the tags and their linker are nearly as unreactive as the bead matrix itself. Two binary-encoded combinatorial libraries have been 30 reported where the electrophoric tags are attached directly to the solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-603 1) and provide guidance for generating the subject compound library. Both libraries were constructed using an orthogonal attachment strategy in which the library member was linked to the solid support by a photolabile linker and the tags were -48- WO 2004/039320 PCT/US2003/033681 attached through a linker cleavable only by vigorous oxidation. Because the library members can be repetitively partially photoeluted from the solid support, library members can be utilized in multiple assays. Successive photoelution also permits a very high throughput iterative screening strategy: first, multiple beads are placed in 96-well 5 microtiter plates; second, compounds are partially detached and transferred to assay plates; third, a metal binding assay identifies the active wells; fourth, the corresponding beads are rearrayed singly into new microtiter plates; fifth, single active compounds are identified; and sixth, the structures are decoded. Toxicological Assessments 10 During the drug development process, potential therapeutic agents or drug candidates must be demonstrated to be both safe and effective for their intended use. In drug development processes, potential drug candidates are subjected to toxicology assessments in an effort to demonstrate safety. In general, following contact of a compound with a population, the effect of the 15 compound on the members of the population is determined. The effect of the compound on the members of the population is generally determined by evaluating one or more of a number of different phenotypic parameters. Phenotypic parameters that are evaluated in a given assay of the subject invention may vary widely depending, at least in part, on the nature of the multi-cellular organisms being employed. Typically, phenotypic parameters 20 that are evaluated in any given assay include one or more of the following: (1) viability; (2) morphological defects; and (3) fecundity. Specific parameters that may be evaluated include one or more of: (1) lethal dose, e.g. LD.sub.50, LD.sub.10 etc.); (2) growth defects; (3) sterility effect dose; (4) developmental defects; (5) neurologic impairment; (5) life-span modulation, e.g. life span enhancing or shortening; and the like. 25 A number of different types of non-mammalian multi-cellular organisms may be employed in toxicological assessments, where these types of organisms include insects, amphibians, fish, and the like. Specific organisms of interest include: Xenopus, Zebrafish, Caenerhabditis, Drosophila and the like. Of particular interest are invertebrate animals, particularly members of the phylum arthropoda, and more particularly members of the class 30 insecta. Of particular interest are flies. For example, flies of the family Drosophilidae, where the animal is often a Drosophila melanogaster. The multi-cellular organisms employed may be at any stage of their life, e.g. in the larval stage, in the adult stage, etc. -49- WO 2004/039320 PCT/US2003/033681 For example, a compound is brought into contact with a population of multi-cellular organisms in a manner such that the compound is capable of exerting activity on at least a substantial portion of, if not all of, the individual organisms of the population. By substantial portion is meant at least 40 number %, usually at least 50 number % and more 5 usually at least 60 number %, where the number % may be substantially higher and can be as high as 80, 90 or 95 number % or higher. Generally, each compound is contacted with the members of the population in a manner such that it is internalized by the organisms. Typically internalization will be by ingestion, i.e. orally, such that that each compound will generally be contacted with the plurality of organisms by incorporating the compound in 10 the nutrient medium, e.g. water, aqueous solution of additional nutrient agents, etc., of the organisms. For example, where the multi-cellular organism is a fly, the candidate agent is generally orally administered to the fly by mixing the agent into the fly nutrient medium and placing the medium in the presence of the fly, (either the larva or adult fly, usually the larva) such that the fly feeds on the medium. 15 In addition to the above parameters, the gene expression levels of the test organisms can be assayed, e.g. gene expression levels in treated larva, pupa, and/or flies can be evaluated. The genes can be from "housekeeping" genes that provide basic metabolic information to developmental and tissue specific genes to gauge which tissue or cell type is affected and when. A variety of different gene expression protocols, including arrays based 20 protocols, are known to those of skill in the art, including those described in: EP 0 328 829 BI and U.S. Pat. Nos. 5,468,613; 5,580,726; 5,599,672; 5,512,462; 5,162,209 and 5,162,209, the disclosures of which are herein incorporated by reference. Methods of analyzing differential gene expression are also described in Maniatis, et al., Molecular Cloning, A Laboratory Manual, (Cold Spring Harbor Press, Cold Spring Harbor, 25 N.Y.)(1989); Nucleic Acid Hybridization, A Practical Approach (Hames, B. D., and Higgins, S. J. eds, IRL Press, Oxford)(1985); WO 95/21944; Chalifour, et al., Anal. Biochem. (1994) 216: 299-304; Nguyen et al., Genomics (1995) 29: 207-216; Pietu et al., Genome Res. (1996) 6: 492-503; and Zhao et al., Gene (1995) 166: 207-213. The effect of a compound on a particular physical parameter or parameters being 30 evaluated may be determined manually or robotically, such that in many embodiments determination of the effect of the compound on the organism is accomplished via an automated procedure. -50- WO 2004/039320 PCT/US2003/033681 The effect of the compound on the phenotypic parameter or parameters is then related to the toxicity of the compound. As such, the effect on the phenotypic parameter(s) is employed to derive a toxicity profile for the assayed compound, where toxicity profile refers to the toxic activity of a given compound, i.e. its collection of one or more toxic 5 activities, such as lethality, sterility causing activity, etc. Fly Modelfor Toxicology A candidate chemical is dissolved in water at or near its saturation point. Serial dilutions of this stock solution are used to rehydrate instant fly media (Fisher Scientific). Specifically, one toxicity assay will comprise of instant fly media rehydrated with pure 10 stock solution of a chemical, while another will be rehydrated with a 10% solution of the chemical (in water). This format will be used to generate data over a 4 to 5 log dose range for each chemical tested. A known quantity of embryos, typically between 40-50, is used as the input. Specifically, 40 to 50 embryos are counted and placed in the receptacle that contains the 15 media/chemical mixture to be tested. The embryos may be counted manually or by automation (e.g., liquid suspension of embryos flowing through a diode). The larva feed on the media/chemical mixture. All aspects of development from larva stage to adult must proceed normally in the presence of the chemical. The only food and water source available to the larva and flies contains the chemical. It is shown that the variability of and intake 20 amount that can be expected using this protocol by feeding the larva chemicals that are easy to assay. Specifically, iron, copper, and zinc have been selected. Sensitive and accurate kits are commercially available to analyze these chemicals down to a concentration of I part per million. This will assign quantitative analyses to determine variability between larva in a test receptacle and between larva in different receptacles. 25 The developing larva and pupa are examined for normal growth and development. Then the adult flies are analyzed for lethality, sterility, developmental defects, and life span alterations. Lethality is determined by dividing the number of adult flies that enclose by the total number of embryos that were placed in the receptacle. Sterility is examined for both males and females by crossing them to normal flies. A physical examination of the adults 30 reveals any visible defects, such as limb defects, tissue formation defects, abnormal coordination etc. Finally the flies are allowed to live the natural span of their life to determine whether an effect occurred to either shorten or lengthen the average lifespan of the fly. -51- WO 2004/039320 PCT/US2003/033681 Exemplification The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit 5 the invention. Example 1 Synthesis of CS1590 and CS1591 A 200 mL three neck round bottom flask, equipped with a stir bar, a thermometer and a gas adapter was charged with 4-methoxyphenylacetonitrile (9.38 g, 63.76 mmol) and benzene 10 (70 mL). The reaction mixture was allowed to cool to 0 *C followed by the addition of sodium amide (4.97 g, 127.5 mmol) and stirred an additional 2 hours at this temperature. After this period of time (R)-epichlorhydrine (5.9 g, 63.76 mmol) was added and the resulting reaction mixture was stirred overnight, the solvent was reduced under reduced pressure and the residue was dissolved in ethanol (50 mL) and aqueous potassium 15 hydroxide (Imol/L, 40 mL). The solution was then heated to reflux overnight followed by addition of concentrated hydrochloric acid to adjust the pH=1. The aqueous phase was extracted with tert.-butylmethylether (200 mL) and ethyl aceate (200 ml). The organic phases were combined, washed with sat. sodium chloride, dried (MgSO4) and the solvent was reduced under reduced pressure to afford crude CS1590 which was purified by column 20 chromatography on silica gel using ethyl acetate/dichloromethane 1:4 as an fluent. The fractions containing the desired product were combined and reduced under reduced pressure to afford CS1590 (5.42 g, 41.7%) as an off white solid. In a similar fashion the desired enantiomer CS1591 was synthesized. Example 2 25 Synthesis of CS1608 and CS1609 A 200 mL three neck round bottom flask, equipped with a stir bar, a thermometer and a gas adapter was charged with n-butyllithium (1.6 mol/L, 29.8 mL, 47.7 mmol), cooled to 0 *C followed by the addition of diethylamine (3.49 g, 47.7 mmol). The solution was stirred for 20 min, allowed to cool to -78 C followed by the addition of a solution of CS1590 (6.08 g, 30 29.8 mmol) in tetrahydrofuran (50 mL). The reaction mixture was allowed to warm to room temperature overnight, followed by quenching the reaction mixture into an aqueous saturated solution of ammonium chloride (200 ml) and subsequent extraction with ethyl acetate. The organic phase was separated , dried (MgSO4) and the solvent was reduced -52- WO 2004/039320 PCT/US2003/033681 under reduced pressure to afford crude CS1608 (8.10 g, 98%) which was used without further purification for the next step. In a similar fashion the desired enantiomer CS1609 was synthesized. Example 3 5 Synthesis of CS1628 and CS1648 A 200 mL three neck round bottom flask, equipped with a stir bar, a thermometer and a gas adapter was charged with CS1608 (5.6 g, 20.19 mmol) and N,N-dimethylformamide (20 mL), cooled to 0 *C, followed by the addition of sodium azide (5.2 g, 80.76 mmol), triethylamine (10.2 g, 100.95 mmol) and methanesulfochloride (4.6 g, 60.57 mmol). The 10 suspension was stirred for 24 hours at room temperature, quenched into water (200 ml-) and extracted with ethylacetate (2 x 200 mL), dried (MgS04) and the solvent was reduced under reduced pressure to afford crude CS1628, which was purified by column chromatography on silica gel using heptane/ethyl actetate 5:1 as an eluent to afford CS1628 (2.2 g, 36%) as an off white solid. 15 In a similar fashion the desired enantiomer CS1648 was synthesized. Example 4 Synthesis of CS1649 and CS1658 A 50 mL three neck round bottom flask, equipped with a stir bar, a thermometer and a gas adapter was charged with CS 1628 (2.2 g, 7.27 mmol) and dichloromethane (200 mL), 20 cooled to -35 *C followed by the addition of a solution borontribromide in dichloromethane (1.0 mol/L, 21.8 mL, 21.8 mmol). The reaction mixture was kept for 48 h at -28 'C, cooled back to -40 'C followed by the addition of methanol. The resulting mixture was poured into water (200 mL), extracted with ethyl acetate (2 x 200 mL), dried (MgSO4) and the solvent was reduced under reduced pressure to afford crude CS1649, which was purified by column 25 chromatography on silica gel using heptane/ethyl actetate 2:1 as an eluent to afford CS1649 (1.39 g, 66.5 %) as an off white solid. In a similar fashion the desired enantiomer CS1658 was synthesized. Example 5 Synthesis of CS1665 and CS1710 30 A 200 mL hydrogenation bottle was charged with CS1649 (1.1 g, 3.81 mmol), methanol (50 ml-) and catalytic amounts of Pd/C. The reaction was subjected to 1 bar of hydrogen pressure until full conversion was observed, the reaction mixture was filtered through a pad of Celite and the solvent removed under reduced pressure to afford crude CS1655, which -53- WO 2004/039320 PCT/US2003/033681 was purified by column chromataograph on silica gel using dichloromethane/methanol/triethylamine 10:0.5:0.25 as eluent to afford CS1665 (0.80 g, 80%) as an of white solid. In a similar fashion the desired enantiomer CS1710 was synthesized. 5 Analytical Data for CS 1665 Appearance: Off white solid 'H NMR (MeOd-4): Figure 12 "C{'H} NMR (apt) (MeOd-4): Figure 13 Optical Rotation la]i2D (c 0.5, Methanol): -89 IR (KBr, Neat, Solvent): N/A HIPLC: Purity: 97% @ 220 nm, 100% @ 254 nm Method: Gradient of 10% acetonitrile to 95% acetonitrile over 8 min, equilibrate 2 min at 95%, 0.1% TFA, Flowrate: 2.0 mL/min Column: Zorbax XDB-C8 Elemental Analysis: N/A Mass Spectrum (ESI): m/z = 263 [C 1 5
H
22
N
2 0 2 +H]* Analytical Data for CS 1710 Appearance: Off white solid 'H NMR (MeOd-4): Figure 16 3 C{lH}-NMR (apt) (MeOd-4): Figure 17 Optical Rotation la]i"D (c 0.5, Methanol): +86.2 IR (KBr, Neat, Solvent): N/A HIPLC: Purity: 98% @ 220 nm, 100% @ 254 nm Method: Gradient of 10% acetonitrile to 95% acetonitrile over 8 min, equilibrate 2 min at 95%, 0.1% TFA, Flowrate: 2.0 mL/min Column: Zorbax XDB-C8 Elemental Analysis: N/A Mass Spectrum (ESI): m/z = 263 [Ci 5
H
22
N
2 0 2 +H]f 10 -54- WO 2004/039320 PCT/US2003/033681 Example 6 Synthesis of CS1713 and CS1714 A 10 mL round bottom flask was charged with CS1665 (0.51 g, 1.71 mmol) and hydrochloric acid in dioxane (5 mol/L, 10 mL). The mixture was stirred for 1 hour at room 5 temperature, followed by removal of the solvent under reduced pressure to afford CS1713 (0.43 g, 84%) as an off white solid. In a similar fashion the desired enantiomer CS1714 was synthesized. Analytical Data for CS1713 Appearance: Off white solid 1H NMR (MeOd-4): Figure 20
'
3 C{'H}-NMR (apt) (MeOd-4): Figure 21 Optical Rotation [a]2 0 D (c 0.1, Methanol): +80 IR (KBr, Neat, Solvent): N/A HPLC: Purity: 97% @ 220 nm, 98% @ 254 nm Method: Gradient of 10% acetonitrile to 95% acetonitrile over 8 min, equilibrate 2 min at 95%, 0.1% TFA, Flowrate: 2.0 mL/min Column: Zorbax XDB-C8 Elemental Analysis: N/A Mass Spectrum (ESI): m/z = 263 [C 1 5
H
22
N
2 0 2 +H]* io Analytical Data for CS1714 Appearance: Off white solid 1H NMR (MeOd-4): Figure 24 1 3 C{'H}-NMR (apt) (MeOd-4): Figure 25 Optical Rotation [al2D (c 0.1, Methanol): -74 IR (KBr, Neat, Solvent): N/A HPLC: Purity: 96% @ 220 nm, 100% @ 254 nm Method: Gradient of 10% acetonitrile to 95% acetonitrile over 8 min, equilibrate 2 min at 95%, 0.1% TFA, Flowrate: 2.0 mL/min Column: Zorbax XDB-C8 Elemental Analysis: N/A Mass Spectrum (ESI): m/z = 263 [CsH 22
N
2 0 2 +H]* -55- WO 2004/039320 PCT/US2003/033681 Example 7 Preparation of CS1814 A 10 mL flask equipped with a magnetic stir bar was charged with CS1665/2 (120 mg, 0.46 mmol), CS1710/1 (120 mg, 0.46 mmol) and hydochloric acid in dioxan (5 mol/L, 5 5 ml). The suspension was stirred for 1 hour, reduced under reduced pressure and the residue was again taken up in hydrochloric acid in dioxan (5 mol/L, 1 mL). The suspension was stirred for another hour and reduced under reduced pressure to afford CS1814 (240 mg, quant) as an off white solid. The solid was dissolved in methanol (10 mL, homogenous solution), transferred to a 20 mL round bottom flask and the flask was washed out with 10 additional 5 mL of methanol, combined with the above solution (total volume of approximately 15 mL, homogenous solution) and reduced under reduced pressure to afford CS1814 (240 mg, quant) as an off white solid. The solid was dried under high vacuum. 50 mg of this material were taken and dissolved in methanol (10 ml-) followed by measuring the optical rotation. The solution was later transferred back to the 20 mL flask 15 (homogenous solution) and the solvent removed under reduced pressure. Analytical Data for CS 1814 Appearance: Off white solid 1H NMR (MeOd-4): Figure 30 "C('H}-NMR (apt) (MeOd-4): Figure 31 Optical Rotation [IID (c 0.5, Methanol): 0 racemic IR (KBr, Neat, Solvent): N/A HPLC: Purity: 97% @ 220 nm, 98% @ 254 nm Method: Gradient of 10% acetonitrile to 95% acetonitrile over 8 min, equilibrate 2 min at 95%, 0.1% TFA, Flowrate: 2.0 mL/min Column: Zorbax XDB-C8 Elemental Analysis: N/A Mass Spectrum (ESI): m/z - 263 [C1 5
H
22
N
2 0 2 +H] Example 8 20 Biological Testing of CS1814 and Reference Compounds The results from the biological testing of CS1814 and various reference compounds are presented in Figures 32-40 and 59. The data in Figure 59 indicate that CS 1814 has an -56- WO 2004/039320 PCT/US2003/033681
IC
50 = 0.22 p.M for inhibition of norepinephrine transporter and an IC 50 value of 12.7 nM for inhibition of serotonin transporter. The binding constants for CS 1814 are Ki = 0.218 pM for norepinephrine transporter and Ki = 6.73 nM for serotonin transporter. The methods employed in this study have been adapted from the scientific 5 literature to maximize reliability and reproducibility. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained. Assays were performed under conditions as described below. Literature reference(s) for each assay are tabulated below and hereby incorporated by reference. Where presented, IC 50 values were determined by a non-linear, least squares 10 regression analysis using Data Analysis ToolboXTM (MDL Information Systems, San Leandro, CA, USA). Where inhibition constants (Ki) are presented, the Ki values were calculated using the equation of Cheng and Prusoff (Cheng, Y., Prusoff, W.H., Biochem. Pharmacol. 22:30993108, 1973) using the observed ICso of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the K 1 of 15 the ligand (obtained experimentally at MDS Pharma Services). Where presented, the Hill coefficient (nH), defining the slope of the competitive binding curve, was calculated using Data Analysis ToolboxTM. Hill coefficients significantly different than 1.0, may suggest that the binding displacement does not follow the laws of mass action with a single binding site. Where IC 50 , K;, and/or nH data are presented without Standard Error of the 20 Mean (SEM), data are insufficient to be quantitative, and the values presented (Ki, IC 50 , nH) should be interpreted with caution. Methods: -57- WO 2004/039320 PCT/US2003/033681 U 178050 CYP45, 1A2 Source: Human recombinant Sf9 insect cells Substrate: 5 pM 3-Cyano-7-ethoxycoumarin Vehicle: 0.1 % DMSO Pre-Incubation Timefremp: None Incubation Time/Temp: 30 minutes @ 37 OC Incubation Buffer: 75 mM Potassium Phosphate buffer, pH 7.5 Quantitation Method: Spectrofluorimetric quantitation of 3-Cyano-7-hydroxycoumarin Significance Criteria: 2 50% of max stimulation or inhibition E 118070 CYP450, 2C19 Source: Human recombinant Sf9 insect cells Substrate: 25 pM 3-Cyano-7-ethoxycoumarin Vehicle: 0.1 % DMSO Pre-Incubation Time/Temp: None Incubation Time/Temp: 45 minutes @ 37 OC Incubation Buffer: 75 mM Potassium Phosphate buffer, pH 7.5 Quantitation Method: Spectrofluorimetric quantitation of 3-Cyano-7-hydroxycoumarin Significance Criteria: 50% of max stimulation or inhibition M 118060 CYP450, 2C9 Source: Human recombinant Sf9 insect cells Substrate: 25 pM 3-Cyano-7-ethoxycoumarin Vehicle: 0.1 % DMSO Pre-Incubation Time/Temp: None Incubation Time/Temp: 45 minutes @ 37 DC Incubation Buffer 75 mM Potassium Phosphate buffer. pH 7.5 Quantitation Method: Spectrofluorimetric quantitation of 3-Cyano-7-hydroxyccumarin Significance Criteria: : 50% of max stimulation or inhibition E 118080 CYP450, 2D6 Source: Human recombinant Sf9 insect cells Substrate: 50 pM 3-Cyano-7-ethoxycoumarin Vehicle: 0.1 % DMSO Pre-Incubation Timeremp: None Incubation Time/Temp: 45 minutes @ 37 OC Incubation Buffer: 75 mM Potassium Phosphate buffer, pH 7.5 Quantitation Method: Spectrofluorimetric quantitation of 3-Cyano-7-hydroxycoumarin Significance Criteria: 2 50% of max stimulation or inhibition -58- WO 2004/039320 PCT/US2003/033681 0 118090 cYP450, 3A4 Source: Human recombinant Sf9 insect cells Substrate: 50 pM 7-benzyloxy-4 (trifluoromethyl)-coumarin Vehicle: 0.1 % DM50 Pre-Incubation Time/Temp: None incubation Time/]emp: 30 minutes @ 37 OC InLcubation Buffer 75 mM Potassium Phosphate buffer. pH 7.5 Quantitation Method: Spectrofluorimetric quantitation of 7-Hydroxy-4-(trifluoromethy) coumarin Significance Criteria: a 50% of max stimulation or inhibition M 200510 Adenosine A, Source: Human recombinant CHO cells Ligand: 1 eM [H] DPCPX Vehicle: 1 % DM50 Incubation Time/Temp: 90 minutes @25 OC Incubation Buffer: 20 mM HEPtS pH 7.4,10mM MgCI, 100 MM NaCI NonSpecific Ligand: 100 pM R(-)-PIA Ka: 1.4 nM* -:2.7 pmole/mg Protein Specific Binding: 85% Quantitation Method: Radioligand Binding Significance Criteria: ;! 50% of max stimulation or inhibition 5 0 200610 A denosine A,, 0 203100 Adrenergic ef,, Source: Human recombinamnt HIEK-2u3 cells Source: Wistar Rat submaillary gland Ligand: D-05 PM C 1 HI CGS.280 Ligand1 0.25 nM (nMH] Praosin Vehicle: 1 % DMS0 Vehicle: 1 % DMSO Incubation Timeremp! 90 minutes @ 25 OC Incubasion Time/renp: 60 minus@ 25 S Incubation Buffer: SB mM Tris-C. p" 7.4. 10 mM Incubation Buffer 50 mM TrisHC. 0.5 mM HDTEAP p1 M MgCl,, I MM E1TA, 2 U/nil m 7N Adenoesine DeaminaRe NonSpecific Ligand: I D.111 Phentolamine NonSpecific Ligand: 50 vM NECA K,.: 0.17 em S0,64 tiM I R: 0.18 pnsolmg Protin 7 Pm2le7mg Proteinp 5mol/migPt Binding 90% Specific Binding: 5% Qantitation method-R Radioliadiriignd Binding Quantitation Method: Radliltsd Binding Signsificance Criteria: a: 50% of aw stimulation or Significance Criteria: a 5of ma stimulatn or inhibition inhibition -59- WO 2004/039320 PCT/US2003/033681 M 203200 Adrenergic a,, Source: Wistar Rat liver Ligand: 0.25 nM [1H] Prazosin Vehicle: 1 % DMSO Incubation Time/Temp: 60 minutes @ 25 OC Incubation Buffer: 50 mM Tris-HCI ,0. 5 mM EDTA. pH 7.4 NonSpecific Ligand: 10 pM Phentolamine Kd: 0.31 nM * Bma: 0.18 pmole/mg Protein* Specific Binding' 90% * Quantitation Method: Radioligand Binding Significance Criteria: Z 50% of max stimulation or inhibition 0 203400 Adrenergic a1, Source: Human recombinant HEK-293 cells ligand: 0.6 nM [3H] Prazosin Vehicle- 1 % DMSO Incubation Time/Temp: 60 minutes @ 25 OC Incubation Buffer: 50 mM Tris-HCI NonSpecific Ligand: 10 pM Phentolamine Kd: 0.58 nM * Bm.: 0.17 pmoLe/mg Protein* Specific Binding: 80% * Quantitation Method: Radoligand Binding Significance Criteria: 2 50% of max stimulation or inhibition -60- WO 2004/039320 PCT/US2003/033681 U 203620 Adrenergic au Source: Human recombinant insect 5f9 cells Ligand: 1 nM P]- MK-912 Vehicle: I % DMSO Incubation Time/Temp: 60 minutes @ 25 *C Incubation Buffer 75 mM Tris-HC, pH 7.4,12.5 mM MgCI, 2 mM EDTA NonSpecific Ligand: 10 pM WB-4101 Kd: 0.6 nM * Bm: 4.6 pmole/mg Protein* Specific Binding: 95% * Quantitation Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition M 203710 Adrenergic a, Source: Human recombinant CHO-KI cells Ligand: 2.5 nM [11H1 Rauwolscine Vehicle: 1 % DMSO Incubation Timefemp: 60 minutes @ 25 CC Incubation Buffer: 50 mM Tris-HCI. 1 mM EDTA, 12.5 mM MgC,, pH 7.4, 0.2% BSA at 25 0 C NonSpecific Ligand: 10 pM Prazosin Kd: 2.1 nM * Bm.: 2.1 pmole/mg Protein* Specific Binding: 90% * Quantitation Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition a 204010 Adrenergic P 204110 Adrenergk 02 Source: Human recombisunt Rex IG ccls Source: Human recombinant CHO-NBR cells Uigand: 0 03 AM [-l CyanopindcAot Ligand: 0.2 nm [H] CGP.12177 Vehicle: I % DA5O Vehicle: 1 % OMSO Incubation Time/Temp: 2 hours @ 25CC Incubation TimeTemp: 60 minutes a 25 Ic Incubation Buffer: 50 mM Tris-HCIL 5 mM EDTA, 1.5 Incubati04 Buffe: 50 mM Trin-HCI. M.mM EOTA. 5.0 mM CaCl,. 120 mM NaCL 1.4 mM mM M&C6.1120mM NaCI. pH 7.4 ascorbic acid. 10 mBA a5A, pH NonSptcif LigandmgA I A pHCI-11551 7A4 Kd 1 0.44 nMI NonSpecific Ligand: 100 pM 5-)-Propranolol 8-r 0.437 pmole/mg Protein* K 0.041 nM *Speific Binding: 95% 0.072 pmole/mg Protein- Quantitation Mthod: Radioligand Binding Specific Binding; 95% * Significance Criteria: k 50% of max timulation or Quantitation Method: Radioligand Binding inhibition Significance Criteria: Z SO% of max stimulation or nnhcbituon 5 -6u1-n uf: 50m rsH~,05mMET .
WO 2004/039320 PCT/US2003/033681 * 212500 Sradykinin B, E 21261 Bracfrkinin 82 Source; Hunkan ls729 Celts Source: Human recombinnt CHO729 cells Ligand: 2,5 nM PhI (Oet-Arg'a)-Kallidin Ligand: 0.2 WI Bradycinin vehicle: 1 % DM50 Vehicle: 1 % DM50 Incubation Timertemp: 60 minutes @ 25 C Icubator Timetmp: 90 minutes @2S C Incubation Buffer: 20 mM HEPES. 125 mM N-methyl-D- Incubation Buffer 24 MTES-NH 4 OH, pH G.8. 1 mM 1, glucamine. 5 mM KCl. I mM 1,1D.I0-phenanthroine0.3% BSA Phenanthroline, pH 7.4 NonSpecifk Ligord: 5 pM Bradykinin NonSpecific Ligand: 10 pM (Des-Are. Leu")-Bradykinin Kd/ 0.29 rM 0-5 nMI B,,, 2 pnole/mg Protnr 0.059 pmole/mg Protein* Specific Binding 905' Specific Binding: 70% Quantitation Method; Radioligarid Binding Quantitation Method: Radiotigand Binding Significance Criteria: a SO% of max stimulation or Significance Criteria: 2 50% of max stimulation or inhibition inhibition * 214510 Cal1cium Channel L- Type, Benzothiarepine U 2 14600 Ca/c/un: Channel t1- Typ, Dihydropyr-idine Source: Wistar Rat biain Source: Wistan Rat cerebral cortex Ligad: 2 eM PH] Dittineem gLiand: 0.1 nA [H] Nitrendipine vehicle: 1 % DM60 Vehicle: 1 % DM50 Incubation Tmimefemjx 3 hours @ 4 'C Incubation Time/Temp: 90 minutes @ 25 IC Incubation Buffer 10 mM Tris-HCI, 0.1% BSA, p" 7.4 Incubation Buffer 50 mM Tri-NHCI, pH 7.7 at 2 m0C at 2MC nrSpecific Ligand: 1 pM Nifedipine NonSpeciic Ligand: 10PM 5iltide: r 0.1 a nM I ; 0.01£ pol , B-: 0.23 pno*e/g Protein' 0.21 pmole/g Protein Specific Binding: 219 Specific Binding: 73%0* Quanitationn Iethod: Radiigand Binding Quantitation method: Radioiiggad Binding Significance Criteria: k 50% of max stimuLatiun or Significance Criteriac 50% of max stimulation or inhibition inhibition 5 * 216000 Calcium Channel N- Type e 219500 opamme 0, Source: Wistar Rat brain frontal lobe Sourcen Human recombinant (IO cel Ligand: 10 PM [' ltiaConotoe a CVA Lipnd: 1z4 eM PHm SCH-23390 Vehicle: 1 % DMSO vehicle 1 % 0145 Incubation lime/Temp: 30 minutes @4 (C Inubation limeiremp: 2 hours @37 C Incubation Buffer 20 mM Tis-ICL, pit 7.4, 0.5% BSA Incubation buffer 50 MM s-MCI p7A 7.4. 150 mM NonSpecific Ligand: 0.1 pM a-Conotoxin GVIA MaCI, 1.4 mM Ascorbic Acid. K 0.01 n ' 0.00pI *SA B,,, 0.1 prole/mrg Protein' NonSpecific tiged: tO pM ft)-Busaclamot Specific Binding 76% * ld: 1,4 nM Quantitation Mthod: Iladiotigand Binding 80.:, 0.53 pmolefmg Protein, Significance Criteria: SOM of mae Stimtulation er Specitic Binding: 90%. inhibition Quantitation method; Radiotigand Binding Signiicince CriterCia a 50 of max stimulation or inhibition -62- WO 2004/039320 PCT/US2003/033681 M 219600 Dopamine D, Source: Human recombinant ClO cells Ligand: 0.16 nM PH] Spiperone Vehicle: I % DMSO Incubation Time/Temp: 2 hours @ 25 IC Incubation Buffer 50mM Tris-HCI, pH 7.4150mM NaCI, 1.4 mM Ascorbic Acid. 0U01% BSA NonSpecific Ligand: 10 pM Haloperidol Kd: 0.08 nM B": 0.48 prnole/mg Protein* Specific Binding: 85% Quantitation Method: Radioligand Binding Significance Criteria: a 50% of max stimulation or inhibition N 219800 Dopamine Dj Source: Human recombinant CHO cells Ligand: 0.7 nM PHI Spiperone Vehicle: 1 % DMSO incubation TimefTemp: 2 hours @ 37 C Incubation Buffer: 50 mM Tris-HCI, pH 7.4, 150 mM NaCl, 1.4 mM Ascorbic Acid, 0.001% BSA NonSpecific Ligand: 25 pM H(-)Sulpiride Kr: 0.36 nM * 01.1 pmole/mg Protein* specific Binding: 85% * Quantitation Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition -63- WO 2004/039320 PCT/US2003/033681 U 219900 Dopamine D... Source: Human recombinant CHO cells Ligand: 0.5 nM [ 3 H] Spiperone Vehicle: 1 % DM50 incubation Time/femp: 2 hours @ 25 UC Incubation Buffer, 50 mM Tris-HCI, pH 7.4, 150 mM NaCl. 1.4 mM Ascorbic Acid, 0.001% BSA NonSpecific Ligand: 10 pM Haloperidol
K
6 : 0.27 nM * Bmx: 1 pmole/mg Protein* Specific Binding: 90% * Quantitation Method: Radioligand Binding Significance Criteria: 50% of max stimulation or inhibition * 224010 Endothelin ET Source: Human recombinant CHO cells Ligand: 0.03 nM [m5l] Endothelin-1 'Vehicle: 1 % DMSO Incubation Time/Temp: 2 hours @ 37 C Incubation Buffer: 50 mM Tris-HCI, pH 7.4, 0.5 mM CaCh. 0.05% Tween-20. 1 mg/mI SA NonSpecific Ligand: 0.1 pM Endothelin-1
K
6 : 0.048 nM * B. : 0.35 pmole/mg Protein' Specific Binding: 90% * Quantitation Method: Radioligand Binding Significance Criteria: 50% of max stimulation or inhibition 5 U 224110 Endothel i E r U 22Y500 Ep/dermal C wth FactTr (,GO Source: Human recombinant CHO.Ki cells ia-Cc HUMaR A4) cel~a Ligand: 0.1 M [-ij Endothetin-1 tiea 0.55 CM [fl EeirfrzA Vehicle: 1 % DM50 F=ier tJGl) tksui) Incubation Time/Temp: 2 hours @ 25 C vehicle I % Incubation Buffer- 50 mM HEPES, I mL CaC, 5 mM IaCUH*0*CruJIen S0minutes m 23LT MgC, 0.5% BSA, pH 7, K4. 1.2MM MMO. 1-2 M Cal. (protease free) 1 "Wnl BSA, OH 7.7 NonSpecific Ligand: 0.1 pM EndotheUn-s NO ift Lkwd: 10 nM (sadrral Growth Fator 4d: 0.085 nM *tUW lhM;n3 4,3 pmole/mg Protein* 0.032 em' Specific Binding: 75% * Qualtitation Method: Radioligand Binding .mulenee frotgn' Significance Criteria: 50% of max stimulation or It Bir sk . inhibition Quaft2ta2L550 Methra: Grwdictid Gndir Ssakni-., Ccrirs ; 5K% af mar Stitrulation, or inibtaO i -64- WO 2004/039320 PCT/US2003/033681 M 226010 Estrogen ERa- U 226500 GA BAA. Agonist Site Source: Hueman recombinant insect 5fo Source: Wiatar Rat brain (minus cells cerebellum) Ligand: 0.5 nM PHI Estradiol Ligami 1 nm [)fl Muscinit Vehicle: I % DM50 vehicle: 1 % DM50 Incubation Time/remp: 2 hours @ 25 It Incubation limeffemp: 10 minutes @4 K Incubation Buffer: 10 mM TriurHCl. pH 7.5, 10% Incubation Buffer 50m Tis-MCI pH 7.4 Glycerol, I rnM D017 1 mgfrnl BSA NanSpecific Uiganld: 0.1 vM muscimnol Nonspecific Ligand: 1 IM DiethytilbestroK 3.8 eM K,,: V. nm* 1.8 pmoleimg Protein p i1400 pmoe/m Proteifi Specific Binding: 9idi Specific Binding: 85% uantitatin Method: Rdioligand Binding QuantitatHon Method: Radioligand Binding Significance Criteria: 5f S% of nax stimulation or Significance Criteria: a' 50% of max stimulation or Inhibition inhiibition * 226600 GA BAA. Beazodiazepine, Central1 0 228510 GA BA#, Non-Selective Source: Wiset Rat brain (minus Source: Wistar Rat brain cerebellum) Ligan: 0.5 nM Pill CGP-54G26 Ligand: 1 nM [1111 Flunitaepam VehicleM P % DM50 Vehicle 1 % hMSO Incubation Tirneemp: 20 minutes @ 2 OC Incubation Time/Temnp: 10 minutes @25 C Incubation Buffer: SB mM Tri-CL 2.5 mU CaC%, Incubation Baffer: 50 MM Na-K Phosphate. pH 7.4 D, 251 Nonpecific Ligand: 10 pM Diaepam NonSpecific tigttndh 100 y bM CGP-54 Kd,: 4A4 nM *K0: 2,3 nM 0p3 PM B/m 1P1 rmole/ng rein 1.2 pmole/mg Protei, Specific Binding: 80%' 8,,:4.1 pniotejmg' Quantitaflon Method: Radiotigand Binding Specific Binding: 91% * Significance Criteria: a 50% of mannstimulation or Quantilation Metd: Radiotigand Binding inhibition Significance Criteria: a 50% of max stimiulatlion or inhibition 3 232010 Glucacorticoid Source 101nan BSU 53 cells Lig~at 6 nM [I'l Deanmehnoine Vehicle I e DM5 e Incubation Tkninemca 2 hturs i5 mC icubarin uffier. RPMI 1640. 10 mM tEPEScbto 7B 2 NonSpecific Ligun±, 20 11M OeNamethasgne 1: ScM I B-.: Fit000 R'celI* Specific plneng: 7in g Qusafitation Metlid R Qadul aniad eithog Significance Ceia: z 50% of lani stimulation or iniibition 5 -65- WO 2004/039320 PCT/US2003/033681 M 232700 Glutamate, Kainate N 232810 Glutamate, NMOA, Agonism Source: Wistar Rat brain (minus source: Wistar Rat cerebral cortex cerebellum) Ligani: 2 nM 1H1 CGP-39653 Ligand: 5 nM ['H] Kainic acid Vehicle: I % DMS0 Vehicle: 1 % DM50 Incubation rime/femp: 20 minutes 0 4 OC Incubation Time/Temp: 60 minutes @ 4 OC Incubation Buffer: 50 mM Tris.ICI. pH 7.4 Incubation Buffer: 50 mM Tris-HCI, pH 7.4 NonSpecific Ligand: 1000 pM L-Glutamate NonSpecific Ligand: 1000 pM L-Glutamate Kd: 0,019 PM * Kd: 0.012 pM *B,,: 2.3 pmoe/mg Protein* B_: 0.35 pmole/mg Protein* Specific Binding 70% Specific Binding: 80% *Quantitation Method: Radioligand Binding Quantitation Method: Radioligand Binding Significance Criteria: a 50% of max stimulation or Significance Criteria: 2 50% of max stimulation or inhibition inhibition E 232910 Gluta7mate, NMDA, Glycne M 233000 Glutamate, NMDA, Phencyclidine Source: Wistar Rat cerebral cortex Source: Wistar Rat cerebral cortex Ligand: 0.33 nM [PHI MDL105519 Ligand: 4 nM [l3 CG TCP Vehicle: 1 % DMSO Vehicle: 1 % DMSO Incubation TimefTemp: 30 minutes @ 4 IC Incubation Time/Temp: 42 minutes @ 25 OC Incubation Buffer: 50mM HEPES. pH 7.7 Incubation Buffer: 10 rM Tris-HCI, pH 7.7 NonSpecific Ligand: 10 pM MDL-1 05519 NonSpecific Ligand: 1 pM Dizolcipine (MK-801) Kd: nM *Kd: 8.4 nM * 3-3-7 pmole/mg Protein- B: 0.78 pmole/mg Protein* Specific Binding 85% Specific Binding: 7% * Quantitation Method: Radioligand Binding Quantitation Method: Radioligand Binding Significance Criteria: a! 50% of max simulation or Significance Criteria: 2 50% of max stimulation or inhibition inhibition -66- WO 2004/039320 PCT/US2003/033681 M 239610 Histamine H, Source: Human recombinant CHO-Ki cells Ligand: 1.2 nM [3H] Pyritamine Vehicle: 1 % DMSO Incubation Timefremp: 3 hours @ 25 OC Incubation Buffer: 50 mM Tris, pH 7.4,2 mM MgCL, 100 mM NaCI. 250 mM Sucrose. NonSpecific Ligand: 1 pM Pyrilamine Kd: 1.1 eM * 13...:6.7 pmole/mg Protein* Specific Binding: 94% Quantitation Method: Radioligand Binding Significance Criteria: a. 50% of max stimulation or inhibition 0 239710 Histamine H 2 Source: Human recombinant CHO-K1 cells Ligand: 0.1 nM [yi Amincapotentidine Vehicle: 1 % DM50 Incubation Timeffemp: 2 hours @ 25 'C Incubation Buffer: 50 mM KH,fOd NaHP0,. pH 7.4 NonSpecific Ligand: 3 pM Tioidine 1d: 0.45 nM * B,,,,: 6.9 pmole/mg Protein* Specific Binding: 90% * Quantitation Method: Radioligand Binding Significance Criteria: 50% of max stimulation or inhibition U 23-9810 Histamine HU 241000/9z7 Hne stmnCenteaH Source: Human recom binant C 0-KI tel Source: Witar Rat cerebral cortex Ligaiid: 3 eM [)HIi R-ca-Methylhistarnini Ligand: 2 eM I11 Idazoan VeAMH) Vehicle: 1 % DM vehicle, 1 % DM50 Incubafon Tinaenbimp: 30 minutes @ 25 C Incubation Timtemp:. 901 mi nutes @ 25 C Incubation Buffer: SO naM Tnin-ilCI, 0.5 m M EDTA. pH Incubation Buffer- 50 mM Iris-HC . pH 7.u 10bmM 7.4 at B5uC MgCt,0.04% BSA NonSpecific Ligand I VM Idazoxun NoaSweific Ligand:L 1 pM R(-)-a-Methylhistamine K 4 flM (RANIH) B, ~0.14 pmolelnsg Protein' p: 2.4 nM e Specific BindingB i5%n g 8-,,: 412 pmole/mg Protein- Quantilation Method: Radioligad Binding Specific Binding: 95% 1 Significance Criteria: 2 50% of max stimulation or Quantitation Method: Radiotigand Binding inhibition Significance Criteria: a 50% of max stimulation or inhibition -67- WO 2004/039320 PCT/US2003/033681 0 243510 Intereukin IL-., Non-Selective 0 250600 Leukatriene L D 4 Source: Mouse 3T3 cells Source: Duncan Hartley derived Guinea Ugand: 10 pM [11] Interteukin-la II.- pig lung I t) tigand: 0.2 nM (I Leukotriene D, (LTD4 Vehide: 1 % DM50 Vehicle: 1 % DM50 incubation TimcfTemp: 2 hours @ 37 UC Incubation Time/Temp: 60 minutes @ 25 'C Incubation Buffer RPMI 1640. 20 mM HE PES, 0.1% Incubation Buffer 50 mM Tr-HClI, 0.01% BSA, 5 mM Sodium Azide. 1% BSA, pH 7.2. Caa. 5 mM MgC[,. 100 pg/mL NonSpecific Ligand: 0.03 PM Interleusn-la (IL-lu) Bacitracin, 1 mM Benamidine, K 6 pMA 1 0.1 mM Phenylmethytwlfonyl B.: 8.2 fmole/mg Protein* Fluoride Specfic Binding: 70%* NonSpecific Ligand: 0.1 pM Leukotriene D, (LTOJ QuOntitation Method: Radieligand Binding Kd: 0.2 nM * Significance Criteria: 2 50% of max stimulation at B.,: 0.24 ptole/mg Ptotein' inhibition Specific Binding: O5%* Quantitation Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition 2U 252700 Muscainic A Source: Human recombinant insect Sf9 Source: Human recombinant insect Sf9 cells cells Ligand: 0.29 nM [3H Methscopolamine Ligand: 0.29 nM PH)~ Metriscopolamine Vehicle: 1 % DM50 Vehicle: 1 % DM50 IncubationnTime/Temp::600minutees@@25 0C Incubation rimeffemp: 60 minuses @ 25 OC Icbto ie~m: 6 iue 5O Incubation Buffer: 50 mM Tris-HCI, pH 7.4, 10 mM MgCI,, 1 mM EDTA MgCI, 1 MM IOTA NonSpecific Ligand: 1 pM Atropine Ku: 0.092 nM * B.: 2.1 pmole/mg Protein* B 4.9 pmolelmg Protein' Specific Binding: 95%, Specific Binding 96% Quantitation Method: Radioligand Binding Quantitation Method: Radioligad Binding Significance Criteria: 2 50% of max stimulation or Significance Criteria: 2 50% of max stimulation or inhibition inhibition -68- WO 2004/039320 PCT/US2003/033681 M 252800 Muscarinic M 3 Source: Human recombinant insect Sf9 cells Ligand: 0.29 nM PH) Methscopotamine Vehicle: 1 % DMSO Incubation Time/Temp: 60 minutes @ 25 DC Incubation Buffer: 50 mM Tris-HCL, pH 7.4, 10 mM MgCly, 1 mM EDTA NonSpecific Ligand: 1 pM Atropine Kd: 0.078 nM * 8-: 3.2 pmole/mg Protein* Specific Binding: 96%* Quantitation Method: Radioligand Binding Significance Criteria: a 50% of max stimulation or inhibition N 257000 Neuropeptide Y, Source: Human SK-N-MC cells Ligand: 0.013 nM (1l] Peptide YY Vehicle: 1 % DMSO Incubation Time/Temp: 45 minutes @ 25 0 C Incubation Buffer: HBSS, 2 mg/mL BSA, 1 mM MgCI2, 1 mM CacI 5 NonSpecific Ligand: 0.1 pM Neuropeptide Y (human, rat) Kd: 0.62 nM Bm.: 5800 R/cell Receptors/cell* Specific Binding 85% * Quantitation Method: Radioligand Binding Significance Criteria: a 50% of max stimulation or inhibition N 257110 Neuropeptide Y 2 8 258590 Nicotinic Acetylcholine Source: Hunan KAN-TS neuroblastoma cell: Source: Human IMR-32 cells tigand: 10 pM [Il Peptide V Ligand: 0.1 nM jIsJ| Epibatidine Vehicle: 1 % DMS0 Vehicle, I % DMs0 Incubation Time/femp: 2 hours @ 37 tC Incubation Time/remp: 60 minutes @ 25 4C Incubation Buffer: 25 mM HEPES. 2.5 mM CaCl. 1 mM Incubation Buffer SO mM Tris-HCI, pH 7.4 M&Cl. 0.1% Bacitracin, pH 7.4 NonSpctific Ligand: 100 pim 0-Nicotine NonSpecific Ligand: I JIM Neutopeptide Y (13-36) Ka: 0.22 nM * (porcine) 5": 0.46 pntote/ng Protein* KI 0.012 nM * Specific Binding- 97%* D_: 0.5 pmole/mg Protein* Quantitation Method: Radioligand Binding Specific Binding: 90%* Significance Criteria: 1 SD% of max stimulation or Quantitation Method: Radiotigand Binding inhibition Significance Criteria: a 50% of max stimulation or inhibition 5 -69- WO 2004/039320 PCT/US2003/033681 * 260110 OpiSte 5 (OPI. DOP) Source: [1rumnr recombinant CHO cels Liand: 0.9 nM P-] NaIltind(e Vehicle: 1 % DMSO InCobation Time/Tremp; 2 hours @ 25 C Incubation auffer: 50 mM Tris4CiL S mM MgCI. pH 7A NcnSpecific Ligand 10 pM Natoxone 0.49 nM * Sp i nn: 8.6 pmole/mg Protein* Speific Bindcing: 00% , Quantitation Method: Radioligand Binding Veificanre criteria: a 50% of max stimulaion or inhibifino N 260210 Opiate K (OP2, KOP) Source: Human recombinant HEK-293 cells Ligand: 0.6 nM PH Diprenorphine Vehicle: 1 % DMSO Incubation TimelTemp: 60 minutes @ 25 OC Incubation Buffer 50 mM Tris-HCI. pH 7.4 NonSpecific Ligand: 10 pM Naloxone Kd: 0.4 nM * Bm.: 1.1 pmole/mg Protein* Specific Binding: 90% * Quantitation Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition * 260410 Opiate p (OP3, MOP) Source: Human recombinant CHO cells Ligand: 0.6 nM [ 3 H) Diprenorphine Vehicle: I % DMSO Incubation Time/Temp: 60 minutes @ 25 DC Incubation Buffer: SO mM Tris-HCl, pH 7.4 NonSpecific Ligand: 10 pM Naloxone Kd: 0.41 nM * Bm_: 3.8 pmole/mg Protein* Specific Binding: 90% * Quantitation Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition -70- WO 2004/039320 PCT/US2003/033681 U 264500 Phorbol Ester U 2650W Platelet Activating Factor (PAF) Source: ICRMousebrain Source: Human platelets Ligand: 3 nM IIHI PDBu Llpnd: 0,12 nM I 1 Hj PAF Vehickl: 1 % DM50 vehicle" 1 % DM50 Incubation Time/Temp: 60 minutes @ 25 4C Incubation Timeffemp 3 hours @ 2S IC lacubation Buffer: 20 mM Tris-HCI containing 5 mM Incubation Buffer 50mM Tris-HO, pH 7. 100mM CaClj, pH 7.5 at 25 0 C KCI. 5 mM EDTA. S mM MgC[, NonSpecific Ligand: 1 pM PD8u 0,25% BSA (wfe). Kit 8.7 nM * Nonpedffk Ligand 1 aM PAF 86 26 pmole/mg Protein" Kd- 0.13 W Specific Binding; 80% * 8 120 Ricell Quantitation Method: Radiatigand Binding Specific Binding 90% Significance Cesjerii: 2: 50% of max timulation or Quantitaiou Method: Radioligand Binding inhibiaon Significance Criteria: z 50% of max stimulation or inhibition N 265600 Potassium Channel [KA rp] Source: Syrian hamster pancreatic beta cells HIT-T1S Lggand: 5 nM n1d Glibenclamide Vehicle: 1 % DMSO Incubation Time/Temp: 2 hours @ 25 QC Incubation Bufferu 50 mM MOPS. 0.1 mM CaCl, pH 7.4 NonSpecific Ligand: 1 pM Glyburide K0.64 nM M B pole/mg Protein1 Specific Binding: 90% * Quantitation Method: Radioliga ni nd Binding Significance Criteria: 50% of max stimulation or inhibition M 268700 Purinergic P [ Source: New Zealand Derived Albino Rabbit urinary bladder Ligand: B nM [H] a, -Methylene-ATP Vehicle: 1 % DM50 Incubation Timeremp: 30 minutes @ 25 OC Incubation Buffer: 50 mM TriS-HCI, pH 7.4 NonSpecific Ligand: 100 pM P. y-Methylene ATP K: 02.2 nM * Kd 2 : 2.2 pM* B-: 2 pmole/mg Protein B-2: 790 pmole/mg Protein* Specific Binding: 80% Quantitation Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition M 26800 Puinergc 71- WO 2004/039320 PCT/US2003/033681 N 268870 Purinergic P 2 , Source: Wistar Rat brain Ligand: 0.1 nM [35] ATP-cr Vehicle: 1 % DMSO Incubation Time/Temp: 60 minutes @ 25 IC Incubation Buffer: 50 mM Tris-HCI, pH 7.4 NonSpecific Ligand: 10 pM ADP-5 Kd: 0.015 pM * Bm_: 16 pmole/mg Protein* Specific Binding: 87% * Quantitation Method: Radioligand Binding Significance Criteria: 50% of max stimulation or inhibition M 271170 Serotonin (5-Hydroxytryptamine) 5-HTIA Source: Human recombinant CHO cells Ligand: 1.5 nM [ 3 H) 8-OH-DPAT Vehicle: 1 % DMSO Incubation Time/Temp: 60 minutes @ 25 0 C Incubation Buffer: 50 mM Tris-HCI, 10 mM MgS04, 0.5 mM EDTA, 0.1% Ascorbic Acid, pH 7.4 NonSpecific Ligand: 10 pM Metergoline Kd: 2 nM * Bm': 1.3 pmole/mg Protein* Specific Binding: 75% * Quantitation Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition -72- WO 2004/039320 PCT/US2003/033681 N 271910 Serotonin (5-Hydroxytryptamine) 5-HT Source: Human recombinant HEK-293 cells Ligand: 0.69 nM PHI GR-65630 Vehicle: 1 % DM50 Incubation Time/Temp: 60 minutes @ 25 C Incubation Buffer 50 mM Tris-HCI. pH 7.5, 1 mM EOTA, S mM MgC 0 NonSpecific Ligand: 10 pM MDL-72222 Ku: 0.2 nM * 81-,: 11 pmole/mg Protein Specific Binding: 90% Quantitation Method: Radioligand Binding Significance Criteria: z! S0% of max stimulation or inhibition *2781 ) 0 si;gma a, Source: Human lurkat cells Ligand: .nM PH] Haloperidol Vehicle: 1 % DM50 Incubation limne6Temp: 4 hours @ 25 OC Incubation Buffer: 5 mM KTHP/KHPO, buffer pH 7.5 NonSpecific Ligand: 10 pM HaLoperidol K0: 5.8 nM * 0.71 pmole/mg Protein* Specific Binding: 80% * Quantitation Method: Radioligand Binding Significance Criteria: a 50% of max stimulation or inhibition a 278200 sigma7 or 2 U 279450 Sodium Canel, Site 1 Source: Wistar Rat brain Source: Wistar Rat brair Ligand: 3 WM 111] Ifenpioslil ligand: 2 eM PHI SsLitoiin Vehicle: 1 % DUSO vehicle: 1 % DM5 Incubation Time/Tesnp: 60 minutes o37 nC Ilcu bltoi Time/emp: 30 minutes @4 Incubation Buffer 50 mM Ti-Cai, pH 7.4 Incubation Buffer: (1) Houogefiation buffer 140 r4onSpecific Ligand 10 pM Ifenprodil mM NaCL 20 mM lisdlCI. PH 7.1, K.4.8 nM' 1 mM PMSF (2) 75 mM Hepes/140 B,,,,: 1.3 csmote/rng Protein, mum NaCI. pH 7.5 (3) Assay Specific Binding: 85% buffer homogenization buffer to Quantitation method: Radioligard Binding buffer (2) is 1:4 Significance Crieria: a 50% of max stimulation or NcrSpecific Ligind: 10 pM Tetrodlotoxin inhibition Kd: 1.4 nM* 8_'.,: 2.7 pmole/mg Protein Specific Binding: M Quantitation Method: Radioligand Binding Significance Criteria: k SO% of max stimulation or inhibition -73- WO 2004/039320 PCT/US2003/033681 U 279510 Sodium Channel. Site 2 U 255510 Tachykinin N/C, Source Wislar Rat brain Source: Human recombinant CHO cdi Ligand: S nM PH] Batrachotoxinin A 20-a. Lgan± 0.25 aM PHI 5R-140333 Renzoate Vehicle: I % DMSO vehicle: 1n T i m/ IncbatioT TiemfTp:p: 90 minutes @25 C incubation TimefTemp: G0 minutes @ 37 KC Incubation Buffer: 20 mM HEPES. pl1 7.4, 1 mM MnIC6, Incubation Buffer: 50 mM Tris-HCt, pH 7.4 at 250C. 0.01% SSA 50 mM NEPES. 130 mM choline-CL NtnSpeific Ligand: 2 PM U703,o6 5,4 mM KCI 0.8 mM Mg50,7tH20 Kd 03 mA* (or MgCb), 5,5 mM Glucose, 40 B ': 10 pmole-g Protein, pg/ml L41a Specific Bindin: 85% NonSpecific Ligand: 100 pM Veratridine Qiaititation Method: Radinligand Binding Ka 0.052 pM * Significance Criteria ' 50% Of max stimulation or 0.7 pmole/mg Protein inhibition Specific binding; 77% Quantitation Method: Radioligand Binding Significance Criteria: a 50% of max stimulation or inhibition N 255010 Testosterone U 220320 rransporte , opkinn e (NAT) Source: Rat recombinlant F, cli SourcE Human recombinant CHO ells igand: 1.S eM PH] miholerue Ligand 0.15 nMM PHIN RTI-55 Vehicle: 1 % DMSO Vehile : 1 % DM50 Incubation 1ine/Tenp: 4 haurs @ 4 OC Incubation Tin Temp: 0 ionurs 4 2C lnr tion Buffet-. 50mAIM Tr-lId (pH 7.). OS M Incubation uffer: 00 mM Na. SmM TiH- , 1 NaCI. 10% Glycero[. 2 mM PM4 Leupeptin 10 pM PMSF. p" 7.4 Dithio-itreitol 1 mng/m ,I l anNonSpecific Ligand: 0 pM 7Noifensine 25 Ethanol :Ka 0.58 nM * NonSpecific Ligand: 10 PM Miboleronle BB 0.047 pmole/mg Protein 3 nM - Specific Binding: 5% * B"i930 pmole/mng Protein* Qua utitation Method: Radioligand Binding Specific Binding: 9ot' Significance Ctitei k SO% of Mar Stimulation or Quantitation method: RadiMligand Binding inhibition Significance Criteria: :t 50% of max stimulation or inhibition 5 -74- WO 2004/039320 PCT/US2003/033681 E 204410 Transporter, Norepinephrine (NET) U 274020 Transporter, Serotonin (5 Source: Human recombinant MoCK ceus Hydroxytryptamine) (SERT) Ligand: 0.2 nM (11 RU55 Source: Human recombinant nEK-293 cells Vehicle: 1 % OMS0 Ligand: Ois nM il) RTSS Incubation Time/Temp: 3 houts @ 4 "C Vehicle: I % DMSO Incubation Buffer SO mM TriS-HCL 1 O mM NaCl, 1 Incubation Timc/Temp: 3 hours @4 -C aM leupeptin. 10 pM PMSF pH 7.4 Incubation Buffer. 100 mM NaCl. 50 mM Ttis HCJ. 1 NonSpecific Ligand: 10 pM Desipramine pM Leupeptin, 10 pM PMSF. pH 7.4 K,: 0.024 aiM * Non5pecific Ligand: 10 pM Imipramine 5ma: 2.5 pmole/mg Protein- K': 0.17 M * Specific Binding- 75% * B0.m 0.41 pmole/nmg Protein* Quantitatiora Method: Radioligand Binding Specific Bindirng 95%* Significance Criteria: 2 50% of max stimulation or Quantitation Method: Radiotigand Binding inhibition Significance Criteria- f 50% of max stimulation or inhibition M 226400 Transporter, GABA Source: Wistar Rat cerebral cortex Ugand: 6 nM [ 3 H] GABA Vehicle: 1 % DMSO Incubation Time/Temp: 20 minutes @ 25 OC Incubation Buffer 10 mM Na-HEPES, 120 mM NaCl. 4 mM Ca Acetate, 10 PM Isogiuvacine. 10 pM (-)Baclofen, pH 7.5 NonSpecific Ugand: 10 pM NO-711 Kd: 0.3 pM * Bm.: 60 pmole/mg Protein* Specific Binding: 80% * Quantitation Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition Historical Values 5 Literature References: CAT. #. Reference 118050. Crespi, C. L., Miller, V. P. and Penman, B.W. (1997) Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal Biochem 248(1): 10 188 190. Gentest Technical Bulletin (Version 4.2: Revised 27 September 2000) A high throughput method for measuring cytochrome P450 inhibition. Gentest Technical Bulletin (Version 4-2. 15 Revised 27 September 2000). -75- WO 2004/039320 PCT/US2003/033681 118060. Crespi, C. L., Miller, V. P. and Penman, B.W. (1997) Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal Biochem 248(1): 188 190. 5 Gentest Technical Bulletin (Version 4.2: Revised 27 September 2000) A high throughput method for measuring cytochrome P450 inhibition. Gentest Technical Bulletin (Version 4-2: Revised 27 September 2000). 118070. Crespi, C. L., Miller, V. P. and Penman, B.W. (1997) Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal Biochem 248(1): 10 188 190. 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(1986) Binding of 7a, 17a-dimethyl-19 nortestosterone (mibolerone) to androgen and progesterone receptors in human and 20 animal tessues. Endocrinology 118(4):_1327 - 1333. Example 9 Biological Testing of CS1814, CS1713, and CS1714 The results from the biological testing of CS1814, CS1713, CS1714, and 25 various reference compounds are presented in Figures 41-58 and 60-62. The methods employed have been adapted from the scientific literature to maximize reliability and reproducibility. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained. Assays were performed under the conditions described below. The literature reference(s) for 30 each assay are are listed below. Where presented, IC 5 0 values were determined by a non-linear, least squares regression analysis using Data Analysis ToolboxTM (MDL Information Systems, San Leandro, CA, USA). Where inhibition constants (K;) are -85- WO 2004/039320 PCT/US2003/033681 presented, the Kivalues were calculated using the equation of Cheng and Prusoff (Cheng, Y., Prusoff, W.H., Biochem. Pharmacol. 22:3099-3108, 1973) using the observed IC 50 of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the KD of the ligand 5 (obtained experimentally at MDS Pharma Sarvices. Where presented, the Hill coefficient (nH), defining the slope of the competitive binding curve, was calculated using Data Analysis ToolboxTM. Hill coefficients significantly different than 1.0, may suggest that the binding displacement does not follow the laws of mass action with a single binding site. Where IC 5 o, K 1 , and/or nH data 10 are presented without Standard Error of the Mean (SEM), data are insufficient to be quantitative, and the values presented (Ki, IC 50 , nH) should be interpreted with caution. CS1713 (Vial #2), CS1714 (Vial #3), and CS1814 (Vial #1) were evaluated for inhibition of cellular Serotonin and Norepinephrine Uptake. In 15 addition, CS1713 (Vial #2) and CS1714 (Vial #3) were evaluated in various radioligand binding assays, and for inhibition of CYP450 3A4 at initial concentrations of 10 gM. As depicted in Figure 60, significant activity ( 50%) was observed for displacement of radioligand from Serotonin Transporter binding sites (Vial #2 Ki = 3.88 nM, Vial #3 Ki = 8.15 nM) and Norepinephrine 20 Transporter binding sites (Vial #2 Ki = 0.112 jiM, Vial #3 Ki = 1.68 IM). As depicted in Figure 61, CS1814 (Vial #1) is approximately equipotent in inhibiting serotonin and norepinephrine uptake (IC 50 = 28.6 nM for norepinephrine, IC 50 = 21.7 nM for serotonin). Interestingly, CS 1713 (Vial #2) is a more potent inhibitor of norepinephrine uptake than serotonin uptake (IC 50 = 25 10.3 nM for norepinephrine, IC 50 = 22 nM for scrotonin). In contrast, CS1714 (Vial #3) is a more potent inhibitor of serotonin uptake compared to norepinephrin uptake (IC 50 = 88.5 nM for norepinephrine, IC 5 0 = 40.3 nM for serotonin). The fact that CS1713 (Vial #2) is a more potent inhibitor of norepinephrine uptake would render it a superior therapeutic agent for treating 30 diseases linked to norepinephrine uptake. In addition, the CS1714 (Vial #3) would useful for treating conditions requiring selective inhibition of serotonin uptake. -86- WO 2004/039320 PCT/US2003/033681 Importantly, no cytotoxicity was observed for CS1713 (Vial #2), CS1714 (Vial #3), orCS1814 (Vial #1) at 10 pM. In addition, CS1814 (Vial #1) is a selective inhibitor of norepinephrine and serotonin transporters. The fact that CS 1814 generally does not bind well to other receptors, as depicted in 5 Figures 32 and 33, substantially reduces the risk of negative side effects associated with administering the compound to a patient. Therefore, it is likely that CS 1713 and CS 1714 will not have detrimental side effects. Methods U 118090 CYP450, 3A4 Source: Human recombinant Sf9 insect cells Substrate: 50 pM 7-benzyloxy-4 (trifluoromethy-coumarin Vehicle: 0.1% DMSO Pre-Incubation TimcTemp: None Incubation Time/Temp: 30 minutes @ 37 0 C Incubation Buffer: 75 mM Potassium Phosphate buffer, pH 7.5 Quantitation Method: 5pectrofluorimetric quantitation of 7-Hydroxy-4-(trifluoromethyl) coumarin Significance Criteria: t 50% of max stimulation or inhibition 10 U 212610 Bradykinin B2 Source: Human recombinant CHO-KI cells Ligand: 0.2 nM PI Bradykinin Vehicle: 1% DMSO Incubation Time/emp: 90 minutes @ 250C Incubation Buffer: 24 mM TES-NH 4 0H, pH 6.8, 1 mM l,10-phenanthroline. 0.3% BSA Non-Specific Ligand: 5 pM Bradykinin K,: 0.29 nM* BMAX. 2 pmolelmg Protein Specific Binding: 90% Quantitalion Method; Radioligand Binding Significance Criteria: 50% of max stimulation or inhibition -87- WO 2004/039320 PCT/US2003/033681 M 214510 Calcium Channel L-Type, Benzothiazepine Source: Wistar Rat brain Ligand: 2 nM PHI Diltiazem Vehicle: 1% DMSO Tncuhation Time/Temp: 3 hours @ 4 0 C Incubation Buffer: 50 mM Trjs-HCI, 0.1% BSA, pH 7.4 at 25 0 C Non-Specific Ligand: 10 pM Diltiazem K,: 0.016 PM. BM : 0.21 pmole/mg Protein Specific Binding: 73% Quanilntion Method: Radioligand Binding Significance Criteria: z SO% of max stimulation or inhibition W204410 Transporter, Norepinephri e (NE Source: Human recombinant MDCK cells Ligand: 0.2 nM ['IHl RTI-55 Vehicle: 1% DMSO Incubation TiniMefl'p: 3 hours @ 4DC Incubation Buffer: 50 mM Tris-HCI, 100mM NaC, 1 pM leupeptin, 10 pM PMSF. pH 7.4 Nous-SpecificLigand: 10 pM Desipramine K,: 0.024 pM * Bm 0 2.5 pmole/mg Protein Specific Binding; 73% * Quaaritationi Method: Radioligand Binding Significance Criteria: 2 50% of max stimulation or inhibition -88- WO 2004/039320 PCT/US2003/033681 * 274020 Transporter, Serotonin (5 Hydroxytryptamine) (SERT) Source: Human recombinant HEK-293 cells - Ligand: 0.15 nM[251] RTI-55 Vehicle: 1% DMSO Incubation Timefremp: 3 hours @ 4 0 C Incubation Buffer: 100mM NaCI, 50 mM Thu HCl 1 iiM Leupeptin. 10t PM PMSF, pH 7A4 Non-Specific Ligand: 10pM Imipranine KD: 0.17 nM 8M : 0.41 pmole/mg Protein 4 Specific Binding: 95% Quantitation Method: Radicigand Binding Significance Criteria: f 50% Of max stimulation or inhibition E 302100 CytotoxiCity, Norepinephrine Uptake Target: Human MDCK cells Dog kidney Vehicle: 0.4% DMSO Incubation Timefrcmp: 30 minutes @ 25 0 C Incubation Buffer: 5 MM Tris-HCl. 7.5 mM HEPES. 120 mM NaC l, 5.4 mM K i. 1.2 mm Cacil, 1.2 MM MgSO 4 . 5 mM Glucose I mM Ascorbic Acid, pH 7.1 Quantitatius Method: Spectrofluonimetnec quantitation of Alamar Blue Significance Criteria-Ag: NA Significance Criteria-Ant: N O% Decrease in fluorescence iVtentity relative to vehicle control _89- WO 2004/039320 PCT/US2003/033681 U 364100 Cytotoxicity, Serotonin (5 Hydroxytryptamine) Uptake Target: Human HEK-293 cells Huamn embryonal kidney Vehicle: 0.4% DMSO Incubation Time/Temp: 30 minutes @ 25*C Incubation Buffer: 5 mM Tris-HC, 7.5 mM HEPES. 120 mM NaCl, 5.4 mM KCL, 1.2 mM Caa, 1.2 mM MgSO4., 5 mM Glucose, 1 mM Ascorbic Acid, pH 7.1 Quantitation Method: Spectrofluorimetric quantitation of Alamar Blue Significance Criteria-Ag: N/A Significance Criteria-Ant: 2 50% Decrease in fluorescence intensity relative to vehicle control * 302000 Uptake, Norepinephrine Target: Human MDCK cells Dog kidney Vehicle: 0.4% DM50 Incubation Time/Temp: 10 minutes@ 25oC Incubation Buffer: 5 mM Tris-HCI, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KCI, 1.2 mM CaCl, 1.2 mM Mgso 4 , 5 mM Glucose. 1 mM Ascorbic Acid. pH 7.1 Quantitation Method: Quantitation of PH]Norepinephrine Significance Criteria-Ag: N/A Significance Criteria-Ant: a 50% Inhibition of PH]Norepinephrine uptake relative to desipramine response * 364000 Uptake. Serotonin (5 Hydroxytryptamine) Target: Human HEK-293 cells Human embryonic kidney Vehicle: 0.4% DMSO Incubation Time/Temp: 10 minutes @ 250C Incubation Buffer: 5 mM Tris-HCI. 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KCI. 1.2 mM Cad 5 , 1.2 mM Mgso 4 ,. 5 mM Glucose, 1 mM Ascorbic Acid, pH 7.1 Quantitation Method: Quantitation of [ 3 H]Serotonin uptake Significance Criteria-Ag: N/A Significance Criteria-Ant: L 50% Inhibition of 3 H]Serotonin uptake relative to fluxetine response 5 Literature References (CAT. #. Reference) -90- WO 2004/039320 PCT/US2003/033681 118090. Crespi, C. L., Miller, V. P and Penman, B.W. (1997) Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal Biochem 248(1): 188 190. 5 Gentest Technical Bulletin (Version 4.2: Revised 27 September 2000) A high throughput method for measuring cytochrome P450 inhibition. Gentest Technical Bulletin (Version 4.2) Revised 27 September 2000). 204410. Galli, A., DeFelice, L., Duke, B.-J., Moore, K. 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Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 30 -92-
权利要求:
Claims (44)
[1] 1. An isolated compound represented by A: R X R 1 R 1 R4 R 3 -N N R 3 X R1 R A wherein 5 X represents independently for each occurrence 0, S, or NR; R represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, formyl, acyl, silyl, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m-R80; 10 RI represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, arylakylamino, sulfhydryl, alkylthio, arylthio, arylakylthio, nitro, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, 15 (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m-R8o; R 2 represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, or -(CH2)m-Rso; R 3 represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, or -(CH2)m-Ro; 20 R 4 is absent or present between one and four times inclusive; R 4 , if present, represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, arylakylamino, sulthydryl, alkylthio, arylthio, arylakylthio, nitro, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, 25 (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, -93- WO 2004/039320 PCT/US2003/033681 (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m Ro; R 80 represents independently for each occurrence an aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl moiety; 5 m is independently for each occurrence an integer in the range 0 to 8 inclusive; and the compound is a single enantiomer; or a pharmaceutically acceptable salt or prodrug thereof.
[2] 2. An isolated compound represented by B: X R R R R4 R2R R3-NR N R 3 X R1 R B 10 wherein X represents independently for each occurrence 0, S, or NR; R represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, formyl, acyl, silyl, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, 15 (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m-Rso; R1 represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, arylakylamino, sulfhydryl, alkylthio, arylthio, arylakylthio, nitro, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyl, 20 (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m-RO; R 2 represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, or -(CH2)m-Rso; R3 represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, 25 heteroaryl, arylalkyl, or -(CH2)m-Rso; R4 is absent or present between one and four times inclusive; -94- WO 2004/039320 PCT/US2003/033681 R4, if present, represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, arylakylamino, sulfhydryl, alkylthio, arylthio, arylakylthio, nitro, azido, alkylseleno, formyl,.acyl, carboxyl, silyl, silyloxy, 5 (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m Rgo; R 8 o represents independently for each occurrence an aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl moiety; 10 m is independently for each occurrence an integer in the range 0 to 8 inclusive; and the compound is a single enantiomer; or a pharmaceutically acceptable salt or prodrug thereof.
[3] 3. The compound of claim 1 or 2, wherein X represents 0.
[4] 4. The compound of claim 1 or 2, wherein R represents H. 15
[5] 5. The compound of claim 1 or 2, wherein R1 represents H.
[6] 6. The compound of claim 1 or 2, wherein R 2 represents H.
[7] 7. The compound of claim I or 2, wherein R 3 represents alkyl.
[8] 8. The compound of claim I or 2, wherein R4 is absent.
[9] 9. The compound of claim 1 or 2, wherein X represents 0; and R represents H. 20
[10] 10. The compound of claim 1 or 2, wherein X represents 0; R represents H; and R' represents H.
[11] 11. The compound of claim I or 2, wherein X represents 0; R represents H; R' represents H; and R 2 represents H.
[12] 12. The compound of claim 1 or 2, wherein X represents 0; R represents H; R, 25 represents H; R 2 represents H; and R 3 represents alkyl.
[13] 13. The compound of claim 1 or 2, wherein X represents 0; R represents H; RI represents H; R2 represents H; R3 represents alkyl; and R 4 is absent.
[14] 14. The compound of claim 1 or 2, wherein X represents 0; R represents H; R' represents H; R 2 represents H; R 3 represents ethyl; and R 4 is absent. 30
[15] 15. A formulation, comprising a compound of claim I or 2; and a pharmaceutically acceptable excipient.
[16] 16. A formulation, comprising a compound of claim 1 or 2; and and a compound selected from the group consisting of analgesics, anti-inflammatory drugs, -95- WO 2004/039320 PCT/US2003/033681 antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators, anti asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastro-intestinal drugs, muscle relaxants, nutritional agents, vitamins, 5 parasympathomimetics, stimulants, antinarcoleptic, and anorectics.
[17] 17. A formulation, comprising a compound of claim 1 or 2; and a compound selected from the group consisting of aceclofenac, acetaminophen, adomexetine, almotriptan, alprazolam, amantadine, amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine, amphetamine, aripiprazole, aspirin, atomoxetine, 10 azasetron, azatadine, beclomethasone, benactyzine, benoxaprofen, bermoprofen, betamethasone, bicifadine, bromocriptine, budesonide, buprenorphine, bupropion, buspirone, butorphanol, butriptyline, caffeine, carbamazepine, carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine, choline salicylate, citalopram, clomipramine, clonazepam, clonidine, clonitazene, clorazepate, 15 clotiazepam, cloxazolam, clozapine, codeine, corticosterone, cortisone, cyclobenzaprine, cyproheptadine, demexiptiline, desipramine, desomorphine, dexamethasone, dexanabinol, dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, dezocine, diazepam, dibenzepin, diclofenac sodium, diflunisal, dihydrocodeine, dihydroergotamine, dihydromorphine, dimetacrine, 20 divalproxex, dizatriptan, dolasetron, donepezil, dothiepin, doxepin, duloxetine, ergotamine, escitalopram, estazolam, ethosuximide, etodolac, femoxetine, fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine, flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan, gabapentin, galantamine, gepirone, ginko bilboa, granisetron, haloperidol, huperzine A, hydrocodone, 25 hydrocortisone, hydromorphone, hydroxyzine, ibuprofen, imipramine, indiplon, indomethacin, indoprofen, iprindole, ipsapirone, ketaserin, ketoprofen, ketorolac, lesopitron, levodopa, lipase, lofepramine, lorazepam, loxapine, maprotiline, mazindol, mefenamic acid, melatonin, melitracen, memantine, meperidine, meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone, 30 methamphetamine, methocarbamol, methyldopa, methylphenidate, methylsalicylate, methysergid(e), metoclopramide, mianserin, mifepristone, milnacipran, minaprine, mirtazapine, moclobemide, modafinil, molindone, morphine, morphine hydrochloride, nabumetone, nadolol, naproxen, naratriptan, nefazodone, neurontin, -96- WO 2004/039320 PCT/US2003/033681 nomifensine, nortriptyline, olanzapine, olsalazine, ondansetron, opipramol, orphenadrine, oxaflozane, oxaprazin, oxazepam, oxitriptan, oxycodone, oxymorphone, pancrelipase, parecoxib, paroxetine, pemoline, pentazocine, pepsin, perphenazine, phenacetin, phendimetrazine, phenmetrazine, phenylbutazone, 5 phenytoin, phosphatidylserine, pimozide, pirlindole, piroxicam, pizotifen, pizotyline, pramipexole, prednisolone, prednisone, pregabalin, propanolol, propizepine, propoxyphene, protriptyline, quazepam, quinupramine, reboxitine, reserpine, risperidone, ritanserin, rivastigmine, rizatriptan, rofecoxib, ropinirole, rotigotine, salsalate, sertraline, sibutramine, sildenafil, sulfasalazine, sulindac, 10 sumatriptan, tacrine, temazepam, tetrabenozine, thiazides, thioridazine, thiothixene, tiapride, tiasipirone, tizanidine, tofenacin, tolmetin, toloxatone, topiramate, tramadol, trazodone, triazolam, trifluoperazine, trimethobenzamide, trimipramine, tropisetron, valdecoxib, valproic acid, venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone, zolmitriptan, zolpidem, and zopiclone. 15
[18] 18. A method of treating a mammal suffering from depression, comprising the step of: administering to said mammal a therapeutically effective amount of a compound of claim 1 or 2.
[19] 19. A method of treating a mammal suffering from fibromyalgia syndrome, comprising the step of: 20 administering to said mammal a therapeutically effective amount of a compound of claim 1 or 2.
[20] 20. A method of treating a mammal suffering from mental disorders including Functional Somatic Disorders, for example, depression, fibromyalgia syndrome, chronic fatigue syndrome, pain, attention deficit/hyperactivity disorder, and visceral 25 pain syndromes (VPS), such as irritable bowel syndrome (IBS), noncardiac chest pain (NCCP), functional dyspepsia, interstitial cystitis, essential vulvodynia, urethral syndrome, orchialgia, and affective disorders, including depressive disorders (major depressive disorder, dysthymia, atypical depression) and anxiety disorders (generalized anxiety disorder, phobias, obsessive compulsive disorder, 30 panic disorder, post-traumatic stress disorder), premenstrual dysphoric disorder, temperomandibular disorder, atypical face pain, migraine headache, and tension headache, comprising the step of: -97- WO 2004/039320 PCT/US2003/033681 administering to said mammal a therapeutically effective amount of a compound of claim 1 or 2.
[21] 21. The method of claim 18, 19, or 20, wherein said mammal is a primate, equine, canine or feline. 5
[22] 22. The method of claim 18, 19, or 20, wherein said mammal is a human.
[23] 23. The method of claim 18, 19, or 20, wherein said compound is administered orally.
[24] 24. The method of claim 18, 19, or 20, wherein said compound is administered intravenously.
[25] 25. The method of claim 18, 19, or 20, wherein said compound is administered 10 sublingually.
[26] 26. The method of claim 18, 19, or 20, wherein said compound is administered ocularly.
[27] 27. The method of claim 18, 19, or 20, wherein said compound is administered transdermally.
[28] 28. The method of claim 18, 19, or 20, wherein said compound is administered rectally. 15
[29] 29. The method of claim 18, 19, or 20, wherein said compound is administered vaginally.
[30] 30. The method of claim 18, 19, or 20, wherein said compound is administered topically.
[31] 31. The method of claim 18, 19, or 20, wherein said compound is administered 20 intramuscularly.
[32] 32. The method of claim 18, 19, or 20, wherein said compound is administered subcutaneously.
[33] 33. The method of claim 18, 19, or 20, wherein said compound is administered buccally. 25
[34] 34. The method of claim 18, 19, or 20, wherein said compound is administered nasally.
[35] 35. A composition comprising a selective serotonin reuptake inhibitor and a compound of claim 1 or 2.
[36] 36. A composition comprising a selective norepinephrine reuptake inhibitor and a compound of claim I or 2. 30
[37] 37. A composition comprising a selective serotonin reuptake inhibitor, a selective norepinephrine reuptake inhibitor, and a compound of claim 1 or 2.
[38] 38. The composition of claims 36 or 37, wherein said selective norepinephrine reuptake inhibitor is milnacipran. -98- WO 2004/039320 PCT/US2003/033681
[39] 39. The composition of any one of claims 35-37, wherein said compound of claim 1 or 2 is CS1713.
[40] 40. The composition of any one of claims 35-37, wherein said compound of claim 1 or 2 is CS1714. 5
[41] 41. The composition of any one of claims 35-40, wherein said composition further comprises CS 1814.
[42] 42. A composition comprising a selective serotonin reuptake inhibitor and CS1814.
[43] 43. A composition comprising a selective norepinephrine reuptake inhibitor and CS1814. 10
[44] 44. A composition comprising a selective serotonin reuptake inhibitor, a selective norepinephrine reuptake inhibitor, and CS 1814. -99-
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法律状态:
2009-07-02| FGA| Letters patent sealed or granted (standard patent)|
2012-05-17| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|
优先权:
申请号 | 申请日 | 专利标题
US42164002P| true| 2002-10-25|2002-10-25||
US60/421,640||2002-10-25||
US42306202P| true| 2002-11-01|2002-11-01||
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US44514203P| true| 2003-02-05|2003-02-05||
US60/445,142||2003-02-05||
PCT/US2003/033681|WO2004039320A2|2002-10-25|2003-10-22|STEREOISOMERS OF p-HYDROXY-MILNACIPRAN, AND METHODS OF USE THEREOF|
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