Indian Patents
Title of Invention

STEREOISOMERS OF P-HYDROXY-MILNACIPRAN AND METHODS OF USE THEREOF
Abstract The present invention relates generally to the enantiomers ofpara-hydroxy- milnacipran or congeners thereof. Biological assays revealed that racemic para-hydroxy- milnacipran is approximately equipotent in inhibiting serotonin and norepinephrine uptake (IC50 = 28.6 nM for norepinephrine, IC50 = 21.7 nM for serotonin). Interestingly, (+) para- hydroxy-milnacipran is a more potent inhibitor of norepinephrine uptake than serotonin uptake (IC50 = 10.3 nM for norepinephrine, IC50 = 22 nM for serotonin). In contrast, (-)­ para-hydroxy-milnacipran is a more potent inhibitor of serotonin uptake compared to norepinephrin uptake (IC50 = 88.5 nM for norepinephrine, IC50 = 40.3 nM for serotonin). The invention also relates to salts and prodrug forms of the aforementioned compounds. In certain embodiments, the compounds of the present invention and a pharmaceutically acceptable excipient are combined to prepare a formulation for administration to a patient. Finally, the present invention relates to methods of treating mammals suffering from various afflictions, 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.
Full Text 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 irom tricycHc 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 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-lbor J. etal., 1996, Int. Clin. Psychopharm., 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 gastrointestinal disturbances such as nausea and dyspepsia are common with these agents (Hindmarch 1., 1997, Human Psychopharmacology, 12:115-119). For example, for widely prescribed SSRl 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).
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 norepinephrine (NE) receptors. Milnacipran is also often referred to as norepinephrine and serotonin reuptake inhibitor (NSRl) 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 (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

Phase II Clinical Trial in Fibromyalgia, Media Release, March 21, 2003, Available from: URL: ht^;//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, 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 stimulant efifects. 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 human clinical trials with tolerabiltty decreasing with increasing dose (Puech A. et al., 1997, Int. Clin. Psychopharm., I2;99-?08). In the double-blind, randomized, multicenter clinical study the most frequent spontaneously reported adverse events for lOO 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 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 (GuelfiJ.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 mihiacipran recipients than for TCA imipramine 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-) 65). 11 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 a!., 1993, Eur. 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

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 efTect is listed if frequency 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 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, 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 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, milnaciprandosageof 200 mg/day was superior to the lower doses (Von Frenckell R et al., 1990, Int. Clin. Psychopharmacology., 5:49-56). Milnacipran dosing regime of ] 00-250 mg daily was recently reported for the treatment of fibromyalgia (US Patent No. 6,602,911). Itwouldbe very difficuh to reach the upper limits ofthe 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.
The (+)-dextroenantiDmer of milnacipran (F2695, (+)-]S,2R-milnacipran) is niughly twice as active in inhibiting norepinephrine and serotonin reuptake as the racemic mixture. See Viazzo et al. Tetrahedron Lett. 1996, 57, 4519-4522; Deprez et al. £wr. /

Drug Metab. Pharmacokinet. 1998, 2i, 166-171. N^oreover, the (-)-levro enantiomer of
milnacipran (F2696, (-)-l R,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 episodes and other mental disorders including Functional Somatic Disorders. Summary of the Invention
One aspect of the present invention relates to the enantiomers ofpara-hydroxy-milnacipran or congeners thereof. Biological assay studies revealed that (+)-/j«rir-hydroxy-milnacipran is an approximately two-fold more potent inhibitor of norepinephrine uptake 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 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 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.
Figure 2 depicts a " H NMR spectrum of lactone CSl 590.
Figure 3 depicts a "^C NMR spectrum of lactone CS1590.
Figure 4 depicts a "H NMR spectrum of amide CS1608.
Figure 5 depicts a "C NMR spectrum of amide CS1608.
Figure 6 depicts a "H NMR spectrum of CSI628.
Figure 7 depicts a "^C NMR spectrum of CSl 628.
Figure 8 depicts a "H NMR spectrum of CS1649.
Figure 9 depicts a "^C NMR spectrum of CSl 649.

Figure 10 depicts a HPLC chromatogram of CSI665 (HPLC Conditions: 10% to 95% acetonitrile within 8 min; 2 min at 95%, LM with 0.1% TFA, Flow: 2.0mL/min, SSuiery Zorbax XDB-C8).
Figure 11 depicts amass spectrum of CSl665.
Figure 12 depicts a "H NMR spectrum of CS1665.
Figure 13 depicts a "^C NMR spectrum of CS1665.
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).
Figure 15 depicts amass spectrum of CSl 710.
Figure 16 depicts a " H NMR spectrum of CS1710.
Figure 17 depicts a "^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: 2.0mL/min, Saule: Zorbax XDB-C8).
Figure 19 depicts a mass spectrum of CS 1713.
Figure 20 depicts a "HNMR spectrum of CS1713.
Figure 21 depicts a "^C NMR spectrum of CSl 713.
Figure 22 depicts a HPLC chromatogram of CS1714 (HPLC Conditions: 10% to 95% acetonitrile within 8 min; 2 min at 95%, LM with 0.1% TFA, Flow: 2.0mL/m!n, Saule: Zorbax XDB-C8).
Figure 23 depicts amass spectrum of CSl 714.
Figure 24 depicts a " H NMR spectrum of CS 1714.
Figure 25 depicts a "^C NMR spectrum of CS1714.
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: 2.0mL/min, Saule: Zorbax XDB-C8).
Figure 27 depicts a LC/MS chromatogram ofCS1814.
Figure 28 depicts a mass spectrum of selected peaks from the LC/MS chromatogram of CS1814.
Figure 29 depicts a mass spectrum of a peak from the LC/MS chromatogram of CS18I4.
Figure 30 depicts a "H NMR spectrum ofCSISU.

Figure 31 depicts a "^C NMR spectrum of CSISH.
Figure 32 depicts biological activity data for CS1814 in assays using receptors from human (hum) and rat.
Figure 33 depicts biological activity data for CSl 814 in assays using receptors from human (hum), mouse, guinea pig (gp), syrian hamster (syh), and rat.
Figure 34 depicts biological activity data for CSl814 in assays using receptors from human (hum) and rat.
Figure 35 depicts biological activity data for CSl 814 in assays using receptors from human (hum).
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 CSI814(Vial#I).
Figure 40 depicts a graph of % inhibition of Serotonin Transporter (SERT) by CS1814(Vial#l).
Figure 41 depicts biological activity data for CS1713 (Vial #2) and CS1714 (Vial #3).
Figure 42 depicts biological activity data for CS17I3 (Vial #2) and CS1714 (Vial #3).
Figure 43 depicts biological activity data for CS1713 (Vial #2) and CS1714 (Vial #3).
Figure 44 depicts biological activity data for CSI713 (Vial #2), CS1714 (Vial #3), andCSISM (Vial ^1).
Figure 45 depicts biological activity data for CS) 713 (Vial #2), CSl 714 (Vial #3), andCSISM (Vial^l).
Figure 46 depicts biological activity data for CS1714 (Vial #3).
Figure 47 depicts a graph of % inhibition of Norepinephrine Uptake by CSl 814 (CEL-l) and Desipramine.
Figure 48 depicts a graph of % inhibition of Serotonin Uptake by CS18I4 (CEL-l) and Fluoxetine.
Figure 49 depicts a graph of % inhibition of Norepinephrine Transporter by CS1713 (CEI.-3) and Desipramine.

Figure 50 depicts a graph of % inhibition of Serotonin Transporter by CSl 713 (CEL-3) and GBR. 12909.
Figure 51 depicts a graph of % inhibition of Norepinephrine Uptake by CSI713 (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 CSI714 (CEL-5) and Desipramine.
Figure54depictsagraphof%inhibitionof Serotonin Transporter by CS1714 (CEL-5) and GBR-12909.
Figure 55 depicts a graph of % inhibition of Norepinephrine Uptake by CSl 714 (CEL-5) and Desipramine.
Figure 56 depicts a graph of % inhibition of Serotonin Uptake by CS1714 (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 CSl 814.
Figure 60 depicts a summary of significant primary results for CSl 713 (Vial #2) andCS1714(Vial#3).
Figure 61 depicts a summary of significant primary results for CS1814 (Via! #1), CS 1713 (Vial Ul) and CS 1714 (Vial #3).
Figure62depictsasummary of secondary results for CSl 814 (Vial #1), CS17i3 (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
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.


Racemic milnacipran
The term "ED50" 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.
Compounds refered to in the specification and figures are identified using a six-character alpha-numeric code. For example, racemic/j-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 isp-hydroxy-milnacipran and the data was taken from batch 1.
The term "LD50" means the dose of a drug which is lethal in 50% of test subjects.
The temi "therapeutic index" refers to the therapeutic index of a drug defined as LD50/ED50.
The term "structure-activity relationship (SAR)" refers to the way in which altering the molecular structure of drugs alters their interaction with a receptor, enzyme, etc.
The term ""agonisf" 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 "antag,onist" refers to a compound that binds to a receptor site, but does 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.
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.

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 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 (o) constant. This well known constant is described in many references, for instance, i. March, Advanced Organic Chemistry. McGraw Hill Book Company, New York, (1977 edition) pp. 251-259. The Hammett constant values are generally negative for electron donating groups (a[P] = -0-66 for NH2) and positive for electron withdrawing groups (o[P] = 0.78 for a nitro group),
tT[P] indicating para substitution. Exemplary electron-withdrawing groups include nitro, acyl, forrayl, alkylsulfonyl, arylsulfonyl, trifluoromethyl, cyano, chloride, and the like. Exemplary electron-donating groups include amino, methoxy, and the like.
The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyi (alicyclic) groups, alkyl substituted cycloalkyi groups, and cycloalkyi substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C]-C3o for straight chain, CyCi^Q for branched chain), and more
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 "aralkyi", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromalJc group).
The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in 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 fi-om one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower 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 temi "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,

ftiran, thiophene, imidazole, oxazole, thiazole, triazole, pyrazoie, 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 aromaticf nng can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyi, alkenyl, alkynyl, cycioalkyi, hydroxyl, alkoxyl, amino, nitro, sulfhydiyl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, siiyl, ether, alkylthio, alkylsulfonyl, arylsulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like. The term "aiyl" also includes polycyclic ring systems having two or more cyclic rings in which two 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 or//io, me/a and/jora apply to 1,2-, 1,3-and 1,4-disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ort/io-dimethylbenzene are synonymous.
The terms "heterocyclyl" or "heterocyclic group" refer to 3- to lO-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, phenoxathiin, pyrrole, imidazole, pyrazoie, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoHne, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, fiirazan, phenoxazine, pyrrolidine, oxolane, 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, aralkyi, alkenyl, alkynyl, cycioalkyi, hydroxyl, amino, nitro, sulfhydryl, Imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, alkylsulfonyl, arylsulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
The terms "polycyclyl" or "polycycl ic group" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocvclvls^ in which two nrmnre

carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that an 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, aralkyi, alkenyl, alkynyl, cycloalkyi, hydroxy], amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, alkylsulfonyl, aiylsulfonyl, ketone, aldehyde, ester, a heterocyclyi, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -CI,
-Br or -1; the term "sulfhydryl" means -SH; the terms "hydroxy" and "hydroxyl" mean -OH; and the term "sulfonyl" means -S02--
The terms "amine" and "amino" are art-recognized and refer to both unsubstifuted and substituted amines, e.g., a moiety that can be represented by the general formula:
wherein R9, Rj Q and R" ] Q each independently represent a group permitted by the rules of
valence.
The term "acylamino" is art-recognized and refers to a moiety that can be represented by the general formula:
wherein Re, is as defined above, and R"{ 1 represents a hydrogen, an alkyl. an alkenyl or -(CH2)ni-R8" where m and Rg 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
H-^^9
I
^10
wherein R9, Rjo are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.
i:

The term "alkylthio" refers to an aikyi group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" moiety is represented by one of-S-alkyi, -S-alkenyl, -S-alkynyl, and -S-(CH2)ni-R8- wherein m and Rg are defmed above. Representative alkylthio groups include methylthio, ethyl thio, and the like. The term "carbonyl" is art recognized and includes such moieties as can be represented by the general formula-.
O O
-Lx-R^j^ , or _j(J-R.^^
wherein X is a bond or represents an oxygen or a sulfur, and R) \ represents a hydrogen, an aikyl, an alkenyl, -(CH2)m-R8 "f" ^ pharmaceutically acceptable salt, R"n represents a hydrogen, an alkyl, an alkenyl or -(CH2)ni"R8. where m and Rg are as defined above. Where X is an oxygen and Rji orR"ji is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R] i is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R ] ] is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen, and R"| ] is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "Ihiolcarbonyl" group. Where X is a sulfur and R^j orR"|] is not
hydrogen, the fonnula represents a "thiolester." Where X is a sulfur and Rj ] is hydrogen, the formula represents a "thiolcarboxylic acid." Where X is a sulfur and R| ]" is hydrogen, the formula represents a "thiolfonnate." On the other hand, where X is a bond, and Rj i is not hydrogen, the above formula represents a "ketone" group. Where X is a bond, and R] i is hydrogen, the above formula represents an "aldehyde" group.
The terms "alkoxy!" 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 covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an elher is or resembles an alkoxyl, such as can be represented by one of-O-alkyl, -O-alkenyl, -O-alkynyl, -0-(CH2)m-R8, where m and Rg are described above.
The terra "sulfonate" is art recognized and includes a moiety that can be represented by the general formula:

0
-S-OR^l
o in which R4] is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.
The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl,/»"toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyi groups, respectively. The terms triflale, tosylale, mesylate, and l\onaflate 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, trifluoromethanesulfonyl, nonafluorobutanesulfonyi, p-toluenesulfonyl and methanesulfonyl, r6Sf>ectively. 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 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:
O
II
—O—S—OR41
II
o
in which R41 is as defined above.
The term "sulfonylamino" is art recognized and includes a moiety that can be
represented by the general formula:
O
II
N—S-R
! 11
" 0
R
The term "sulfamoyi" is art-recognized and includes a moiety that can be represented by the general formula:

0
11 /R
S-N
it O R_
The term "sulfonyl", as used herein, refers to a moiety that can be represented by the general formula:
O
II —S—R44 (I 0
in which R44 is selected fi-om ttie group consisting of hydrogen, alkyl, alkenyl, alkynyi, cycloalkyi, heterocyclyl, aiyl, or heteroaryl.
The term "sulfoxido" as used herein, refers to a moiety that can be represented by the general formula:
0
II
in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyi, cycloalkyi, heterocyclyl, aralkyl, or aryl.
A "selenoaikyi" 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-alkyI, -Se-alkenyl, -Se-aikynyl, and -Se-(CH2)ni"R7:. m ^nd R7 being defined above.
Analogous substitutions can be made to alkenyl and alkynyi 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 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 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

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 invention, the heleroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein vi"hich 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 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 prctecling group chemistry has been reviewed (Greene, T.W.; Wuls, P.G.M. Protective Groups in Organic Synthesis, l""^ ed.; Wiley: New York, 1991).
Certain compounds of the present invention may exist in particular geometric or stcreoisomeric forms. The present invention contemplates all such compounds, including cis- and (rans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-i5omers, 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 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 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 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

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 ^" 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. Synthesis of Individual Enanleriomers ofp-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 lithium diethylamide, generated from n-butyllithium and diethylamine, furnished CS1608 and the corresponding enanliomer, 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 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 CS1665 and CS1710. Preparation of the corresponding hydrochloric acid salts was accomplished by using hydrochloric acid in dioxane and subsequent removal of the 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 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

foUowing parameters (nature and concentration of neutral CD, concentration of S-p-CD) on many separation factors (electropborelic mobility, selectivity, effitciency, 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 . 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. Biolopcal Activity Analysis
The results from the biological testing of CS1814, CS1713, CS17i4, and various reference compounds are presented in Figures 32-62. CSl 814 (Vial #1), CSI713 (Vial #2), and CS1714 (Vial #3) were evaluated in various radioligand binding assays, and for inhibition of CYP450 3A4 at initial concentrations of 10 [iM. As depicted in Figures 59 and 60, significant activity (350%) was observed for displacement of radioligand from Serotonin Transporter binding sites (Vial #1 Ki = 6.73 nM, Via! Ml Ki = 3.88 nM, Via! #3 iCi = 8.15 nM) and Norepinephrine Transporter binding sites (Vial #1 Ki = 0.218 jiM, Vial #2 Ki = 0.112 nM, Vial #3 Ki = 1.68 ^M).
In addition, CSHH (Vial #2), CSHM (Vial #3), and CS1814 (Vial #1) were evaluated for inhibition of cellular Serotonin and Norepinephrine Uptake. As depicted in Figure 61, CS1814 (Vial #1) is approximately equipotenl in inhibiting serotonin and norepinephrine uptake (IC50 = 28.6 nM for norepinephrine, ICso = 21.7 nM for serotonin). Interestingly, CS1713 (Vial #2) is a more potent inhibitor of norepinephrine uptake than serotonin uptake (IC50 ^ 10.3 nM for norepinephrine, IC50 = 22 nM for serotonin). In contrast, CSI714 (Vial #3) is a more potent inhibitor of serotonin uptake compared to norepinephrin uptake (ICso = 88.5 nM for norepinephrine, IC50 = 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 diseases linked to norepinephrine uptake. In addition, the CS1714 (Vial #3) would useful for treating conditions requiring selective inhibition of serotonin uptake.

Importantly, no cytotoxicity was observed for CSI7I3 (Vial #2), CS17I4 (Vial #3), or CS1814 (Vial #1) at 10 nM. In addition, CS18I4 (Vial #]) is a selective inhibitor of norepinephrine and serotonin receptors. The fact that CS1814 generally does not bind well to other receptors, as depicted in 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 CS17U will not have detrimental side effects. Compounds & Methods of the invention
In certain embodiments, a compound of the present invention is an isolated compound represented by A:

wherein
X represents independently for each occurrence O, S, or NR;
R represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, ar heteroaryl, arylalkyl, formyl, acyl, silyl, (alkyloxy)carbonyl, (aryloxy)carbonyl, (aryIa[kyloxy)carbony!, (alkylaraino)carbonyl, (arylaminojcarbonyl, (arylalkylamino)carbonyl, alkylsulfonyi, arylsulfonyl, or -(CH2)tn-R-8o;
R" represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, ai heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, aryiamino, arylakylamino, sulfhydryl, alkylthio, arylthio, arylakylthio, nitro azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyi, (aryloxy)carbonyl, (arylaikyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalky!amino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)m-Rfio;
R^ represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, ar heteroaryl, arylalkyl, or -(CH2)m-R8o;

R represents independently for each occurrence H, alkyl, cycioaikyl, alkenyl, aryl, heleroaryl, aiylalkyi, or -(CH2)m-R8o;
R is absent or present between one and four tin\es inclusive;
R , if present, represents independently for each occurrence H, alkyl, cycioaikyl, alkenyl, aryl, heteroaryl, arylalkyi, cyano, halogen, hydroxy), alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, arylakylamino, sulfhydryl, alkylthio, aryithio, arylakylthio, nitro, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arySalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or-(CH2)ni-
Rso;
Rao represents independently for each occurrence an aryl, cycioaikyl, cycloalkcnyl, 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 pharmaceutical ly acceptable salt or prodrug thereof.
In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents O.
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 A and the attendant definitions, wherein R" represents H.
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 A and the attendant definitions, wherein R^ represents alkyl.
In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein R"" is absent.
In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents O; and R represents H.
In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents O; R represents H; and R" represents H.

^

In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents O; R represents H; R" represents H; and R^ represents H,
in certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents O; R represents H; R" represents H; R^ represents H; and R^ represents alkyl.
In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents O; R represents H; R" represents H; R^ represents H; R^ represents alkyl; jwd R* is absent.
In certain embodiments, the compounds of the present invention are represented by A and the attendant definitions, wherein X represents O; R represents H; R" represents H; R^ represents H; R^ represents ethyl; and R"" is absent.
In an assay based on a mammalian GPCR, certain compounds according to structure A have IC50 values less than 10 yxM, more preferably less than 1 jiM, even more preferably less than 100 nM, and most preferably less than lOnM.
In an assay based on a mammalian GPCR, certain compounds according to structure A have EC50 values less than ] 0 ^iM, more preferably less than I fiM, even more preferably less than 100 nM, and most preferably less than 10 nM.
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 ftom fibromyalgia syndrome.
In certain embodiments, compounds according to structure A are effective in the treatment of a mammal suffering from mental disorders including Functional Somatic Disorders, for example, depression, fibromyalgia syndrome, chronic fatigue syndrome, pain, attention deficitAyperactivity 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 depression) and anxiety disorders (generalized anxiety disorder, phobias, obsessive compulsive disorder, panic disorder, post-traumatic stress disorder), premenstrual dysphoric

disorder, teraperomandibular disorder, atypical face pain, migraine headache, and tension headache.
In certain embodiments, a compound of the present invention is an isolated compound represented by B;


R^

-N-
R^

R^-

R
/
B
wherein
X represents independently for each occurrence O, S, or NR;
R represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, ajyl, heteroaryl, arylalkyl, formyl, acyl, silyl, (alkyloxy)carbonyl, (aryloxy)carbonyl, (aryia!kyloxy)carbonyl, (a(kylamino)carbonyl, (arylamino)cart>onyl, (arylalkyiamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -{CH2)m-^0"
R 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, (alkylam ino)carbony], (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2WR80;
R"^ represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, or -{CH2)m-R8o;
R^ represents independently for each occunence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, or-(CH2)m-Rgo;
R"" is absent or present between one and four times inclusive;
R\ 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, alkylseieno, formyi, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyl, (aryloxy)carbony!, (aryialkyloxy)caTbonyl, (alky)amino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsuifony], or-(CH2)m-
Rso;
Rgo represents independently for each occurrence an aryl, cycloailtyl, cycloalkenyl, heterocyclyl, or polycyelyl 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 pharmaceutical ly acceptable salt or prodrug thereof.
In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents O.
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" represents H.
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^ represents alkyl.
In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein R"" is absent.
In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents O; and R represents H.
In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents O; R represents H; and R" represents H.
In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents O; R represents H; R" represents H; and R^ represents H.
In certain embodiments, the compounds of the present invention are represented by B and the attendant definitions, wherein X represents O; R represents H; R" represents H; R^ represents H; and R^ represents alkyl.

In certain embodiments, the compounds of the present invention are represented b; B and the attendtuit definitions, wherein X represents O; R represents H; R" represents H; R^ represents H; R^ represents alkyl; and R"* is absent.
tn certain embodiments, liie compounds of the present invention are represented b> B and the attendant definitions, wherein X represents O; R represents H; R" represents H; R^ represents H; R"* represents ethyl; and R"" is absent.
In an assay based on a mammalian GPCR, certain compounds according to structure B have IC50 values less than 10 ^M, more preferably less than 1 liM, 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 B have EC50 values less than 10 ^M, more preferably less than 1 |iM, 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 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 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 vuivodynia, 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, 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 1S,2R l-(4-Methoxy-phenyl9-3-oxa-bicyclo[3.1.0]hexan-2-one(CSI590), IR,2S ]-(4-Methoxy-phenyi9-3-oxa-bicyclo[3.1.0]hexan-2-one (CS1591),IS, 2R 2-Hydroxymethyi-l-(4-methoxy-phenyl)-cyclopropanecarboxyIic acid diethylamide (CSI608), IR, 2S 2-Hydroxymethyl-I -(4-methoxy-phenyl)-cyclopropanecarboxylic acid diethylamide

(CSI609), IS, 2R 2-A2idomethy!-I-(4-m6thoxy-phenyi)-cyclopropanecarboxylic acid diethylamide (CS1628>,lR,2S2-Azidometiiyl-l-{4-melhoxy-phenyl)-cyclopropanecarboxylic acid diethylamide (CS1648), 1S, 2R 2-Azidomethyl-] 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, 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.
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, 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, desipramine, desomorphine, dexamethasone, dexanabinol, dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, dezocine, diazepam, dibenzepin, diclofenac sodium, diflunisal, dihydrocodeine, dihydroergotamine, dihydromorphine, dimetacrine, divalproxex, dizatrtplan, dolasetron, donepezil, dothiepin, doxepin, duloxeti"ne, ergotamine.

escitalopram, estazolam, ethosuximide, etodolac, femoxetine, fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine, flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan, gabapentin, galanlamine, gepirone, ginko bilboa, granisetron, haloperidol, huperzine A, hydrocodone, hydrocortisone, hydromorphone, hydroxyzine, ibuprofen, imipramine, indiplon, indomethacin, indoprofen, iprindole, ipsapirone, ketasenn, ketoprofen, ketorolac, lesopitron, levodopa, lipase, lofepramine, lorazepam, loxapine, maprotil"me, maz"indo], mefenamic acid, melatonin, melitracen, memantine, meperidine, meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone, 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, nomifensine, nortriptyline, olanzapine, olsalazine, ondansetron, opipramol, orphenadrine, oxaflozane, oxaprazin, oxazepam, oxitriptan, oxycodone, oxymorphone, pancrelipase, parecoxib, paroxetine, pemoline, pentazocine, pepsin, perphenazine, phenacetin, phendimctrazine, 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, 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, vatdecoxib, valproic acid, venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone, zolmitriptan, Zolpidem, zopiclone and isomers, salts, and combinations thereof.
By adjunctive administration is meant simultaneous administration ofthe compounds, in the same dosage form, simultaneous administration in separate dosage forms, and separate administration ofthe compounds.
In certain embodiments, the present invention relates to ligands for a GPCR, e.g., a receptor for a neurotransmitter, wherein the ligands are represented by a structure outlined above, and any ofthe sets of definitions associated with a structure. In certain embodiments, the ligands ofthe present invention are antagonists, agonists, partial agonists or inverse agonists of a GPCR. In certain preferred embodiments, the ligands ofthe present

■■y

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 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.
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 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 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 fi"om exposure to the inflammatory agent carrageenan; moreover, administration of certain kinase inhibitors diminishes hyperalgesia 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 af^er an 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.

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 diseases, trigeminal neuralgia, rheumatoid arthritis, toxins, cancer (which can cause direct or remote (e.g. pamneoplastic) 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 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 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 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 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 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

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 manufacturt of other compounds of the present invention also form part of the invention. Pharmaceutical Composilions
In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeuticatly-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 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, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a control led-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 "therapeutical ly-effective amount" as used herein means that amount of 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 ceils 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 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 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

examples of materials which can serve as pharmaceutical ly-acceptable carriers include: (I)
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)
excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil,
cottonseed oil, safTloweroil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, ■
such as propylene glycol; (II) 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; (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 pharmaceutically-acceptable salts with pharmaceutical ly-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 Cfm 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 with a suitable organic or inorganic acid, and isolating the sah 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, {umarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (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, 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-

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 flinctional groups and, thus, are capable of forming pharmaceutical ly-acceptabie salts with pharmaceutically-acceptabie bases. TTie term "pharmaceutical ly-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, carbonate or bicarbonate of a pharmaceutically-acceptabie metal cation, with ammonia, or with a pharmaceutically-acceptabie 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, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Bergeetal., 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 compositions.
Examples of pharmaceutically-acceptabie 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 ascorbyi palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, 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 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

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 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, [n certain embodiments, an aforementioned 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 witii 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 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¬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.
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 pharmaceutical ly-acceplable 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, 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

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 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 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 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 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 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 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"

(20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). A difliision 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 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 carboxymethylcellulose, and carbopol 934, 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 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 of either applying an immediate release layer on top of the extended release core using coating or compression process or in a muhiple 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 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 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 svnlhetic eums. inciiiflino

acacia, alginates, methylceliulose, and polyvinylpyrrolidine can also be used. Polyethylene glycol, hydrophilic polj-mers, 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 slippeiy solids as talc, magnesium and calcium 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.
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 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 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 polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylceliulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl raelhacrylate, and other methacrylic resins that are commercially available under the tradename Eudragit®. (Rohm Pharma; Westerstadt, Germany), including Eudragit®. L30D-55 and LI 00-55 (soluble at pH 5.5 and above), Eudragil®. L-lOO (soluble

ai pH b.u and above), Eudragit*. S (soluble at pH 7.0 and above, as a result of a higher degree of estenfication), and Eudragits®. NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability); vinyl polymers and copolymers such as polyvinyl pyrroUdone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chltosan, 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 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, 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, 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 sorbilan 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 effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may alsd be used-Small quantities of an anti-fbaming 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 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 acryiate, and vinyl or enzymatically degradable polymers or copolymers as described above. These hydrophilic

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 witti the matrix material, the mixture can be compressed into tablets.
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 carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, 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 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, microcrystailine cellulose, aluminum metahydroxide, bentonite, agar-agar and 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 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 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

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, parafTms, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, laic 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 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 C2inbe made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can 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.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutical ly-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, 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, 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.

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, 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 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 parenteral ly-adminjstered drug form is accomplished 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 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 combination with a pharmaceutical ly 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 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

by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
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.
These compoimds 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, intracistematly and topically, as by powders, ointments or drops, including buccally and sublingually.
Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitebie hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutical ly-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 may be varied so as to obtain an amount of the active ingredient which Is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
TTie 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 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 and prescribe the eifective 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

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 an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebrovenlriculai and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 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 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).
[n another aspect, the present invention provides pharmaceutical ly acceptable compositions which comprise a therapeuticaily-effective amount of one or more of the subject compounds, as described above, fomiulated 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 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 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 pharmaceuticals.
The term "treatment" is intended to encompass also prophylaxis, therapy and cure.

The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equlnes, cattle, swine and sheep; and pouUry and pets in general.
The compound of the invention can be administered as such or in admixtures with 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.
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 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" O and B books, Corvallis, Ore., U.S.A., 1977). Combination Therapy
The compounds of the invention may be administered to a patient in combination with one or more therapeutic agents. TTie 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 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 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

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 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, iaitidepressants, 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.
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 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. 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 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 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

combination therapy comprises at least one of the compounds of the invention, a SSRI, and a SNR]. In certain embodiments, a SNRI is milnacipran.
Incertainembodiments, the combination therapy comprises CSl 713 and a SSRI. In certain embodiments, the combination therapy comprises CS1713 and a SNRl. In certain embodiments, the combination therapy comprises CSl 713, a SSRJ, andaSNRl. In certain embodiments, the invention relates to the above-mentioned combination therapies of CS1713 which further comprise CSI814.
In certain embodiments, the combination therapy comprises CSl 714 and a SSRI. In certain embodiments, the combination therapy comprises CSl 714 and a SNRI. In certain embodiments, the combination therapy comprises CSl714, a SSRI, and a SNRI. In certain embodiments, the invention relates to the above-mentioned combination therapies of CS17I4 which further comprise CS1814.
In certainembodiments, the combination therapy comprises CSl 814 and a SSRI. In certain embodiments, the combination therapy comprises CS1814 and a SNRI. In certain embodiments, the combination therapy comprises CSl 814, a SSRI, and a SNRI. Combinalorial Libraries
The subject compounds readily lend themselves to preparation using the methods of combinalorial chemistry, providing access to combinatorial libraries of compounds for the screening of pharmaceutical, agrochemical or other biological or medically-related activity 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 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 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, Biondelle et al. (1995) Trends Anal. Chem. 14:83; the Afiymax U.S. Patents 5,359,115 and 5,362,899: the Ellman U.S. Patent

5,288,514: the Still et al. PCX publication WO 94/08051; Chen et al. (1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092, WO93/09668 and WO91/07087; and the Uraer et al. PCT publication WO93/20242). Accordingly, a variety of libraries on the order of about 16 to 1,000,000 or more diversomers can be 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. PCTpublication WO 94/08051, 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 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 difluse across the membrane to an 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-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 MALDl can be used to release a 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
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 polyaciylic acid-grated polyethylene ptns arrayed in the microtitre plate format. The Geysen technique can be used

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) Tetrahedron Lett 31:5811-5814; Valerioetal. 0991) Anal Biochem 197:168-177; Bray et al. (1991) TetrahedronLett32:6l 63-6166).
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) 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.
[n 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 appropriale reaction solutions, while all common steps such as resin washing and deprotection are performed simultaneously in one reaction vessel. At the end of the synthesis, each ba^ contains a single compound-
D) Combinatorial Libraries fay Light-Directed, Spatially Addressable Parallel Chemical
Synthesis
A scheme of combinatorial synthesis in which the identity of a compound is given 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 etal. (1991) Annu Rep MMChern26:27I-280;Fodor, S.RA. (1991) Science 251.-767; Pirrungetal. (1992) U.S. Patent No-5.143.854; Jacobs et al. f 1994) Trends Biotechnol 12:19-26). The spatial resolution of photolithography affords miniaturization. This technique can be carried out through the use protection/deprotection reactions with photolabile protecting groups.
Thekey points of this technology are illustrated in Gallop etal. (1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared for coupling through the covalent

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 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 r^ictants define the products and their locations. Since this process utilizes photolithography techniques, the number of compounds ibat 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 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 with an encoded tagging system. A recent improvement in the identification of active compounds from combinatorial libraries employs chemical indexing systems using la.gs 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 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
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 7^ (= 823,543) peptides composed

of ail combinations of Arg, Gin, 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 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 NH2 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 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 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-synlhesis of the tag and the library member. Furthermore, the chemical lability of the tag, particularly the phosphate and sugar anomeric linkages, may limit the choice of reagents and conditions that can be employed for the synthesis of non-oHgomeric 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 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 (Nikolaievet al. (1993) Pept Res 6:161-170). branched linkers are employed so that the coding unit and the test compound can both be attached to the same fiinctional 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 fPteket al. (1991) Tetrahedron Lett 32:3891-

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 ^" " 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 aUemative form of encoding the test compound library employs a set of non-sequencable electrophoric tagging molecules that are used as a binary code (Ohimeyer et al. (1993) PNAS 90:10922-10926). Exemplary tags are haloaromatic allcyl 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 alky) 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 ^^ g^ upwards of 10" 2) different molecules. In the original report (Ohimeyer 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 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 etal. (1994) j_0rg 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.
Although several amide-linked libraries in the art employ binary encoding witii 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 neariy as unreactive as the bead matrix itself. Two binary-encoded combinatorial libraries have been reported where the electrophoric tags are attached directly to the solid phase (Ohimeyer et al. (1995) PNAS 92:6027-6031) 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 phoiolabile linker and the tags were

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 microliter 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 microliter plates; fifth, single active compounds are identified; and sixth, the structures are decoded. Toxicohgical Assessments
During the drug development process, potential therapeutic agents or drug candidates must be demonstrated to be both safe and effective for Uieir 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 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 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.lO 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.
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 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.

For example, a compound is brought into contact with a population of muiti-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 mdividual organisms of the population. By substantial portion is meant at least 40 number %, usually at least 50 number % and more 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 the nutrient medium, e.g. water, aqueous solution of additional nutrient agents, etc., of the organisms. For example, where the muhi-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.
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 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, N.Y.)(1989); Nucleic Acid Hybridization, A Practical Approach (Hames, B. D., and Higgins, S. J. eds, IRL Press, Oxford)(l 985); 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 a!., Gene (1995) 166-. 207-213.
The effect of a compound on a particular physical parameter or parameters being evaluated may be determined manually or robolically, such that in many embodiments determination of the effect of the compound on the organism is accomplished via an automated procedure.

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 activities, such as lethality, sterility causing activity, etc. Fly Model for Toxicology
A candidate chemical is dissolved in water at or near its saturation point. Serial dilutions of this stock solution arc used to rehydrate instant fly media (Fisher Scientific). Specifically, one toxicity assay will comprise of instant fly media rehydrated with pure 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 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 amount that cart 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.
file 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 tbat were placed in the receptacle. Sterility is examined for both males and females by crossing them lo normal flies. A physical examination of the adults 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 eJTect occurred to either shorten or lengthen the average lifespan of the fly.

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 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.S8 g, 63.76 mmol) and benzene (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 hydroxide (Imol/L, 40 mL). The solution was then heated to reflux overnight followed by addition of concentrated hydrochloric acid to adjust the pH=l. The aqueous phase was extracted with tert.-butyimethylether (200 mL) and ethyl aceate (200 ml). The organic phases were combined, washed with sat. sodium chloride, dried (MgS04) and the solvent was reduced under reduced pressure to afford crude CS1590 which was purified by column chromatography on silica gel using ethyl acetate/dichloromethane 1:4 as an eluent. 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
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, 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 (MgS04) and the solvent was reduced

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 CSt609 was synthesized.
Example 3
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
suspension was stirred for 24 hours at room temperature, quenched into water (200 ml-) and
extracted with elhylacetate (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 CSl628 (2.2 g, 36%) as
an off white solid.
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 CS1628 (2.2 g, 7.27 mmol) and dichloromethane (200 mL),
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 (MgS04) and the solvent
was reduced under reduced pressure to afford crude CS1649, which was purified by column
chromatography on silica gel using heptane/ethyl actetate 2:1 as an eluent to afford CSI649
(1.39 g, 66.5 %) as an off white solid.
In a similar fashion the desired enantiomer CSI658 was synthesized.
Example 5
Synthesis of CS1665 and CS1710
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 CSI655, which

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.
Analytical Data for CS1665

Appearance: Off white solid
"H NMH (MeOil-4):
"^C{" Hi NMR (apt) (MeOd-4)-. Figia-e 12 Figure 13
Optical Rotatton [a\\ (c 0.5, Methanol): -89
IR (KBr, Neat, Solvent): N/A
HPLC: Purity: 97% @ 220 nm, 100% @ 254 nm
Method: Gradient of 10% acetonitrile to 95% acelonitrile over 8 min, equilibrate 2 rnin at 95%, 0.1% TFA, Flowrate: 2.0 ml./min
Column: Zorbax XDB-C8
Ekmental Analysis: N/A
Mass Spectrum (ESI): m/z = 263 [CiiHsiNjOs+Hf
Analytical Data for CSI710

Appearance: Off white solid
"H NMR (MeOd-4): "C("Hj-NMR (apt) (MeOd-l): Figure 16 Figure 17
Optical Rotation |a|^""D (c 0.5, Methanol): +86.2
IR (KBr, Neat, Solvent): N/A
HPLC: Purity: 98% @ 220 nm, 100% @ 254 nm
Method-. Gradient of iO% ac«tonilrile to 95% acetonitrife 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,3H22N;Oj+H]""

Example 6
Synthesis of CS1713 and CS17I4
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
temperature, followed by removal of the solvent under reduced pressure to afford CS17I3
(0.43 g, 84%) as an off white solid.
In a similar fashion the desired enantiomer CS17I4 was synthesized.
Analytical Data for CS1713

Appearance: Off white solid
"H NMR (McOd-4): "^C{"H}-NMR (apt) (MeOd-*): Figure 20 Figure 21
Optical Rotation (al"^ (c 0.1, Methanol): +80
IR (KBr, Neat, SoKent): N/A
HPLC: Purity: 97% @ 220 nin, 98% @ 254 nm
fMethod: Gradient of 10% acetonilrile to 95% acetonitrile over 8 min, equiiibrale 2 min at 95%, 0.!% TKA, Flowrate: 2.0 mL/min
Column: Zorbax XDB-C8
Elemental Analysis: N/A
Mass Spectrum (£S)): m/z = 265 [CiiHs^NjOj+l-ll*
Analytical Data for CSI714

Appearance: OfT white solid
"HNMR{MeOd-4): "C{"H}-NMR (apt) (MeOd-4): Figure 24 Figure 25
Optical Rotation [al^o (c O-I, Methanol): -74
IR (KBr, Neat, Solvent): N/A
HPLC: Purity: 96% @ 220 nm, 100% @ 2S4 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: 2«rbax XDB-Cg
Elemental Analysis: N/A

Mass Spectrum (ESI): m// = 263[C,5H22N,0,+H]"
Example 7 Preparation of CS1814
A 10 mL flask equipped with a magnetic stir bar was charged wilhCS1665/2 (120 mg, 0.46 mmol), CS1710/1 (120 mg, 0.46 mmol) and hydochloric acid in dioxan (5 mol/L, 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, I 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 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 (homogenous solution) and the solvent removed under reduced pressure.
Analytical Data for CS1814

Appearance: Off while solid
"HNMR(MeOd-4>: "C{"Hj-NMR (apt) (MeOd-4): Figure 30 Figure 31
Optical Rotation |a|"„ (c O.S, Methanol): 0 racemic
IR (KBr, Neat, Solvent): N/A
HPLC: Purity: 97% @ 220 nm, 98% @ 254 nm
Method: Gradient of 10% acelonitrile to 95% acetonitrile over 8 min, equilibrate 2 min at 95%, 0.1% TFA, Flowrate: 2.0 mlVmin
Column: Zorbax XDB-C8
Elemental Analysis: N/A
Mass Spectrum (ESI): m/z = 263 [C,sHMNiOi+Hr
Example S
Biological Testing of CS1814 and Reference Compounds

The results from the biological testing of CSl 814 and various reference compounds are presented in Figures 32-40 and 59. The data in Figure 59 indicate that CSl 814 has an [Cso = 0-22 fiM for inhibition of norepinephrine transporter and an /C50 value of J2.7 nM for inhibition of serotonin transporter. The binding constants for CSl 814 are Ki = 0.218 jiM for norepinephrine transporter and Ki = 6.73 nM for serotonin transporter.
The methods employed in this study 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 conditions as described below. Literature reference(s) for each assay are tabulated below and hereby incorporated by reference.
Where presented, IC50 values were determined by a non-linear, least squares regression analysis using Data Analysis TooiboXTM (MDL Information Systems, San Leandro, CA, USA). Where inhibition constants (Ki) are presented, the Kj 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 Kj of the ligand (obtained experimentally at MDS Pharma Services). Where presented, the Hill coefficient (n^), defining the slope of the competitive binding curve, was calculated using Data Analysis Toolbox""^"*". Hili 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 IC50, K,, and/or UH data are presented without Standard Error of the Mean (SEM), data are insufficient to be quantitative, and the values presented (K,, IC50, nn) should be interpreted with caution. Methods:

VaO50CyP450. !A2 Source:
Sub si rate:
Vehicle:
Pre-lnciibatinn rime/TemR:
Incubation Time/femp:
incubation Buffer:
Ouamitatiofi Method:
Significarte Criteria"
118070 CYP450.2C 19 Source:
Substrate: Vehicle:
Prp-lrcubalion Time/Temp: Incubation Time/Temp" incubation Buffer
QLantitation Method:
Significance Criteria

Human recombinant Sf9 insect
cells
5 IJM 3-Cyano-7-ethDxycoLmarin
0.1 1, DMSO
None
30 minutes @J7">C
7S mM Potassium Phasphale buffer,
pH7.S
;;pertrQfluDrimetric quanlilalion
of 3-Cyann-7-hydro«ycoumarin
? 50% of maK slimulation or
inhibition
Human recombinant Sf9 insect
cells
25 pM 3-CyanD-7-ethoiycoumarin
0.1 K DWSO
None
45 minut« @ 37 "C
75 mM Potassium Phosphate buffer,
pH7.S
Spectrofluorimeliic quantitation
of 3-CyanD-7-hydroiycoumarin
z 50* of mai stimulalian or
inhibition



1]8060CYP4SD.2C9 Source
Substrate:
Vehicle:
Pre-lncubation Time/Temp:
l^£ubalJDn TiraeTenip;
IncLbation F5Lffer
Quantitation Method:
Significance Criteria:
118080 CYP450. 2D6 Source:
Substrate:
Vehicle:
Pre-lncubalion Time/Temp:
Incubation Time/Temp:
Incubation Buffer:
Quantitation Method
Significance Crrterra-

Humai! recombinant 5f9 insect
cells
25 |JM 3-Cyano-7-ethojycoumarin
0.1 % DMSO
None
« mOiMlei @ 37 K
75 mM Potassium Phosphate buffer.
pH7S
Spectrofluorimetrjc quantitation
of 3-Cyano-7-hydroxycQumarin
>. 50% of ma inhibition
Human recombinant 5fl3 insect
cells
50 pM 3-Cyano-7-sthoiycoumatin
0.1 » DMSO
None
45 minutes @ 37 "C
75 mM Potassium Phosphate buffer,
pH 7.5
Specttofluorimetnc quantitation
of 3-Cyano-""hydroiiycoumarin
? !0i6 of mair itimutstiaa or
inhibition

B 118090 CYP4S0, 3A4 Source-
S libit rate.
Vehicle:
Pre-lncubador fime/femp: Incubation Time/Temp: Intubalion Buffer
QuantitatiQii Method
Significance Ci-iiena"

HLman recombinant Sf9 insect
cells
50 iiW J-benzyloxy-4-
Jifi f! ucromelbyO-CDUni win
0 1 •%, &MSO
None
30 minutes @ 37 ■<:> 75 mM PolassiLim Phoiphate buffer,
pH 7.B
Specttafluarimetric quantitation
of 7-Hydroiy-4- couriidrin
J 50% ot man stimulation or
inhibition



200570 Adenosine A,
Source" Ligand"
Vehids:
Incubation Time/Temp"
Incubation Buffer
Nonspecific Ugand: K„.
Specjfic Binding" Quantitation Melliod: Significance Criteria-

Human recombinant CMO cells 1 nM [=H] DPCPX
1 % DMSO
90 minutes @ 75 °C
20 mM HEPES pH 7.4.10 mM UgCI,.
lOOmMNaC!
1D0liMR|-l-PIA 1.4 nM •
2.7 pmole/mg Protein"
Radialigand Birding
2 5GS of max slimulation or
inhibition


20O6W Adenosine /l„ ■ 203100 Adrenergic ClA
Source: Human recombinant HE:K-2S3 cells Source; Wistar Rat submaicHlarv Eland
Ligand: 0-05 |iM[iHlCGS-21se3 LiEMd-. O.JSnMI"HlfrijoSin
v»ide: 1 SDMSO Vebicle: 1 X DMSD
incubMion limetemp: 90 minutEiS 2S t Incubation iims/remp: 60 minutes # 25 "C
tncubaiion Buffer SD mM Tris-HCI, pH T.H, 10 mM Incubation Buffiii; 50 mM Tris-HO. 0.5 mM IDTA. pM
MgClf. 1 mM TDTt 2 U/inl 74
Adenosine Cteaminaw Nonspecific LiBaitd: ID iiM Phtnlolamine
nonSpecitie l«^a," SIf yM W£CA Kj." 0.)7nM-
x.: 0064 wW 6^ 0.16 pmole/iT^ Ptotan*
Bw ? pmofertnE Frnwin" Sptxifit Binding; 90*-
Sp«ific Sirtdinj" BSi- Qiianliiation Method. Radioiigand Binding
Oumtilalion Meihcxi: i"aaiOl!fW"() B"nding Significance Criteria: s 505 Of m» Slimulalion or
Signidcance Cnteria: k SOS of m,u iiirriolaiion or inhibftiQn inhibition

203200 Adrenergic a,s
Source:
Ligard:
Vehicle:
Incubation Time/Temp:
Incubalion Buffer:
Nonspecific Ligand:
Specific Binding: Quantitation Melfiod Significance Criteria.

Wiilar Rat iiver
0.25 nM \>»] Praiosin
1 %QMSO
60 minutes @ 25 "C
SO mM Tri5-ilCI .0. 5 mM EDTA, pH
7,4
10 [iM PheMoiamine
0.31 nM*
O.IB pmole"mg Protein"
90ft ■
Radioligand Binding
? SDm of maK slimulation or
inliibttion



203400 Adrenergic a,o
Source:
ligand.
Vefiicle
IncjbatinnTimeiTemp.
Incjbatior Bjffer
Nonspecific Ligand;
Specific Binding: Quantitalion Method: Significance Criteria:

Human recombinant HEK-293 cells
0.6 nM I"M] Prazosin
1 % DMSO
50 minutes @ 2S °C
SO mM Tris-HCl
10 )jM Phentolamine
0 SB nU •
0 17 pmole/mg Protein"
8CB."
Radioligand Binding
? SOS of ma» 5timijlation or
inhibition

Human recombinant iniecl Sf9
cells
1 rM["HlMK-512
1 % DMSO
60 mmutei i? 25 °C
7SmMT|-i5-Ha pK 7.4. 12.5 mM
MgCfj, 2 mM EDTrt
IOilMWB-4101
0,6 rM ■
4.6 pmoie/mg Protein"
9SX"
Kodioligand Binding
? SO* of max stimulation or
inliibiiion
Human cecombinant CHO-Kl cells
2.S tiM pH) Rauivolscine
\ % DM50
50 minutes ^ 2S "C
50 mM Tris-HCl, 1 mM EllTA. 12.5
mM MgCI,. pH 7.4. 0.2X BSA at
10 pM Prarasin
7A nM "
2.1 pmole/mg Protein*
90% ■
Radioligaiid Binding
> SOS of mss stimulation or inhibition
■ 203620 Adrenergic a,j,
Source:
lieand;
Vehicle;
Inrubailor Time/Temp:
Incubation Buffer
nonspecific Ligand:
Bmi,:
Specific Binding: Quanlilalion Mel hod: Significance Criteria:
■ 2D3710 Adrenergic a.~i
Source:
Ligand. Vehicle:
(ncubation Time/Temp. Incubalion Buffer
Nonspecific ligand:
Specifc Bi:iding: Quanlilatior^ Welliod Sign if can ce Criteria:

204Q10 A dtenergic §t
iojrce:
l«jnd:
vehicle;
incubsWm TimwTcmp:
IrvtubalKjn Buftw:
fJMSp«ifie tiBjPd.
Spetilic Binding-Quantitaiion MctbmJ: SiEnificance Ciiicria:

Human retomlsirdri ttei tiS cdh 0.D3 fiM [I"ll! Cyjnocmitolnl
1 KDMSa
2 houri If 25 "^
SOmMTris-HCI.SmMlDlA, 1.^ mM CaClj, 120 mM NaCI. l.t nM MCQrtilc acid. 10 mg/|, nyi. pM 7.4 lDOpMS(-)-P^opraniilol
O.O"ll nM "
0.0?1 pmole"mg Frotan"
95«-
Radioligand Binding
i VH, of rnai ilimulation or
inliibition

204 no Adrenergic ft
Source:
Litand
Vehicle:
Incubation Tima"Temp
incubaiiCBi uuftc:
Non5i)etifi<: l> a™-
Specific ^irdins:
QuBntilation Method: SignificanM Criteria:

Huiosii recombinant CHO-NBRf cells
0.2 nM PHIC6P-I;I77
I % DMSO
&)niiinijtei!I>2S"C
ED mM Tris-HO. 0 5 mM EDTA, 5.0
mMMgClj, l2nmMNaCl.pH7.4
10pMICI-nS55l
D.*l nM •
0,a3J pmola"iTig Protein"
551 •
Radiotigard Birvling
i 50S of mas stimulation or
inhihilion

212SO0 Bradykinin B,
Source;
LigiSid:
VthiCle"
incuMiofiTimoTemp:
fnuithalton Buf^":
MonipecirK: Lig^ind:
Specific Binding: Quantitation Method: Si^ificante Criteria.

Human Hi7i9 ceUs
2,5 riM I"H) (Dei-ArE"TKaltidir
1 t DMSQ
6D minutei g 2S "C
JO mM HEPES. 525 mW N-malrtyl-D-
Elucamine. 5 mw KCI. ! niM 1,10-
Phwwrthroiine, pH 7.^
10 |JM (D«-APE», Lcji^-Oradykinin
O.S nM ■
0.059 pmole/mE Pralein"
RadldiEard Bindme
£ SDK of max Ibmutation or
inhibition

212610 Bradykinin ft
Source
L^and:
VeluFli?:
Intubjtion Tiine/Ttmp
incuPaiion Buffer
NonSpwiiftc L«*>d;
Spetific BinainR OjantiUTion Method . SiBiTlicmce Criteria:

HLiTian rKond«nanl CMO-Kl cdli 0.: nM I"Hl Bradykirjn
1 SDM50
9Q minutes g 25 °C
24 niM TES-NH.OH, pH 6,8.1 irM 1,
JO-phenarthrolin*. 0.)* 6SA
S iiifl Bradyfcijiin
0,i9 nM -
2 pmote/nig Prolei"i"
9QS ■
RatJioiigand Bmdir^
■£ SDK of mat stimuiatkin or inhibition



2I4SJ0 Calcium ChannelL-Type. Benzothiszepine

214600 Calcium Channel L - Type. Oihydropyridine


Sail fee: WiRiir Rat bi^ain Source; Wislar Rat cerebral cortEi
L^ard: 2 nM phl| Oiitiaiem Ligand; 01 iiM pM) Niircivdipiiic
Vehicle: 1 SDMSCi MtttfUr- 1 icDMSO
loail>31ion Time/Temp: 3 houn @ ^ "C iiicubatran Time/Teinp: 90 minuies @ 25 ^C
lnait»tion Buffer 5n mM T(i!-MCI. 0.1SBSA,pH 7A InculMlion Su"fw: SOmMTris-HCl.pH7.7al2S"^
aliS^C NnnSpecific Ligand; 1 tjW NifediDine
FJonSp«:if c Ugand: lOpMOillimem 0J31&PM" K,: aiB nM •
0 2J pmolE/tng Protein"
Bon.; QJi pmole/mgl^otein" Spei^fic Binding: 91S-
Specific Rinding" Quantiiaiian Mettiod: 73*"
RadiC^igand Smding Quanijlalion MMhod: Significance f.nteris" Radio^and Si riding
£ SO* of mai stmiitaticft or
Significance Criteria: 2 SDK of mis itimiiljlioo or ■nhit»[ion inhibition

216000 Calcium Channel N-Type ■ 2J9500 Dopamine D,
Source: wistar Rat btairi ftontat lobe Source:
ligand: 10 pM ["^Hi (i).C«nowim GVIA ligand:
Vehicle. 1 S DMSO i/ehicLc:
infub;»iion lime/Temp; 3C minutes g 4 "C intruhalion Time/Tenio;
incubation Buffers ?OmM Trii-IICI. p"7.a.0.51ias^ Incubation Buffer
NonSpecrfic Ligand; 0,1 pM io-ConotO»in GVIA
u O.OSl nM ■
Dniii; 0,88 DmolC"rng, Piotcin" Nonspecific Li&and:
specific Binding: 96* * K.:
Quantitation MeihiKJ; Radioligand Binding "rm^
SigniSf ance Ciileria: ;: SO* of ma" slsniuljtion cr inhibition Sp«i(ic Binding: QuwHitaiion Method:
Significsnce Criteria:

Humai" recombinant CHO cells
l.*nM["H)SCH-3S390
1 % DMSO
J hours i? 37 "C
!0 nlM Tlis-MCl, pH 7,4. 150 mM
NaCl, 1.4 mM Ascorbic Aod,
Q.DQ1K BSA
!0|jM{-)-BulBtlamQl
1,4 nM •
0 53 pmole"nig prottin"
ftadiotigand Binding
J 50% of ma inhihiticxi

219600 Dopamine D^i
Source
Llgard:
Vehicle:
Incubation Time/Temp.
IncuBation Buffer
Nonspecific Ligand:
Specific BinQing: Quanttlation Method:
Sign I finance Criteria:

Human recombinant CMO ceils 0.1S rM I"H] Spiperone
1 % DM50
2 hours @> 25 =C
50 niM Tri5-HCl, pH 7.4, 150 mM
Natl, 1,4 mM Ascorbic Acid,
a.ODlX B5A
10 pM HalPperldol
0.08 nM •
O.Ag pmofa"mg Prolein*
85%"
Radioligand Binding
^ 50% of mai stimulation or
inhibition



219800 Dopamine Dj Source:
Ligand:
Veliicle:
Incubation Time/Temp
Incubation Buffer:
Nonspecific Ligand: K,:
Specific Binding: Quantitation Method: Significance Criteria

Human recombinant CHO cells 0.7 nM ["HI Spiperone 1 % DMSO ; iiours @ 37 »C
50 mM Tris-HCl, pH 7.4, I5D mM NaCI. 1.4 mM Ascorbic Acid, 0.001% BSA 25 pM Si-)-Sulpiride 0.36 nM •
1.1 pmole/mg Protein■ BE5t•
Radioligand Binding J 50* of max stimulation or inhiliition

219900 Dopamine D,j
Source:
Ligand.
Vehicle;
Ircubarion Time/Temp"
Incubation Buffer:
Nonspecific Ligand:
Specific Binding: Quartit^lon Method: Significance Criteria:

Human recombinant CHD cells O.S nM pH) Spiperone
1 % DMSO
2 hours ^ 25 "C
50 mM Tris-HCI, pH 7.4, 15D mM
NaCf, 1 4 mU Ascorbic Acid,
D.001% BSA
10 \iM Haloperidol
QJ7 nM ■
1 pmcie/rng Protein*
go*-
Radioligand Binding
i 50* of mai stifnutation or
inhibition



224010 Endothelm
Source:
Ligand:
Vehicle:
Inculcation Time/Ternp
Incubation Buffer
Nonspecific Ligand:
Bm>i:
Specific Binding" Qjantitation Method Significance Criteria:

ET.

Human recombinant CNO cells 0.Q3 nM ["^U Endolhelin-1
1 S 0M5O
2 hours @ 37 fC
SO rriM Tris-HCI, pH 7.4. 0 5 mM
CaCI„ 0 OSS Tween-20, 1 mg/ml
BSA
0,1 (iM Endothelin-1
OOiSnM ■
Q.35 pmole/tng Protein*
90%*
Radioligand Binding
2 SO!t of mai stimulation or
inhibition


224UQ Entiothelin ET, ■ Z2550a iptHcrrmi Growth Factor ff6"7
Snwct
Ligvid"
vehicle:
ifiCobitiOh TirTie/Temp:
Incubation Buffer: Human recnmoinant CHO-KI Cfll5 0.1 i!h4{"M| [ndothelin-l
1 » DMSO
2 houri @ 2S «C
SOmMHlPES. 1 inMCarb.S mM Soiarr
nesw
wiiicle:
hcLbJti^n rime/Tcmg
l"icul]an:Hi Ru^r hunian A431 tvW^
a.Oi nM ["»ll ipi*inii| ^^mwh
fa-nr ^ItF) (t/unrri
s) iiinui« B ;; t
55 ml* HErfS. (M niM Sa^. 5 niM
MgOj. 0.^» BSA. DM 74 ICO 1.2 mM MCC. U mr/ Ca:L
WonSp«it.c Liganii: (pioieaw hec)
0.1 pW Endolhelin-l
n.DBS nM •
4,J pmole"nig pioiem" )Jon! la nrri F;id?rra8l Siomh Fjcio"
{ICtft flxirnar^
0.D3: nM •
0.) 1M ■
Specific Binding; 7SX* B^i. 1 UBiole/fnj rroisn*
Ouaniitaimn Mtlhod: Rsdii^iEiod Binding S...,. ■a.k jmolp/m£ f rul^i-i-
SignifiMnce cnlera- a SW of mai !iimulalion or in^vibiticn OuJI^iiaion Mcthnd"
S^nifkjncc Crilcria f so* or rrtii" HirrwJBtijT 0.

226500 GABA,. Agonist Site
236010 Estrogen £Rcr Ligand:
Incubatjon Tirnc/Tcmp:
liKoluiion Buffei:
NorSpecrfic Ugand: «»:
5[)«ific Binding: Quanittaiian Met)^od: SiEnificance Cflteria.

Human recwnbmant inscin SfS
cells
0-5 "M |!H! Eilradiol
J K DMSO
2 houfs @ !5 "C
ID fTlM IritHCl. pH 7.5. 10*
Glycerol, l mM DTT. 1 mg/mt B5A
1 liM Dieihylstilbwirol
0.i nM -
1100 [imcle/BiB fratwn*
BS«"
Radiol^and Sindlng
£ SDX of mat cumulation cr
inhibiljon

lij^nd^
IncLballon TimefTemo; intubsthon Buffer. tJnn5p«<:ilk: l> Specifif Binding; Quant Italian Melhixl; ^igntficwce Crttcns

Winar Rat biain (minus
cwebellum)
1 nM ["HI Muscimol
IS DMSO
10 minulES g- fl "C
lO mM Trrt-HCl pH 7.4
0.1 uM MuKimcil
3.8 IM •
I.Bpmole"nig Protein"
90* •
Radioligand Binding
S 50% Dt man ilimuJJtion cw
inhibition


Z?6fi(?£? G/^Mi. Ben. zodiazepine. CmtrsI ■ 2285iO GABA,. Non-Selective
Snurte: Wiiur Rat brain (minus Source: WistarRat brain
cerebellum) Ligand. D.6rK1|"HlCGi"-5*62&
LiEand: 1 nM ["HJ Fluniuarepam Vehicle; lltDMSO
Vehicle. ISOMSO Inasbxiari JinK/Temp: 20 mioutct @ 251
Intubatlan Timen"Biia: 60 minoies ^ 35 "C Incubation BuHcr: SO mM TrJi-HCl. 2.6 mM CaCI,. [
incubMion BuHer: SO mM Wa-K Ph05phale, pH 7.4 Jl 25°C
MonSpeicirc Lipnd: 10 pM DisiEpam Nonspecific Llgand loatiMCGP-54616
Kjv 4.»nM« K(; 2 3 nW
Kdt; O.itlM" 6"a^ 1.1 pmole/mE Protein"
BJUIT" 1.2 pinoie/n^ Protein* Specific Binding: BOS"
&mjii: 4.1 pmofe/n^" Quijntilstion Method. Radioligand Binding
5p«iric Binding: em- Signiftance Cnteria: ^ SQK of man itimulalffin or
Quiniitaiioo Met^tt: Radioligand Binding rnhibition
Sisnificance Criterij. > 50% of ma* stimulation cr
inhibit ion
2S20W Glucocoitko"id

Socrce
lie***"
WehKle
I"lcubat"ijn Tmr/Temp:
lncu6M>on duller.
fJonSpKifii lijaid;
Specific Blndmg. Qtiwlitilioit Method, S(grviScance aiieria.

Hu^nan Htta 53 ceiii
I^Dr^SO
2 hours S> 75 "C
RPM1 10«P. 10 mM MEMS. pH 7.!
2D pM Oeiampthiione
SnM*
SI 000 Ileit¬is*. •
RadiCHiKiBKi Bin*ry.
i K)»crfnia>S("nu(a(ionor
in"iibi^itin

232700 Glutamate, Sojrte:
Ligand:
Vehide;
Incubation Time/Temp
Incubation Buffer
NonSpeciOc Ligand:
"iT»ai-
Spacific Binding: Quantitation Method Significance Criteria:

Kainate
Wistar Ral brain (minus
cerebellum)
5 nM PH| Kainicacid
1 % OMSD
60 minutes @ 4 «C
SO mM Tris-HCI. pH 7.4
lOODliML-Glutamate
0.012 [iM ■
0.35 pmole/mg Protein"
BOS"
Radioligand Binding
k SOS of mai stimulation oi
inhiibiton

232810 Glutamate.
Source:
Ligand:
Vehide:
incubation Time/Temp:
Incubation Buffer,
Nonspecific Ligand:
K„:
Specific Binding. Quantitation Melhiod: Significance Criteria:

NMDA, Agonism
Wistar Rat cerebral Corten
2 nM !"H1 CGP-SgfiF;?
1 % DMSO
20 minutes @ 4 "C
SQmMTiis-HCLpH".4
1000 |JM L-Glularriate
0.019 kiM-
2,3 pniole/mg Protein"
70S-
Radioligand Binding
a SOS of rna> stimulation or
inhibition



232910 Glutamate,
Source:
Ligand:
Vehicle:
Incubation Time/Temp:
Incubation Buffer:
Nonspecific Ligand:
Specific Binding: Quantitation Method"
Significance Crileria:

NMDA, Glycine
V^star Rat cerebral cortex
0,33 nM |"H|MDL-10SS19
1 % 0M5O
30 minutes @ 4 «C
50 mM HEPES, pH 7 7
10 [iM wnL-10S5l9
6 nM ■
3.7 pmole/mg Protein"
8SS-
Radioligand Binding
£ 50% of rnaii stimulation or
inhibition

233000 Glutamate. Source:
Ligand:
Vehicle:
Incubation Time/femp"
Incubation Buffer.
Nonspecific Ligand"
K„:
Bnu.:
Specific Binding:
Quantitation Method:
Significance Criteria:

NMDA. Phencydidine Wistar Rat cerebral cortei 4 nM CH] TCP 1 S DMSO 45 minules @ 25 °C 10 mM Tris-HCI, pH 7.7 1 iiMDiiQlflpine(WK-801) B.4 riM "
0.78 pmole/mg Protein" 94S-
Radioligand Binding ? SOS of mai stimulation or inhibition

■ 2396W Histamme H,
Source" Human lecombinant CHO-Kl Celi5
LiEsnd; 1.3 nM pHj fyilamine
Vehicle: 1 % DMSO
Incubalion Time/Temp- 3 liours @ JS "K:
In cubsl ion Suiter 50 mM Tris, pH 7,4. 2 mM MgCI,,
too mM NaCI, 25D mM Sucrose.
Nonspecific Ligand: I jiM Pyrilarriine
"0^ 1 1 nM ■
B™.: S 7 pmole/mg Protein"
Specific Binding, 94% ■
Quantilatiop Method. Radioligand Binding
5ignific»>ce Criteria. a 5D* of msi. stimulation or
inhibition
■ 239710 Histamine Hi
Source: Human recombinant CH0-K1 cells
Ligand: 0.1 rM |""l| Aminopotentidine
Vehicle: 1 S DMSO
Incubation Time/Temp: 3 liout! @ 25 "C
Incubation Buffer: 50 mM KHjPO,/ NaiHPO,. pH 7,4
Nonspecific Ligand: 3 pM Tiotidine
K^ 0.4S nM •
B™.: 6 9 pmole/mg Protein"
Specific Binding: 90*-
Quantitation Melliod" Radioligand Binding
Significance Criteria: a S0% of ma> stimulation or
inhibHior

H 239810 Histamine H, ■ 241000 imiiSszoiine I,, . Central
Source: Human rerambinani CHO-Ki cd Eotirce: Wlstar !(al cerfWsl coriei
Ligand: 3 nM [JM] ii(-)-a-MeitiylliiStimini Lieand: 2 nM I"Ht Idamun
(ftAWH) vehicle: 1 % DMSO
Vfi Incuiwtion TimEyTcmp: 90 minutes 0 15 "C Incubation Buffer: SOmMTris-Ha.Q.BmM EDTA, pH
tncubttion Outfci: SO mM Tril-HCl, pH 7A ID liM 7.4 31 \y MgQ,, 0.041 as A nonspecific Ligand, 1 pM lda?o Non5f>«ifiC Ligiind 1 pM R(-5-a-MEtliy!hi!taminG 1^ 1 rM ■
(RAIUIH) B-i^: 0.14 pmole/mg Protein"
x.- 7 A nM • 5peatic Binding: 85% *
»->«: 4.2 pmoie/mE P"oicin" Quantiwiion Method: Radioligand flinding
Specific Rinding: 95«- Significance Criteria. f. 50\ of man slimulaiinn or
Quawiiiiion Mciiiod. ;tadictig.and Bitvling inhibitksn
Significance Ciileria: 2 501 of man stimulation or nhitiiliort

2435}0 (nterleukin It-
liRand, Vct>ide: incLbaliO" Bulfei:
NORSpccirc Ligjnd:
lO
r>j3eciit Binding: Oiiam*flt>on Mel had: Sifinificante Cnteria:

1, Non-Selective Mouse 3T3 ctlls
10 pM (i"l| Interleukin-la ([L-
1 %DMSO
7 lioiirsig37"C
HPMI 1640, 20 mM HEP£S, 0.1S
sodium Aiide, 1% BSA. pH ?.!.
0-03 MM lnlerteukm-10 (IL-IcO
fapM"
3.? fmoWiTiE ft-olein"
70*-
RadioIiEdnd Binding
2 50* of ma« i^mulilion at
l^^lbllir^"^

iJCftfJC Leukotriens Source
lijand:
Vehicle:
IncijbBtio" Time/Temp:
rncubilion Buffer
NonSPN"fn ligand:
SpMifii: Binding: Qujnlitabon McihiKJ: Significance Criltru:

□uncan rtaftlcy dcnvcd Guit^ [ME lyng
0 2 nM ["HI Leuholrient D, ri.TD 1 i, DMSO
&D mimites^ JJ^C
5D mM Tr-i-Hd, 0,01% 8SA. $ mM
Caa,. S mM MBCU "OO Hg^niL
Bacitracm, 1 mM eera.vnidine.
0,1 mM Phenyimelh/Hilfnryl
fiLWride
D,l tiM LeuktHfirprie O^ (l!l>.)
0 2nM"
0,24 ptnole/mg Pfoiem"
85%-
Radiolieard Sinding
£ SOX of mat itiRiul^tloii or
inhitjition



252600 Muscarinic M, Source"
Ligand:
Vehicle:
Incubslion Time/Temp:
IncubittiQii Buffer
NonSpecifii: Ligand:
8 ma"
specific Binding: Quarlitaiion Method: Srgnifrcance CrrWria"

Humsn recombinari inseci 5f9 cells
0 29rM I"H] Metliscopolamine
1 % DMSO
60 mimjteE @ ;S °C
50 mM Tri5-na pH 7,4,10 mM
MECb. I iriM EDTA
1 pM Atropine
0.092 fiM ■
2.1 pmole/mg Protein"
95%*
Radioligand Binding
i 50* o" mai stimulation of
inhibition

252700 Muscarinic Mi So Lice:
Ligsnd:
Vehicle;
Incubation Time/Temp:
Incubation Buffer
Nonspecific Ligand;
K-
Specific Binding, Quantitation Method: SiEniticance Cute ha:

Human lecombinant insect 519
cells
0.39 rM [iH] Methscopolamine
1 % DMSO
GO minutes @ 25 =C
50 mM Trii-HCl, ijH 7,4 10 mM
MgCI,, 1 mM EDTA
1 |iM Atropine
0.16 nM -
4.9 pmole/mg Protein"
95*.-
Radioligand Binding
? 50S of mai stiinulation or
inhibition

252800 Muscarinic M^
Source:
Ligand:
Vehicle:
Incubation Time/Temp:
Irituballon Buffer:
Nonspecific Ligand-
Specific Binding Qjantilalion Melfiod" Significance Criteria.

Human recombinant insect 5f9
cells
0.29 nM I"H] Methscopolamine
1 % DMSO
60 minutes @> 25 "C
SO rnM Tris-HCI, pH 7.4.10 mM
MgCI,, I mU EDTA
1 (iM Atropine
0.07B rM ■
3.2 pmole/mg Protein"
96*-
Radioligand Binding
a SOS of mai stimulation or
inhibition



■ 257000 Neuropeptide
source: Ligard:
Vehicle.
incubation Time/Temp:
Incubation Suffer
Nonspecific Ligand"
Bn«j:
Specific Binding: Quantitation Meth oil " Significance Criteria:

Yt
Human SK-N-MC cells
0.0)3 nM|i"!) Peptide VY
1 % DMSO
4S minutes @ 25 "C
HBSS, 2 mg/mL B5A. 1 mM MgCI,. 1
mM CaCI,
0,1 nM Neuropeptide V (human,
rat)
0.62 nM -
5BOO R/cell Receptors/cell"
85%"
Radioligand Binding
a 5DX of mai stimulation or
inhibition



2571W Neuropeptide
Source:
Ligand:
Vfhicte
IntutBtion Time/Temp;
ineubslion Dufie":
NQnSpKific tigand:
Spccihc Sindins; Qiwulitmion Method: SiKoiftcanCB Ortetia;

Hujni.1 ICAN-TS neujohlastoma cell:
lOpM I""!] Peptide YY
1 S DMSO
; hooH ® ^t "C
3S nM HEPES, 2.S mM CiCI,. 1 mM
MKCIJ. CIS Batitracin. pH 7.*
1 uM Meufoptptidc Y 113-36)
(porcine)
0.012 nM •
0,5 pmolyiTie Protein-
90X-
RKJiolJglnd Binding
S 5IB. of ma» Stimulation or
inhibition

258590 Nicotinic Acelytcholine

5oiirtt Human tMit-3J tells
t igand: Q.l nMi"SiJEpibatiriine
Vehicle: 1 K DMSO
liwubilion TirRft/(*;flp: 6Brninutc392^"C
Incubation Buffra: 5D niM Tos-MO, pH 7,4
Nonspecific L^and: JOB pM (-)"Nicoliiie
K, QJJ nM "
B™.: 0,46 pmote/mg Protein*
Specific Sitiaing: 97K-
Qu9n[>l3li«rt Method: Radiohgand Binding
Signitkance Criteria: E 50* of mil stimutatiim or
irthibition

J60110 Opiate 5 Vcnidc.
i(« 1^5.1 lioii T"liip/Trmpt
OujntitaiKxi Udlisil !-^lrTi(i«rKf Criteria

OOP}
HLman retcrabinani DHO C^S
) sfflwSO
1 hogn i?> Ji "C
SO mM Trij4(CL S nA( MEa> pM
yi
0.49 nM "
8-6 pMDlWmg Prolton"
BO*"
RMiOi^nd Brndidg
i hsit, of mai iiimuijimn or
irhjbrjnn

260210 Opiate K (0P2. KOP)

Source:
Ligarid:
Vehicle:
Incubation nme/Temp;
IF"cuballon BjHer
Nonspecific lieand:
SppcifiC Binding: QLantilation Method:
SLgnificance Criteria

Human recombmant KEK-293 ceils
0 6 nM i^HI Dipfenofpbinc
1 S DM50
60 minutes @ 25 ^C
SO mM Tris-HCl, pH 7.4
10 (JM Waloione
0 4nM-
1.1 pmole/mg Piotein"
50%"
Radioligand Binning
J 501i of max stimiilation or
inhibition



260410 Opiate p (OP3,
Sojrce-
Ligand:
Vehicle:
IhCiitiation Time/Temp.
incubation Buftef;
Nonspecific ligand:
K„.
Bra.:
Specific Binding:
Quarititalion Method
Significance Criteria:

MOP)
I luman recombinant CHO cell;
0 6 nM [-H] Diprenorphine
1 1, DMSO
50 minjtes @ 25 °C
SOmMTris-Ha, pH7.4
10 pM Waloione
□ 41 nM*
3 8 pmolc/me Protein"
90*-
RadiQligand Emdine
S 50S of msj stimulation or
irihibition

2650iO Platelet Activating Factor (PAF)
264S00 Phorbol Bsler
Source:
Vehicle
Imubation Time/Tcmp:
IncoUatlod Buffei:
Nonspecific Lisaral.
^pecrfic Binding Quarliution Itlettiiid: Signilkdnce CiilF"ij:

ICRMouie&rsifi i (iM i"Hl POBu 1 % DM50
JQ mM Tris-HG conl*ring S fnU
C»Ci). lai 7,5.11 J5«r:
R7 riM •
25 pmrte/mg Prolein"
80* ■
RldiDliE^nd BindinE
e 50* of mw siimuiaiion o( inhitHtion

Sowce
Vfhkl*:
Inciibarian Time/l"emp:
Ihcubatkin Buffer
^on5pecif>c LiB*id:
6ini.:
Sp(!cifk: Binding: QusrKtaUofl Mcthcd: iiRfiificance CfilC"i.i:

Human platdeu
t % DM5D
J hours 01 J5 K:
50 mMTris-HCI. pH 7,4 JDOmM
KCI. S mU EOTA.S mM MgClj,
D.25%BSA{w/v).
1 pMPAF
0.13 ilM-
i;o R"cer
90S-
Kadioiigand Binding
^ SOX of itiai siimujs^n or
lAhtibiTion

265600 Potaisium Channel[K.jp]

Source Syrian hamster pancreatic twta
CeilsHIT-T!5
Ligand: 5 nM pHl Glil)er5clamWe
Vehicle- 1 t. DM50
Incubation Time/Temp: 2 hours @ 2S "C
Incubation Buffer 50 mM MOPS, 0.1 mM CaCI,, pH 7,4
Nonspecific Lieand: 1 JJM GlyburidE
K,: Q.64 nM •
B™." 1 pmoie/mg PrOlein"
Specific Binding: 90% ■
Quantitation Metliod" Radioligand Binding
Significance Criteria: ? soft pf nai Mimutation or
inhibition
268700 Purinergk Pi,
Source Mew Zealand Derived Albino
Rabbjt urinary liladder
Ligand: B nM ["H] o, E-Methylene-ATP
Vehicle 1 S DMSO
Incuoation Time/Temp: 30 minutes # 25 °C
Incubation Buffer: 50 mM Tns-HCI, pH 7.4
Nonspecific Ligand: lOOpMp. v-MelhyleneATP
Krt,: 2.2 nM ■
Ka.-: 2,2 pM •
Emi,!. 2 pniole/mg pfotein"
B"».: 790 omoie/mg Protein"
Specific Binding" SDS-
Quantitation Method: Radioligand Binding
Significance Criteria: > SOX of mai stimulation or
inhibition

268810 Purinergic P;,
Source:
Ljgand:
VEfiicIf
Incubation TLrne/Temp:
Incubation Buffer
Nonspecific Ligand:
Specific Binding: (Quantitation Method: Significance Criteria:

Wistar Rat brain
D.l nM ["51 ATP-oS
1 % DMSO
60 minute? @ 2S "C
SO mM Tns-HCl, pH 7,4
10 fiW ADP-PS
O.0!5pM"
15 pmole/mg Protein"
B71"
Radioligand Binding
a 50% of man stimulation or
iniiibiticjn



271110 Serotonin
Source;
Ligand:
Vehicle:
incubation Time/Temp:
Incubation Buffer
fJon Sped fit Ligand
Specific Binding: Quantitation Method: Significance Criteria:

(S-Hydroxyiryptamine) 5-HT,^
Human recombinant CHO cell?
l.SnM["H)8-0H-DPflT
I % DMSO
SO minutei @ JS "C
SO mM Tns-HCl, 10 mM MRSO.. D 5
mM EDTA, 0.1« Ascorbic Acid, pH
7,4
10 |jM Metergoline
? nw
1.3 pmole/mg Protein"
75S-
Radioligand Binding
£ 50% of mai stimulation or
inliibiiion

271910 Serotonin
Source:
IJgand:
Vehitle:
Incubaton Time/Temp:
Incubation Suffer
Nonspecific Lieand:
Ka:
Specific Binding: Quantitation Method: Significance Criteria:

(5-Hydroxytryptamine) S-HTj Human reccmbinant HEK-293 cells 0.5S nM [!Hj GJf-£5630
I 16DMS0
60 minutes @ is "Z
5D mM Tris-HCI, pH 7.5. 1 mM
EDTA. 5 mM MgCI,
10 tJM MDL-72i22
0 2 nM-
II pmole/rrg Protein"
90%"
Radioligand Binding
> 50* of ma* stimulation or inNbitiQn



278110 Sigma a.
Source.
Ligand:
Veliicle.
Incubation Time/Temp:
incubation Buffer
Nan Specific Ligand:
K=:
Brriii:
Specific Binding. Quantitation Metliod. Significance Criteria.

Human Jurkat cells 8 nM ["HI HalOfieridol
1 1, 0M5O
4 hours @ 2S "Z
5 mM K,h("0,/KH,F"0, buffer pH 7.5 10 UM Halopendol
5.S nM ■
0.71 pmole/mg Protein-
ao**
Radioligand Binding
2 SOS of man stjmulalion or
inhibition



■ 279450 Sodium Channel. Site 1
278209 Sigma Cj
S^Ul^^e:
ligant
Vehirie.
Inajtelion Time/Temp:
Incubation Buffer.
MonSpecific Ligand:
9r-ij*:
Specific Binding: Quant I t»l ion Method; Sie"ircance Crrttria"

WiSlar Rat t>rair.
J nM I"M] iftnprodil
IJUJMSO
&0 minulese37 iO mM Tri!-nQ. pH 7,4
10 \AA Itenprodtl
^SnM-
1,3 pmole/mB Protein*
est"
Radioligand Bnndir^
± S0% of max itimulation or
inhibition

Source:
t.iQancl;
Vehicle:
incubiifion Tiiiie/ienip"
Incubation Suffer:
MonSpecilic Ligand:
K,:
^p^cjfc Binding: Quantitation Mmhodi Srjiiiricjnce Criteria:

kViiUr Rat brain
InW [-"HI SMitonn
l»DMSO
3D mirutti @ 4 t
11) Homogeriz3iion buffer: 140
mMNaCI. ;0mMJrii.HCI.pH71,
1 mM PMSF 0> 75 mM HepesySflO
mM NJCI. pK 7.S (3) Asiay
buffer, homoEenu"aiion buffer to
buffert;)i5l:4
lO^iMTebodtJIoiin
1.4 iiM -
3.7 pmole/mg Protein"
ws-
Rsdioligand Binding
^ sot of TTUX stimuiation or
inhibition

2555W Tachykimn NK,
Hunan fecotnbinsnt CHO cells
0.2SnMpH|5R-l*D333
t %DMSO
90niinjtei@!S"C
30 mM H£PES, pH 5.4, I ml^ MnCl,.
0.01? BW
2 |1M 1-703,606
O.S nM •
10 pmole/mg Croiein*
B51"
ftadiollEaid Binding
2 ^[W of mai stimulation □>
inhibiliMi
279510 Sodium Channel. Site 2

Source Winar Rat bnin source;
llg^nd: 5 nM i^H) BjlrscliO"miiin A 20-a- ijeamd.
Bsiioate Vehicle:
Vehicle. 1 SDMSO IncubnliOn riineiTcmp:
incubalfon Tifne/Temp: &0minut«@37"C InculMiisn Buffei:
Irtfubatkift Buffer: 50 mM Tnf-HCl, pri lA at IVK..
WJ mM MfPES. no rnM cholme-CL NraiSpecific Ligand:
S.4mMKCI.0.8mMM(SO.."HiO (or MgClj), 5,5 mM GiuCOW, 40 Bnup-
HE"""! LqT» SpecifK binding;
NooSpttific tigand: 10aHlvlUC"" •u 0.052 pM ■ Significai^ce Criteria:
a™: 0.7 pmole/n^ Protein*
Specific Binding: T7S*
Quantitation Method: Radiorigand Binding
SiBnificance CitcriK £ un of max nimulaiiofl cr intiiliition

28S0J0 Testosterone
source:
Liesnij.
Vehicle:
Incubation Time/Tans
lnciibalk»i Buffer
NonSptcJtic Ligand: Specific SindinE: Duantiwtion Method: Significance Criteria:

Hal recomblnait [. ccAI.S nW ["HI Mibde(0!ie
1 S DMSO
4 hours @ 4 "C
50 mM Tni-ltCI tpH 7.5), 0.B M
NaG, IQI Glycerol. 2 mM
Dithidihreitol. i mg/ml B"JA and
2X Etiianol
10 pM M.holeione
3nM-
9J0 pmole/mg Protein"
90%"
Rad^liganQ Binding
!; 50S of mai slimulMion nr
inhibilion

220320 Transporter.
Source.
L^aiid:
Vrtiick
Incutation Titne/Temp:
IncubBlion Buffer-
Nonspecific uigana:
•ipKific BJfidinE-
Qujntitaiion Method: Significance Ciitera

Dopamine (DAT) Muman cetombinant CHO crili 0.15 nM (""If Rn-55 1 XDMSO 3 houfi S? 4 ! too mM mC\. 50 rtwul Tri(-HCI. 1 pMLcupeprin. lOpMPMSF. pH7.4 EO |JM NumifFn^inE D.58 nM -
0M7 pmole/mR PrOiern" 90S"
Radiusigand Binding S S0% ot mai itimulation or inhiCihion

204410 Transporter.
Vchitle:
Fncut»[iDr> Time/Temp: liMTulHrion Buffer
NnnSppcific Ligand:
ipeciiic Bindins Qu^tiUtKin Mclh
Norepinephrine INEJ}
Human lecombinant MDCK cell!
I «QMSO
SQmM T(ii-MCL lOT rnM NaCI. 1
|iM lEupcplin. TO |l^^ ""MSF. pH 7.4
10 fM Deiiipramine
D.024 (If^ •
2.5 prnrfe"ins Prolein*
J5!t •
RadiDli^nd Bind^g
i SW o( mai stiiiiulalioii or
itihibilKxi

274020 Trarjiporter. HydroxytrypUmme)
Ligand:
Vch"CliK
Incubation rimf/Temp: Incubaiion Bu"lei:
NoiiSpccific ligsnd:
a™..-
5pt(>rit ainding: Quantknion Method; 5ignlficin(e Ciiterix

Serotofjin (5-
(SfRT? • -
Humin tKorntjinwn H[K-191 ttlis
D.t5nM[""(| Rn-K
1 * DM50
3 houn @ 4 ic
100 mW NaCI. 50 BVM Trii HC!, }
|iM I.eui>e[Kiii. 10 (iM PMSF. pH i>i
10 pM iniipfsmrne
017rM"
D,41 pmole/mj Protein"
95*"
RadioLrgand Binding
f 505 Ol Biai itimutation or
inhibttiod



226400 Transporter,
Source:
Ligsnd:
Veiijcie;
InculJation Time/Temp,
IncuBation Buffer
Nonspecific Ligand: K,:
Specific flinding: QuantilatJQn Method: Significance Criteria-

GABA Wistar Rat cerelirai cnriei 6 nM pH] GABA 1 t DMSO 20 minutes @ 25 "C 10 mM Na-HEPES, 120 mM NaCl, 4 mM Ca Acetate, 10 UM isoguuacine, 10 [JM (-JBsclofen, pH 7 5 10 IJM NO-71] 0,3 pM •
50 pmole/mg Proteir-80* ■
Radioligand Binding £ SOS of ma
" Historical Valii^?
Literature Refereaces:
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):
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.
Revised 27 September 2000).
118060. Crespi, C.L., Miller, V. P. and Penman, B.W. (1997) Microtiter plate assays
for inhibition of human, drug-metabolizing cytochromes P450. JKnal Biochem 248(1):

188-
190.
Gentest Technical Bulletin (Version 4.2: Revised 27 September 2000)
A high throughput method for measuring cytochrome P450 inhibition. Genlest
Technical Bulletin (Version 4^: 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):
188-
190.
Gentest Technical Bulletin (Version 4.2: Revised 27 September 2000)
A high throughput method for measuring cytochrome P450 inhibition. Genlest
Technical Bulletin (Version 4-2: Revised 27 September 2000).
118080. 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.
Genlest 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).
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 248M:
188-190.
Gentest Technical Bulletin (Version 4.2: Revised 27 September 2000) A high
throughput method for measuring cytochrome P450 inhibition. Genlest Technical
Bulletin (Version 4^:
Revised 27 September 2000).
200510. Libert, F., Sande, J.V., Lefort, A., Czemilofsky, A., Dumont, J.E., Vassart, G.,
Ensinger, H.A. and Mendla, K.D. (1992). Cloning and funct"iotxal characterization of a.
human Al adenosine receptor. Biochem. Biophys. Res. Commun. 187:919- 926.
200610. Varani, K., Gessi, S., Dalpiaz, A. and Borea, P. A. (1996) Pharmacological
and biochemical characterization of purified A2A adenosine receptors in human platelet
membranes by [3H]CG521680 binding. Br. J. Pharmacol. 117:1693-1701

203100. Michel, A.D., Loury, D.N., Whiting, R.L. (1989) Identification of a single alA- "
adrenoceptor corresponding to the alA-subtype in rat submaxillary gland. Br. J.
Pharmacol. 98:883-889.
203200. Garcia-Sainz, J.A., Romero-Avila, M.T., Hernandez, R.A., Macias-Silva, M.,
Olivares-Reyes, A., Gonzalez-Espinosa, C. (1992) Species heterogeneity of hepatic al-
adrenoceptors: at A-, al B-, and at C-subtypes. Biochem. Biophys. Res. Comm. 186:760-
767.
Michel, A.D., Loury, D.N., and Whiting, R.L. (1989) Identification of a single al A-
adrenoceptor corresponding to the al A subtype in the rat submaxillary gland. Br.
J. Pharmacol. 98:833-889.
203400. Kenny, B. A., Chalmers, D. H., Philpott, P. C. and Naylor A. M. (1995)
Ciiaracferization of an ai D-adrenoceptor mediating the contractile response of rat aorta
to noradrenaline. British Journal of Pharmacology. 1X5;. 981 - 986
203620. UhlSn, S., Porter, A.C., Neubig, R.R. (1994) The novel alpha-2 adrenergic
radioligand [3H]MK912 is alpha-2C selective among human aipha-2A, alpha-2B and
alpha-
2C adrenoceptors. J. Pharmacol. Exp. Ther. 271:1558-1565.
203710. Lhlen S., Dambrova, M., Nasman, J., Schioth, H.B., Gu, Y., Wikberg-
Matsson, A., Wikberg, J.E., (1998) Alpha 213- and alpha 2C- adrenoceptors.
comparison with MK9I2, RX821002, rauwolscine and yohimbine. Eur. J. Pharmacol.
343 {\): 93-101.
204010. Feve, B., Elhadri, K., Quignard-Boulange, A., Pairault, J. (1994)
Transcriptional down-regulation by insulin of the b3-adrenergic receptor expression in
3T3-F442A adipocytes: a mechanism for repressing the CAMP signalling pathway.
Proc. Natl Acad. Sci. USA 91:5677-5681.
204110. McCrea, K.E. and Hill S.J. (1993) Salmeterol, a long-acting b2-adrenoceptor
agonist mediating cyclic AMP accumulation in a neuronal cell line. Bril. J. Pharmacol.
110:619-626.
204410. GaKi, A., De Fe)ice, L., Duke, B.-J., Moore, K., Blakely, R. (1995) Sodium
dependent norepinephrine induced currents in norepinephrine transporter transfected
HEK293 cells blocked by cocaine and antidepressants. J. Exp. Bio\. 198:2197-2212.
212500. Menke, J., Borkowski, J.A., Bierilo, K.K., MacNeil, T., Derrick, A.W.,
Schneck, K.A., Ransom, R.W. Strader, CO., Linemeyer, D.L., Hess, J.F. (1994)

Expression cloning of a human B1 bradykinin receptor. J. Biol. Chem. 269:21583-
21586.
212610. Eggerickx, D., Raspe, E., Bertrand, D., Vassart, G., Parmentler, M. (1992)
Molecular cloning, functional expression and pharmacological characterization of a
human bradykinin B2 receptor gene. Biochem Biophys Res Commun 187 (3): 1306 -
1313.
214510. Schoemaker, H. and Langer S.Z. (1985) [3H]Diltiazem binding to calcium
channel antagonist recognition sites in rat cerebral cortex. Eur. J. Pharmacol 1 ] 1:273-
277.
214600. Ehlert, F.J., Roeske, W.R., Itoga, E., and Yamamura, H.I. (1982) The binding
of [3Hlnitrendipine to receptors for calcium channel antagonists in the heart, cerebral
cortex and ileum of rats. Z,//e 5c/. 30:2191-2202.
Gould R.J., Murphy, K.M.M., Snyder, S.H. (1982) [3H]nitrendipine-labe!ed calcium
channels discriminate inorganic calcium agonists and antaggonists. Proc Natl.
Acad. Sci. USA 79:3656-3650. 216000. Moresco, R.M., Govoni, S., Battaini, F., Trivulzio, S., Trabucchi, M. (1990) Omegaconotoxin binding decreases in aged rat brain. Neurobiol. of Aging 11:433-436. 219500. Dearry, A., Gingrich, J.A., Falardeau, P., Fremeau, R.Tjr., Bates, M.D., Caron, M.G. (1990) Molecular cloning and expression of the gene for a human Dl dopamine receptor. Nature 347:72-76.
Sunahara, R.K., Niznik, H.B., Weiner, D.M., Stormann, T.M., Brann, M.R., Kennedy, J.L., Gelemter, J.E., Rozmahel, R., Yang, Y., Israel, Y., Seeman, P., and O"Dowd, B.F, (1990)
Human Dopamine Dl receptor encoded by an intronless gene on chromosome 5. Nature 347:80-83.
Zhou, Q.-Y., Grandy, O.K., Thambi, L., Kushner, J.A., Van To[, H.H.M., Cone, R., Pribnow, D., Salon, J. Bunzow, J.R., and Civelli, O. (1990) Cloning and expression of human and rat Dl dopamine receptors. Nature 347:76-80.
219600. Bunzo, J.R., Van To[, H.H.M., Grandy, D.K., Albert, P., Salon, J., Christie, M., Machida, C.A., "Neve, K.A., and Civelli, O. (1988) Cloning and expression of rat D2 dopamine receptor cDNA Nature 336:783-787.

Grandy, D.K., Marchionni, M.A., Makam, H., Stotlco, R.E., Alfano, M., Frothingham,
L, Fischer, J.B. Burke-Howie, K.J., Bunzow, J.R., Seiver, A.C., Civelli, O. (1989)
Cloning of the cDNA and gene for a human D2 dopamine receptor. Proc. NatL Acad.
Sci. USA 86:9762-9766.
Hayes, G., Biden, T.J., Selbie, L.A., and Shine, J. (1992) Structural subtypes of the
dopamine D2 receptor are functionally distinct: Expression of the clone D2A and D2B
subtypes in a heterologous cell line. Molec. Endocrin. 6:920-926.
219800. Sokoloff, P., Giros, B., Martres, M.P., Bouthenet, M.L., Schwartz, J.C. (1990)
Molecular cloning and characterization of a novel dopamine receptor (D3) as a target
for neuroleptics. Nature 347:146-151.
219900. Van Tot, H.H.M., Bunzow, J.R., Guan, H.C., Sunahara, R.K., Seeman, P.,
Niznik, H.B., Civelli, 0. (1991) Cloning of the gene for a human dopamine D4 receptor
with high affinity for the antipsychotic clozapine. Nature 350:610-614.
Van To[, H.H.M., Wu, CM., Guan, H.-C, Ohara, K., Bunzow, J.R., Civelli, 0.,
Kennedy, J., Seeman, P. Niznik, H.B., and Jovanovic, V. (1992) Multiple dopamine D4
receptor variants in the human population. Nature 358:149-1 52.
220320. Giros, B. and Caron., M.G. (1993). Molecular characterization of the
dopamine transporter. Trends. Pharmacol. Sci. 14: 43-49.
220320. Gu, H., Wall, S., Rudnick, G. (1994) Stable expression of biogenic amine
transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence. /
Biol. Chem. 269fl0):7l24-7130.
224010. Pharmacological characterization of a potent nonpeptide endothelia receptor
antagonist, 97 - 139. The Journal of Pharmacology and Experimental Therapeutics.
268: 1122-1127.
224110. Cain, M.J., Garlick, R.K. and Sweetman, P.M. (1991) Endothelia-I receptor
binding assay for high throughput chemical screening. J Cardiovasc Pharmacol 17
SuppI 7:
5150-151
Chiou, W.J., Magnuson, S.R., Dixon, D., Sundy, S., Opgenorth, T.J. and Wu-Wong,
J.R. (1997) Dissociation characteristics of endothelia receptor agonists and antagonists
in cloned human type-B endothelia
225500. Dittadi, R., Gion, M., Brazzale, A., Bruscagnin, G. (1990) Radioligand binding
assay of epidermal growth factor receptor: Causes of variability and standardization of

the
assay. Clin. Chem. 36:849-854.
Massague, J. (1983) Epidermal growth factor-like transforming growth factodr: II.
Interaction with epidermal growth factor receptors in human placenta membranes and
A43I cells. / BIO/. C/tem. 258:13614-13620.
226010. Oboum, J. D., Koszewski, N. J. and Notides, A. C. (1993) Hormone-and
DNA-binding mechanism of the recombinant human estrogen receptor. Biochemistry.
32: 6229 - 6236.
226400. Shank, R.P., Baidy, W.J., Matucci, L.C., Villani, F.J. Jr. (1990) Ion and
temperature effects on the binding of gamma-aminobutyrate to its receptors and the
high-affinity transport system. J. Neurochem. 54:2007-2015.
226500. Enna, S.J., and Snyder, S.H. (1976) Influences of ions, enzymes and detergents
on gamma-aminobutyric acid-receptor binding in synaptic membranes of rat brain. Mol.
Pharmacol. U:A42-453.
Martinin, C, Rigacci, T., Lucacchini, A. (1983) [3H]muscimol binding site on purified
benzodiazepine receptor. J. Neurochem. 4X"A 183-1185.
Snodgrass, S.R. (1978) Use of [3H]muscimol for GAB A receptor studies. Mature
273:392-394.
226600. Damm, H.W., Mueller, W.E., Schlaefer, U., Woliert, U. (1978)
[3H]flunitrazepam: Its advantages as a ligand, for the identification of benzodiazepine
receptors in rat brain
membranes. Res. Comm. Chem. Pathol. Pharmacol. 22:597-600.
Speth, R.C., Wastek, G.J., and Yamamura, H.I. (1979) Benzodiacepam receptors:
temperature dependence of [3H]flunitra2epam binding. Life Sci. 24:351 -357.
228510. Fackiam, M. and Bowery, N., G. (1993) Solubiliaztion and characterization of
GABAB receptor binding sites from porcine brain synaptic membranes. Br. J.
Pharmacol. 110: 1291-1296
228510. Mathivet P., Bemasconi, R., Barry, J. D., Marescaux, C., Bittiger, H. (1992)
Binding characteristics of y-hydroxybutyric acid as a weak but selective GABAB
receptor agonist. Eur. J. Pharmacol. 321: 67-75
232010. Cidlowski, J. A.andCidlowski, N.B.(1981) Regulation of glucocorticoid
receptors by glucocorticoids in Cultured HeLa S3 Cells. Endocrinology 109: 1975 -
1982.

232700. London, E.D. and Coyle J.T. (1979) Specific binding of E3H]kainic acid to
receptor sites in rat brain. Mol. Pharmacol. 15:492-505.
232810. Sills, M. A. Fagg, G. Pozza, M. Angst, C. Brundish, D. E. Hurt, S. D. Wilusz,
E. J. and Williams, M. (1991). [^H]CGP 39653: a new N-methyl-D-aspartate antagonist
radioligand with low nanomolar affinity in rat brain. European Journal of
Pharmacology \92: 19-24.
232910. Seifel, B.W., Sreekrishna, K., Baron, B.M. (1996) Binding ofthe radiolabeled
glycine antagonist [3H]MDS105,519 to homomeric NMDA-NRla receptors. Eur. J.
Pharmacol. 3\2:357"365.
233000. Goldman, M.E., Jacobson, A.E., Rice, K.C., Paul, S.M. (1985) Differentiation
of [3H] phencyclidine and (+)-[3H]SKF-l0047 binding sites in rat cerebral cortex.
FEES Lett.
190:333-336.
239610. De Backer, M. D., Gommeren, W., Moereels, H., Nobels, G., Van Gompel, P.,
Leysen, J. E. and Luyten, W. H. (1993) Genomic cloning, heterologous expression and
pharmacological characterization of a human histamine H1 receptor. Biochemical and
Biophysical Research Communications. 1601 - 1608
239710. Ruat, M., Traiffort, E., Bouthenet, M. L., Schwartz, J. C, Hirschfeld, J.,
Buschauer, A. anad Schunack, W. (1990) Reversible and irreversible labeling and
autoradiographic localization of the cerebral histamine H2 receptor using ["^^l]iodinated
probes. Proceedings ofthe National Academy of Sciences ofthe United Slates of
America. 87(5): 1658- 1662.
239810. Yanai, K., Ryu, J. H., Sakai, N., Takahashi, T., Iwata, R., Ido, T., Murakami,
K. and Watanabe, T.(1994) Binding characteristics of a histamine H3-receptor
antagonist, [3H]S-methylthiaperamide: comparison with [3H](R)a melhylhistamine
binding to rat tissues. Japanese Journal of Pharmacology. 65(2): 107 - 112.
Zhu, Y., Michalovich, D., Wu, H., Tan, K. B., Dytko, G. M., Mannan, 1. J., Boyce, R.,
Alston, J. Tiemey, L. A., Li, X., Herrity, N. C, Vawter, L., Sarau, H. M., Ames, R.S.,
Davenport, C. M., Hieble, J. P., Wilson, S., Bergsma, D. J. et al. (2001) Cloning,
expression, and pharmacological characterization of a novel human histamine receptor.
Molecular
pharmacology. 59(3): 434 - 441, 2001.

241000. Brown, CM., Mackinnon, A.C., McGrath, J.C., Spedding, M., Kilpatrick, A.T.
(1990) a2-Adrenoceptor subtypes and imidazoHne-Hke binding in the rat brain. Br. J.
Pharmacol. 99:803-809.
243510. Chin, J., Cameron, P.M., Rupp, E., and Schmidt, J.A. (1987) Idenfification of
a high affinity receptor for native interleukin-1 a and interleukin-1 b on normal human
lung
fibroblasts. J. Exp. Med. 165:70-86.
250600. Bruns, R.F., Thomsen, W.J., Pugsley, T.A. (1983) Binding of leukotrienes C4
and D4 to membranes from guinea pig lung: regulation by ions and guanine nucleotides.
ii/e 5c/. 33:645-653.
Mong, S., Wu, H.-L, Hogabaoom, G.K., Clark, M.A., Crooke, S.T. (1984)
Characterization of the leukotriene D4 receptor in guinea pig lung. Eur. J. Pharmacol.
102:1-11.
252600. Buckley, NJ., Bonner, T.I., Buckley, CM., Brann, M.R. (1989) Antagonist
binding properties of five clonal muscarinic receptors expressed in CHO-Kl cell. Mot.
Pharmacol. 35:469-476.
Luthin, G.R. and Wolfe, B.B. (1984) Comparison of [3H]pirenzepine and
3Hjquinuctidinyl-benzilate binding to muscarine cholinergic receptors in rat brain. J.
Pharmacol. Exp. Ther. 228:648-665.
Watson, M., Yamamura, H.I., and Roeske, W.R. (1983) A unique regulatory profile and
regional distribution of [3H]prienzepine binding in the rat provide evidence for distinct
Ml and M2 muscarinic receptor subtypes. Life Sc\. 32:3001-3011.
252700. Buckley, N.J., Bonner, T.I., Buckley, CM., Brann, M.R. (1989) Antagonist
binding properties of five clonal muscarinic receptors expressed in CHO-Kt cell. Mol.
Pharmacol. 35:469-476.
Delmendo, R.E, Michel, A.D., and Whiting, R.L. (1989) Affinity of muscarinic receptor
antagonists for the three putative muscarinic binding sites. Br. J. Pharmacol. 96:457-
464.
252800. Buckley, NJ., Bonner, T.I., Buckley, CM., Brann, M.R. (1989) Antagonist
binding properties of five clonal muscarinic receptors expressed in CHO-Kl cell. Mot.
Pharmacol. 35:469-476.
255510. Patacchini, R. and Maggi, CA. (1995) Tachykinin receptors and receptor
subtypes. Arch. Int. Pharmacodyn. 329:161-184.

257000. Fuhlendorff, J., Gether, U., Aakerlund, L., Langeland-Hohansen, N.,
Thogersen, H., Melberg, S.G., Olsen, U.B., Thastrup, 0., and Schwartz, T.W. (1990)
[Leu3l,Pro34]neuropeptide Y: a specific Yl receptor agonist. Proc. Nail. Acad. Sci.
[/&4 87:182-186.
Sheikh, S.P., O"Hare, M.M., Tortroa, 0., Schwartz, T.W. (1989) Binding of
monoiodinated neuropeptide Y to hippocampal membranes and human neuroblastoma
celt line. J. Biol.
Chem. 264:6648-6654.
257110. Rose, P.M., Fernandes, P., Lynch, J.S., Frazier, S.T., Fisher, S.M., Kodukuta,
K., Kienzle, B., and Seethala, R. (1995) Cloning and functional expression of a cDNA
encoding a human type 2 neuropeptide Y receptor. J. Biol. Chem. 270(39):2266l -22664.
258590. Davila-Garcia, M. 1., Musachio, J. L., Perry, D. C, Xiao, Y., Horti, A.,
London, E. D., Dannals, R. F. and Kellar, K. J. (1997) [125I]IPH, an epibatidine
analog, binds with high affinity to neuronal nicotinic cholinergic receptors. The journal
of pharmacology and experimental therapeutics. Z821: 445 - 451.
258590. Whiteaker, P., Jimenez, M., Mcintosh, J. M., Collins, A. C. and Marks, M.J.
(2000)
identification of a novel nicotinic binding site in mouse brain using [(125)1]-
epibatidine. British journal of pharmacology. 131(4) : 729 - 739,
260110. Simonin, F. et al. (1994) The human d-opioid receptor: Genomic organization,
cDNA cloning, functional expression, and distribution in human brain. Mol. Pharmacol.
46:1015-1021.
260210. Patricia, M., et al.. (1992) Pharmacological profiles of fentanyl analogs as +
and + opiate receptors. Eur. J. Pharmacol. 213: 219-225.
Simonin, F., et al. (1995) Kappa-opioid receptor in humans: cDNA and genomic
cloning, chromosomal assignment, functional expression, pharmacology and expression
pattern in the central nervous system. PNAS U.S.A. 92 15 : 1431-1437.
260410. Wang, J.B., Johnson, P.S., Persico, A.M., Hawkins, A. L., Griffin, C. A., and
Uhl, G.R. (1994) Human mu opiate receptor: cDNA and genomic clones, pharmacologic
characterization and chromosomal assignment. FEBS Lett. 338:217-222.
264500. Ashendel, C.L. (1985) The phorbol ester receptor: a phospholipid-regulated
protein kinase. Biochem. Biophys. Acta 822:219-242.

■- !,

265010. Herbert, J. M., Castro-Faria-Neto, H. C, Harbosa-Filho, J. M., Cordeiro, R. S. B., Tibirica, E. (1997) Pharmacological evidence for the putative existence of two different subtypes of PAF receptors on platelets and leukocytes; studies with yangambin. J. Lipid Mediat. Cell Signal 17: 1-14.
265600. Gaines, K.L., Hamilton, S. Boyd, A.E. 3rd (1988) Characterizatrion of the sulfonylurea receptor on beta cell membranes. J. Biol. Chem. 263:2589-2592. 268700. Bo, X., and Bumstock, G. (1990) High- and low-affinity binding sites for [3H]-a,b-methylene ATP in rat urinary bladder membranes. Br. J. Pharmacol. 101:291-296.
Ziganshin, A.U., Hoyle, C.H., Bo, X., Lambrecht, G., Mutschler, E., Baumert, H.G., Bumstock. G. (1993) PPADS selectively antagonized P2X-purinoceptor-mediated responses in the rabbit urinary bladder. Br. J. Pharmacol. 110:1491-1495. 268810. Boyer, J. L., Cooper, C. L. and Harden T. K. (1990) [""P]3"-0-(4-Benzoyi)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-Purinergic receptor. J. fi(o/Cftem. Vol. 265 No. 23: pp. 13515 - 13520. 271110. Martin, G.R. and Humphrey, P.P.A. (1994) Receptor for 5-hydroxylryptamine: current perspectives on classification and nomenclature. Neuropharm. 33:261-273. 271910. 1. Millerk, W. E., Fletcher, P. W., and Teitler, M. (1992) Membrane-bound and solubilized brain 5-HT3 receptor: improved radioligand binding assay using bovine area postrema or rat cortex and the radioligand [3H]GR65630, [3H]BRL43694, and [3H]LY278584 Synapase, n.:58 - 66.
Boess, F. G., Steward, L. J., Steele, J. A., Liu, D., Reid, J., Glencorse, T. A. and Martin, 1. L. (1997) Analysis of the ligand binding site of the 5-HT, receptor using site- directed mutagenesis: importance of glutamate 106. Neuropharmacology, 3^: 637 - 647. 274020. Gu, H., Wall, S., Rudnick, G. (1994) Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence. J. BioL Chem. 269(10):7124-7130.
278110. Ganapathy, M. E., Prasad, P. D., Huang, W., Seth, P., Leibach, F. H. and Ganapathy, V. (1999) Molecular and iigand-binding characterization of the s-receptor in the Jurkat human T lymphocyte cell line. Pharmacol Exp, Ther 289: 251 -260. 278200. Hashimoto, K., and London, E.D. (1993) Further characterization of [3H]Ifenprodil binding to sigma receptors in rat brain. Eur. J. Pharmacol. 236:159-163.

279450. Doucette, G.J. Logan, M. M., Ramsdell, J. S. and Van Dolah, F. M. (1997)
Development and preliminary validation of a microtiter plate-based receptor binding
assay for paralytic shellfish poisoning toxins. Toxicon, 35 (5}: 625 - 636.
279510. Catterall, W.A., Morrow, C.S., Daly, J.W., Brown, G.B. (1981) Binding of
batrachotoxin A 20-alpha-benzoate to a receptor site associated with sodium channels in
synaptic nerve ending particles. J. Biol. Chem. 256:8922-8927.
285010. Chang, C. and Liao, S. (1987) Topographic recognition of cyclic hydrocarbons
and related compounds by receptors for androgens, estrogens, and glucocorticoids. J.
SteroidBiochem. 27(1-3): 123 - 13L
Traish, A. M., Muller R. E. and Wotiz, H. H. (1986) Binding of 7a, 17a-dimethyl-19-
nortestosterone (mibolerone) to androgen and progesterone receptors in human and
animal tessues. Endocrinolo^ 118(4): 1327 - 1333.
Example 9
Biological Testing ofCSlSM.CSl 713. and CS1714
The results from the biological testing of CS1814, CS1713, CS1714, and 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 each assay are are listed below.
Where presented, IC50 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 (Ki) are presented, the K.values were calculated using the equation of Cheng and PrusofT (Cheng, Y., Prusoff, W.H., Biochem. Pharmacol. 22:3099-3108, 1973) using the observed IC50 of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the KD of the ligand (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 Toolbox^^. 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 IC50, K|, and/or nH data are presenled without Standard Error of the Mean (SEM), data are insufficient to be quantitative, and the values presented (Kj, IC50, nu) should be interpreted with caution.
CS17I3 (Vial #2), CSI7I4 (Vial #3), and CS18H (Via! #1) were evaluated for inhibition of cellular Serotonin and Norepinephrine Uptake. In addition, CS1713 (Via! #2) and CS1714 (Vial #3) were evaluated in various radioligand binding assays, and for inhibition of CYP450 3A4 at initial concentrations of 10 \iM. As depicted in Figure 60, significant activity (350%) 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 Transporter binding sites (Vial #2 Ki = 0.112 nM, Vial #3 Ki - 1.68 ^iM).
As depicted in Figure 61, CS1814 (Vial #1) is approximately equipotent in inhibiting serotonin and norepinephrine uptake (IC50 = 28.6 nM for norepinephrine, IC50 = 21.7 nM for serotonin). Interestingly, CSI713 (Vial #2) is a more potent inhibitor of norepinephrine uptake than serotonin uptake (IC50 = 10.3 nM for norepinephrine, IC5o^22nM for serotonin). In contrast, CS17t4 (Vial #3) is a more potent inhibitor of serotonin uptake compared to norepinephrin uptake (IC50 = 88.5 nM for norepinephrine, IC50 = 40.3 nM for serotonin). The fact that CSl 713 (Vial #2) is a more potent inhibitor of norepinephrine uptake would render it a superior therapeutic agent for treating diseases linked to norepinephrine uptake. In addition, the CS1714 (Vial #3) would useful for treating conditions requiring selective inhibition of serotonin uptake.
Importantly, no cytotoxicity was observed for CSl 713 (Vial #2), CS17l4(Vial#3),orCSI814(Vial#l)atl0tiM. In addition, CS18I4 (Vial #1) is a selective inhibitorof norepinephrine and serotonin transporters. The factthatCS1814 generally does not bind well to other receptors, as depicted in 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 CSl 714 will not have detrimental side effects.
Methods

118090 CyP450, 3A4
Source: Suhslialc:
Pre-lntubaUon Tiinc/Tcmp: Incuhntion Time/Temp-Incuhalion Buffer:
Qjanlilalkm Method: Signifioance Cnicria:

Human recoiubmarl: Sf9 insect cells
50 |iM 7-benzyloiy-4-(Irifluoromethyl)-coumarin
0.1% DM50
None
3Q minutes @ 37"C
75 mM PolasiiLjm Phosphate buffer. pH 7.5
5 pectrofl nan metric quantitation of 7 -HydiDij-«-ttiiHuorDineth>l)-
coumarin
£ 50% of ma" stimulation or inhibition

212610 Bradykinin B,

Stmrcc:
Ligand:
Vi^hiclc:
Incubation TiineyTemp:
Incubacion Biiffcf
Non-Spccifii: L.tganJ"
K„:
SpcL:Ll"ic Binding:
QujnciijIiLiT] Mt^ihixl. Signihciintc CrjJtri.i"

Human tecomtJinant CHO-Kl cells
0.2 nM PHI BracJyIfinin
la DMSO
90 minutes @ 75°C
2i mM TES-NH.OH, pH 6.8, 1 mM l.lD-phenanthrolirie, 0.3% B5A
5 uU Bradykinin
0 29 nM •
2 pmoie/mg Protein ■
90%-
Radioligand Binding
2 50% of maK stimulation or
inhibition

214510 Calcium Channel L-Type, Benzothiazepine
Source: Wiilar Rat brain
LIgand" 2fiM PH) Diltiaiem
Vcliic-lc: ISDMSO
Inciibalion Time/Temp: i liours i^ 4K:
Intubation Buffer: 50 mM Tr«-HCI. 0 IS B5A, pH 7A
at25°C
Non-Specilk Ligand: Ko:
Spccific liindiny; QuanTiiuriOLi Mcihnd: Signifleaner Cnlctia
10 |JM Dfltiajem
0 016pW*
0 21 pmole/mg Prateir ■
73% -
RaOiolfgand Binding
a 5D%of mai 5iinijlatron or
inhibilion
204410 Transporter, Norepinephrine (NET)

SouTcc: I.igiinJ: Vcliklt: [ncubaiiun Tlme/Tcnip:
Incu ball Oil Buffer:
Ni)ii-Spctirit LigaiKl
BMM:
Specific Dindiri:: QmiLjlilaliifn Method-^ignifiojiic^c Cnfcria:

Human recombinanl MDCK tells
0.2 nM [""I) RTI-55
l%DMSO
3 tiou"s @ 4"C
50 mW Tris-HCI. 100 mM fJsCI. I jiM [eupeptin, 10 iiM PUSr, pH 7.4
10 pM Deiipramine
0.024 |JM •
2.S prnoie/mg Protein "
7E%"
Radioligard Binding
^ iol of ma inhibilion

inhibiljon
302100 Cytotoxicity. Norepinephrine Uptake
274020 Transporter, Hydroxytryptamine)
Source;
Ligand:
Vehicle:
Incubaiion TimcJTcmp-
Incuhation liuffei:
Non-Specilk LIgand. Ko:
Spccific Binding: Quantiiaiion Mel hod-SigmfitancE Crilcria:

Serotonin (5-(SERTl
Human rpcomhifiant HEK-29i cells
0.1SnM|""ilRTI-S5
ttDMSO
3 hours @ AK
100 mW NaCl, SB mM Trii HCl, 1 iiM Leupepim, IDjjM PUSF. pH 7.4
10 |JM Imipramirie
0.17 nM"
0.41 pmole/mg protein ■
95S"
Radioligand Bindirg
f 50K of mai slinulation or



T^gei:
Vehicle;
Incubaiion Time/Tcmp:
Incubaiion Buffer:
Quanlilation MeLhod:
Significance Criieria-Ag: Significance Critcria-AnL

Human MOCK cells Dog kidney
0,4X OUSO
30 minulM @ ;s°C
5 mM Tris-HCL 7.5 mM HEPES. 1J0 mM NaO, S.4 mM KCl. 1.2 mM CaCI,, 1.2 mM MgSO,, 5 mM Glucose. 1 mM Ascorbic Acid. pH 7.1
Spectratluo rime trie quanlitalior of Alamar Blue
N/A
2 5051 Decrease in fluorescence intensity relative to vehicle control

■ 364100 Cytotoxicity. Serotonin (5-Hydroxytryptamine) Uptake

TaJgel:
Vehicle:
Incubalion Timc/Temp:
Incubation Buffer:
Quantitation Method:
SignifKance Criiciia-Ag". SigniUcance Criteria-Anl:

Human HEK-;93 cells Huamn
embryonal Itidney
Q.4% DMSO
30 minutes @ 2S°C
5 mM Trii-HCI. 7.5 mM HIPES, 120 mM NaCI, 5.4 mM »tCL 1.3 mM CaCi,. 1.2mMMg50„5mM Glucose, 1 mM Ascorbic Acid, pH 7,1
SpcctrofluDrirTietnc quantitation of Alamar Blue
i sot Decrease in lluorescence intensity relative to vehicle controf

302000 Uptake, Norepinephrine

Target:
Vehicle:
Incubalion Tiine/Temp:
Incubation BufTer:
Human K/DCK cells Dog kiijney
an DUSO
lO minutes @ 2S°C
5 mM Tris-HCI, 7.5 mM HEPES, )20 tnM NaCI, 5.4 mM KCl, 1.2 mM CaCL 1.2mMMgSO., SmM Glucose, I nrM Ascorbic Acid, oH 7.1
Quantitation Method: Quantitation ol I"HjNofepinephrine
Signiricancc Critcria-Ag: N/A
Significance Ctittiia-Anf, i 50\ iTrtiibitiw of
(>H|Norepinephrine uptake relative to desipramine response
364000 Uptake. Serotonin (5-Hydroxytryptamine)

Target:
Vehicle:
Incubalion Timc/Temp:
Incubation Buffer:
Quantitation Method:
Signiricance Criteria-Ag: Significance Criteria-Am:

Human HEK-293 Cells Human
embryonic kidney
0A% DMSO
10 minutes 0> 2510
5 mM Tris-HCI, 7.5 mM HEPE5, 120 mM NaCL 5.4 mU KCL 1.2 mW CaCI,, 1.3 mM MgSO„ 5 mW Glucose, t mM Ascorbic Acid. pH 7.1
Quantitation of |"H|Serotonin
uptake
N/A
£ SOX Inhibition of I>H]Serolonin uptake relative to fluietine response

Literature References (CAT. #. Reference)

118090. Crespi, C. L„ Miller, V. P and Penman, B.W. (1997) Microliter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal Biochem 248(1): 188-190.
Gentest Technical Bulletin (Version 4.2: Revised 27 September 2000) A high throughput method for measuring cytochrome P450 inhibition. Genlesi Technical Bulletin (Version 4.2) Revised 27 September 2000).
204410. Galli, A., DeFelice, L., Duke, B.-J., Moore, K. Blakely, R. (1995) Sodium dependent norepinephrine induced currents in norephinephrine transporter transfected HEK293 cells blocked by cocaine and antidepressants. J. Exp. Biol. 198:2197-2212.
212610. Eggerickx, D., Raspe, E. Berlrand, D., Vassarl, G., Parmentier, M. (1992) Molecular cloning, functional expression and pharmacological characterization of a human bradykinin B2 receptor gene. Biochem Biophys Res Commun 187 (3): 1306 - 1313.
214510. Schoemaker, H. and Langer, S.Z. (1985) [3H]DiItiazem binding to calcium channel antagonist recognition sites in rat cerebral cortex. Eur, J. Pharmacol. 111:273-277.
274020. Gu, H., Wall, S., Rudnick, G. (1994) Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence. J. Biol. Cfew.269fl0):7124-7130.
302000. Galli, A., DeFelice, L. Duke, B.-J., and Blakely, R. (1995) Sodium dependent norephinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants. ./. Exp. Biol. 198:2197-2212.
302100. Page, B., Page, M. and Moel, C. (1993) A new iluorometric assay for cytotoxicity measurements in vitro. Ing. 73:473-476, 1993.
364000. Gu, H., Wall, S., Rudnick, G. (1994) Stable expression of biogenic amine transporter reveals differences in inhibitor sensitivity, kinetics, and ion dependence. J. Biol. Cfew. 269n):7124-7130.

364100. Page, B., Page, M. and Noel, C. (1993) A new fluorometric assay for cytotoxicity measurements in vitro. !nt. J. Oncology 3:473-476, 1993.
Additional Patents and Publications Cited
1. United States Patent 4,478,836.
2. United States Patent 5,034,541.
3. United States Patent 5,621,142.
4. Moret, C. et al. Neuropharmacology 1985, 2 5. Bonnaud, B. et al. J. Med. Chem. 1987, 30, 318-325.
6. Shuto, S. et al. /. Med. Chem. 1995, 38, 2964-2968.
7. Viazzo, P. et al. Tetrahedron Lett. 1996, 37, 4519-4522.
8. Shuto, S. et al. Tetrahedron Lett. 1996, 37, 641-644.
9. Shuto, S. et al. J. Med. Chem. 1996, 39, 4844-4852.
10. Shuto, S.etal.y.Met^. Chem. 1998, •#/, 3507-3514.
11. Deprez, D. et al. Eur. J. Drug Metab. Pharmacokinel. 1998, 23, 166-171.
12. Puozzo, C. et al. Eur. J. Drug Metab. Pharmacokinet. 1998, 23, 273-279.
13. Puozzo C. et al. Eur J. Drug Metab. Pharmacokinet. 1998, 23, 280-286.
14. Shuto, S. et aX.Jpn. J. Pharmacol. 2001, 85, 207-213.
15. Doyle, M.P. et al. Adv. Synth. Catal. 2001, 343, 299-302.
16. Kazuta, Y. et al. Bioorg Med Chem. 2002,10,1777-1791.
17. Ubat, L. et al. /. Chromatogr. B 2002, 773, 17-23.
18. Grard, S. et al. Electrophoresis 2000, 21, 3028-3034. Incorporation by Reference
All of the patents and publications cited herein are hereby incorporated by reference. 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.


We claim:
1. An isolated compound represented by A:
wherein
X represents independently for each occurrence O, S, or NR;
R represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl,
R is absent or present between one and four times inclusive;

R , if present, represents independently for each occurrence H, alkyl, cycloalkyl, allcenyl, aryl, heteroaiyl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aiyloxy, arylalkyloxy, amino, alkylamino, arylamino, arylakylamino, sulfhydiyl, alkylthio, arylthio, arylakylthio, nitre, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)in-R8o;
Rso 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
i
a
wherein
X represents independently for each occurrence O, S, or NR;
R represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, formyl, acyl, silyl, (alkyloxy)carbonyl, (aryloxy)carbonyl, (arylalkyIoxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyl, alkylsulfonyl, arylsulfonyl, or -(CH2)ni-R8o;

R" represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, arylamino, aryiakylamino, sulfliydryl, alkylthio, arylthio, arylakylthio, nitro, azido, alkylseleno, formyl, acyl, carboxyl, silyl, silyloxy, (alkyloxy)carbonyI, (aryloxy)carbonyl, (aryla!kyloxy)carbonyl, (alkylamino)carbonyl, (arylamino)carbonyl, (arylalkylamino)carbonyi, alkylsulfonyl, arylsulfonyl, or -(CU2)m-^io",
R^ represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, or -(CH2)ni-R8o;
R^ represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, or -(CH2)ni"R*o;
R"" is absent or present between one and four times inclusive;
R"", if present, represents independently for each occurrence H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, arylalkyl, cyano, halogen, hydroxyl, alkoxyl, aryloxy, arylalkyloxy, amino, alkylamino, arylamirio, aryiakylamino, sulfhydiyl, 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-Rao;
Rgo represents independently for each occurrence an aryl, cycloalkyl, cycloalkeiiyl, 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.
3. The compound of claim 1 or 2, wherein X represents O.
4. The compound of claim 1 or 2, wherein R represents H.

5. The compound of claim 1 or 2, wherein R" represents H.
6. The compound of claim 1 or 2, wherein R^ represents H.
7. The compound of claim 1 or 2, wherein R^ represents alkyl.
8. The compound of claim 1 or 2, wherein R"" is absent.
^ 9. The compound of claim 1 or 2, wherein X represents O; and R represents H.
10. The compound of claim 1 or 2, wherein X represents O; R represents H; and R" represents H.
11. The compound of claim 1 or 2, wherein X represents O; R represents H; R" represents H; and R represents H.
12. The compound of claim 1 or2, wherein X represents O; R represents H; R" represents H; R^ represents H; and R^ represents alkyl.
f 13. The compound of claim 1 or 2, wherein X represents O; R represents H; R" represents H; R^ represents H; R^ represents alkyl; and R"" is absent.
14. The compound of claim 1 or 2, wherein X represents O; R represents H; R" represents H; R^ represents H; R^ represents ethyl; and R^ is absent.
15. A formulation, comprising a compound of claim 1 or 2; and a pharmaceutically acceptable excipient.

». A composition comprising a selective serotonin reuptake inhibitor and/or a selective norepinephrine reuptalce inhibitor and ; a compound of claim I or 2.

Documents:

1003-chenp-2005 abstract.pdf

1003-chenp-2005 claims-duplicate.pdf

1003-chenp-2005 claims.pdf

1003-chenp-2005 correspondence-others.pdf

1003-chenp-2005 correspondence-po.pdf

1003-chenp-2005 description (complete)-duplicate.pdf

1003-chenp-2005 description (complete).pdf

1003-chenp-2005 drawings-duplicate.pdf

1003-chenp-2005 drawings.pdf

1003-chenp-2005 form-1.pdf

1003-chenp-2005 form-18.pdf

1003-chenp-2005 form-26.pdf

1003-chenp-2005 form-3.pdf

1003-chenp-2005 form-5.pdf

1003-chenp-2005 pct search report.pdf

1003-chenp-2005 pct.pdf

1003-chenp-2005 petition.pdf


« Previous Patent
Next Patent »
Patent Number 220468
Indian Patent Application Number 1003/CHENP/2005
PG Journal Number 30/2008
Publication Date 25-Jul-2008
Grant Date 28-May-2008
Date of Filing 24-May-2005
Name of Patentee COLLEGIUM PHARMACEUTICAL, INC
Applicant Address 400 Highland Corporate Drive, Cumberland, RI 02864,
Inventors:
# Inventor's Name Inventor's Address
1 HEFFERNAN, Michael
2 RARIY, Roman, V
3 BUCHWALD, Stephen, L
4 SWAGER, Timothy, M
PCT International Classification Number C07C 233/58
PCT International Application Number PCT/US2003/033681
PCT International Filing date 2003-10-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/423,062 2002-11-01 U.S.A.
2 60/421,640 2002-10-25 U.S.A.
3 60/445,142 2003-02-05 U.S.A.