Sydnonimines-specific dopamine reuptake inhibitors and their use in treating dopamine related disorders

Abstract

Derivatives of Sydnonimine and its analogues, which bind selectively to dopamine transporter (DAT) proteins are useful for treating and delaying the progression of disorders and illnesses that are alleviated by inhibiting dopamine reuptake.

Claims

1. A method of managing and/or delaying the progression of a disorder alleviated by inhibiting dopamine reuptake in a patient in need thereof, wherein said disorder is autism, the method comprising administering to said patient a therapeutically effective amount of at least one compound having the formula: ##STR00013## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6, independently of one another, are radicals selected from H, C.sub.1-C.sub.6 alkyl, OH, halogen, C.sub.5-C.sub.14 aryl, C.sub.6-C.sub.20 aralkyl, C.sub.1-C.sub.6 alkylthio, C.sub.1-C.sub.6 alkoxy, SH, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, CN, NO.sub.2, carboxy, carbalkoxy, carboxamido, alkylsulfonyl, alkylsulfonyloxy, aminosulfinyl, monoalkylaminosulfinyl, dialkylaminosulfinyl, aminosulfonyl, monoalkylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino, hydroxysulfonyloxy, alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl, aminosulfonylalkyl, monoalkylaminosulfonylalkyl, dialkyaminosulfonylalkyl, aminosulfinylalkyl, monoalkylaminosulfinylalkyl, dialkylaminosulfinylalkyl, said alkyl, alkenyl, alkynyl or cycloalkyl radical being optionally substituted by at least one halogen, OH, SH, NH.sub.2, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, COOH, CN, NO.sub.2, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy group, said aryl and aralkyl radical being optionally substituted by at least one halogen, OH, SH, NH.sub.2, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, COOH, CN, NO.sub.2, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy group; R.sub.a, R.sub.band R.sub.c, independently of one another, represent radicals selected from H, C.sub.1-C.sub.4 alkyl, phenyl or phenyl C.sub.1-C.sub.4 alkyl, said alkyl radical, said phenyl radical and said phenyl C.sub.1-C.sub.4 alkyl radical being optionally substituted by at least one halogen, OH, SH, NH.sub.2, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, COOH, CN, NO.sub.2, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy group; m, n and k are independent integers from 0-4, except that m+n 0, and R.sub.b alkyl when m+n=2; or a pharmaceutically acceptable salt of said compound.

2. The method according to claim 1, wherein the compound administered is selected from the group consisting of 3-(p-methylbenzyl)-sydnonimine-N-phenylcarbamoyl and 3-(phenylpropyl)-sydnonimine-N-phenylcarbamoyl, and a pharmaceutically acceptable salt of said compound.

3. The method according to claim 1, wherein said compound is administered in dosage unit form, said dosage unit containing from about 0.01 to about 200 mg of said compound per kilogram of patient body weight per day.

4. The method according to claim 3, wherein said dosage unit includes a pharmaceutically acceptable vehicle.

5. The method according to claim 1, wherein said compound is administered orally.

6. The method according to claim 1, wherein said compound is administered parenterally.

7. A method of managing and/or delaying the progression of a condition or disease state by modulating dopamine reuptake activity, wherein the condition or disease state is autism, said method comprising administering to a patient having the condition or disease state a therapeutically effective amount of at least one compound having the formula: ##STR00014## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6, independently of one another, are radicals selected from H, C.sub.1-C.sub.6 alkyl, OH, halogen, C.sub.5-C.sub.14 aryl, C.sub.6-C.sub.20 aralkyl, C.sub.1-C.sub.6 alkylthio, C.sub.1-C.sub.6 alkoxy, SH, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, CN, NO.sub.2, carboxy, carbalkoxy, carboxamido, alkylsulfonyl, alkylsulfonyloxy, aminosulfinyl, monoalkylaminosulfinyl, dialkylaminosulfinyl, aminosulfonyl, monoalkylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino, hydroxysulfonyloxy, alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl, aminosulfonylalkyl, monoalkylaminosulfonylalkyl, dialkyaminosulfonylalkyl, aminosulfinylalkyl, monoalkylaminosulfinylalkyl, dialkylaminosulfinylalkyl, said alkyl, alkenyl, alkynyl or cycloalkyl radical being optionally substituted by at least one halogen, OH, SH, NH.sub.2, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, COOH, CN, NO.sub.2, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy group, said aryl and aralkyl radical being optionally substituted by at least one halogen, OH, SH, NH.sub.2, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, COOH, CN, NO.sub.2, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4alkoxy group; R.sub.a, R.sub.b and R.sub.c, independently of one another, represent radicals selected from H, C.sub.1-C.sub.4 alkyl, phenyl or phenyl C.sub.1-C.sub.4 alkyl, said alkyl radical, said phenyl radical and said phenyl C.sub.1-C.sub.4 alkyl radical being optionally substituted by at least one halogen, OH, SH, NH.sub.2, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, COOH, CN, NO.sub.2, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy group; m, n and k are independent integers from 0-4, except that m+n 0, and R.sub.b alkyl when m+n=2; or a pharmaceutically acceptable salt of said compound; said compound having dopamine transporter protein-specific inhibitory activity and locomotor suppressant activity.

8. A method of easing the symptoms and/or effects of autism by inhibiting dopamine reuptake transporter (DAT) activity in a patient in need thereof, the method comprising administering to said patient a therapeutically effective amount of a selective DAT inhibitor; wherein the selective DAT inhibitor exhibits DAT binding inhibition of greater than or equal to 90% at 10 M and a norepinephrine transporter (NET) binding inhibition of less than or equal 15% at 10 M, wherein the selective DAT inhibitor is selected from the group consisting of 3-(p-methylbenzyl)-sydnonimine-N-phenylcarbamoyl and 3-(phenylpropyl)-sydnonimine-N-phenylcarbamoyl, and a pharmaceutically acceptable salt of said selective DAT inhibitor.

9. A method of managing and/or delaying the progression of a disorder alleviated by inhibiting dopamine reuptake in a patient in need thereof, wherein said disorder is autism, the method comprising administering to said patient a therapeutically effective amount of at least one compound selected from the group consisting of 3-(p-methyl-benzyl)-syndonimine-N-phenylcarbamoyl, 3-(p-carboxyl-benzyl)-sydnonimine-N-phenylcarbamoyl, 3-(p-fluoro-benzyl)-sydnonimine-N-phenylcarbamoyl, and 3-(phenylpropyl)-sydnonimine-N-phenylcarbamoyl, and a pharmaceutically acceptable salt of said compound.

10. The method of claim 1, wherein the compound administered comprises 3-(phenylpropyl)-sydnonimine-N-phenylcarbamoyl or a pharmaceutically acceptable salt of said compound.

11. The method of claim 8, wherein the selective DAT inhibitor is 3-(phenylpropyl)-sydnonimine-N-phenylcarbamoyl or a pharmaceutically acceptable salt of said selective DAT inhibitor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graphical representation of the IC.sub.50/K.sub.i profile of representative examples of sydnonimine derivatives used in the practice of this invention, namely, FIG. 1A is 3-(benzyl)-sydnonimine-N-phenylcarbamoyl; FIG. 1B is 3-(p-methyl-benzyl)-sydnonimine-N-phenylcarbamoyl; FIG. 1C is 3-(phenylpropyl)-sydnonimine-N-phenylcarbamoyl; FIG. 1D is 3-(p-carboxyl benzyl)-sydnonimine N-phenylcarbamoyl); FIG. 1E is 3-(p-fluoro-benzyl)-sydnonimine N-phenylcarbamoyl; FIG. 1F is 3-phenethyl-sydnonimine-N-(3-4-dichloro-phenyl)carbamoyl and FIG. 1G is 3-(p-nitrophenethyl)-sydnonimine-N-(3,4-dinitro-phenyl)carbamoyl.

(2) FIGS. 2A-2D are graphs of a pharmacokinetic profile comparison showing that the compound of Example 6 (see below; -.square-solid.-), exhibits slightly higher bioavailablilty than the compound of Example 3 (-.diamond-solid.-) at the same dosage using different routes of administration.

(3) FIG. 3 is a graph of a pharmacokinetic profile comparison showing that the compound of Example 6 (-.square-solid.-=10 mg/kg; -.circle-solid.-=30 mg/kg) demonstrates better oral bioavailabilty than the compound of Example 3 (-.diamond-solid.-=10 mg/kg; --=30 mg/kg) at different dosages.

(4) FIGS. 4A and 4B are graphs showing that the compound of Example 3 demonstrates a dose dependent suppression of spontaneous locomotor activity (NIDA).

(5) FIGS. 5A and 5B are graphs showing that the compound of Example 6 demonstrates a dose dependent suppression of spontaneous locomotor activity (NIDA).

(6) FIGS. 6A and 6B are graphical representations of test results demonstrating that the compound of Example 3 and the compound of Example 6, respectively, are capable of suppressing spontaneous locomotor activity for at least 3 hours (-.circle-solid.-=DAT inhibitor and --=vehicle in FIGS. 6A and 6B).

(7) FIGS. 7A-7C are a series of graphs showing that the compound of Example 3 induces significant behavioral effects in an Irwin Behavioral Battery (FIG. 7A: Spontaneous activity; FIG. 7B: Grip strength; FIG. 7C: Limb tone).

(8) FIG. 8 is a graph showing that the compound of Example 3 reduces locomotor activity in Open Field testing (-.square-solid.-=vehicle; -.Math.-=test compound, 10 mg/kg; -.diamond-solid.-=test compound, 90 mg/kg; -.box-tangle-solidup.-=buspirone, 6 mg/kg).

(9) FIGS. 9A-9D are a series of graphs showing that the compound of Example 6 induces anxiolytic effects in Open Field testing (-.square-solid.-=vehicle; --=buspirone, 6 mg/kg; -.Math.-=test compound, 10 mg/kg; --=test compound, 90 mg/kg).

(10) FIG. 10 is a graph showing that the compound of Example 3 induces an anxiolytic effect in novel environment-induced feeding suppression (-.square-solid.-=vehicle; -.circle-solid.-=buspirone, 6 mg/kg; -.Math.-=test compound, 10 mg/kg; -.diamond-solid.-=test compound, Ex. 3, 90 mg/kg)

(11) FIG. 11 is a graph showing that the compound of Example 3 did not affect Rotarod Performance (-.square-solid.-=vehicle; -.circle-solid.-=EtOH; -.Math.-=Ex. 3, 10 mg/kg; -.diamond-solid.-=Ex. 3, 90 mg/kg).

DETAILED DESCRIPTION OF THE INVENTION

(12) As previously noted, the present invention includes compounds of Formula I, above, pharmaceutical compositions comprising such compounds and methods of using such compounds for treating various disorders and illnesses alleviated by inhibiting dopamine reuptake, or preventing or delaying the progression of those disorders and illnesses.

(13) It should be appreciated that compounds of Formula I, above, may have one or more asymmetric centers and thus exist as stereoisomers, including enantiomers and diastereomers, which are usually named according to the Cahn-Ingold-Prelog system. Although the structure of Formula I is represented without regard to stereochemistry, it is intended to include all possible stereoisomers, which may be racemic mixtures or other mixtures of R and S stereoisomers (scalemic mixtures which are mixtures of unequal amounts of enantiomers), as well as resolved, substantially pure optically active forms, and pharmaceutically acceptable salts thereof.

(14) Stereoisomers of the compounds of formula (I), above, can be selectively synthesized or separated into pure, optically-active form using conventional procedures known to those skilled in the art of organic synthesis. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic forms, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers.

(15) All of the various isomeric forms of the compound of Formula I, above, are within the scope of this invention.

(16) As used herein, the alkyl refers to saturated straight and branched chain hydrocarbon radicals, having 1-6 and preferably 1-4 carbon atoms. The term alkenyl is used to refer to unsaturated straight and branched chain hydrocarbon radicals including at least one double bond, and having 2-7 and preferably 2-5 carbon atoms. Such alkenyl radicals may be in trans(E) or cis(Z) structural configurations. The term alkynyl is used herein to refer to both straight and branched unsaturated hydrocarbon radicals including at least one triple bond and having 2-7 and preferably 2-5 carbon atoms.

(17) The term cycloalkyl as used herein refers to a saturated cyclic hydrocarbon radical with one or more rings, having 3-14 and preferably 5 or 6-10 carbon ring-atoms.

(18) Any alkyl, alkenyl, alkynyl or cycloalkyl moiety of a compound described herein may be substituted with one or more groups, such as halogen, OH, SH, NH.sub.2, C.sub.1-C.sub.4 monoalkylamino, C.sub.1-C.sub.4 dialkylamino, COOH, CN, NO.sub.2, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy.

(19) The term aryl as used herein refers to an aromatic hydrocarbon radical composed of one or more rings and having 5 or 6-14 carbon atoms and preferably 5 or 6-10 carbon atoms, such as phenyl, naphtnyl, biphenyl, fluorenyl, indanyl, or the like. Any aryl moiety of a compound described herein may be substituted with one or more groups, such as halogen, OH, SH, NH.sub.2, C.sub.1-C.sub.4 monoalkylamino, C.sub.1-C.sub.4 dialkylamino, COOH, CN, NO.sub.2, C1-C4 alkyl or C1-C4 alkoxy. The aryl moiety is preferably substituted or unsubstituted phenyl.

(20) The term arylalkyl or aralkyl as used herein refers to radicals having 6 to 20 carbon atoms that combine both an aryl and an alkyl group, as defined above. Any aralkyl moiety of a compound described herein may optionally be substituted with one or more of the same substituent groups mentioned above in reference to the aryl radical.

(21) The term halogen or halo as used herein refers to Fl, Cl, Br and I.

(22) The term alkoxy refers to alkyl-O, in which alkyl is as defined above.

(23) The term alkylthio refers to alkyl-S, in which alkyl is as defined above.

(24) The term carboxy refers to the moiety C(O)OH.

(25) The term carbalkoxy refers to the moiety C(O)O-alkyl, in which alkyl is as defined above.

(26) The term carboxamido refers to the moiety C(O)ONRR, in which R and R, each independently represents H, alkyl, aryl or aralkyl, all as previously defined.

(27) The term alkylsulfonyl refers to the moiety S(O).sub.2-alkyl, in which alkyl is as previously defined.

(28) The term alkylsulfonyloxy refers to the moiety OS(O).sub.2-alkyl, wherein alkyl is as previously defined.

(29) The term amino(monoalkylamino-, dialkylamino-)sulfinyl refers to the moiety S(O)NRR in which R and R each independently represents H, alkyl, aryl or aralkyl, all as previously defined.

(30) The term amino(monoalkylamino-, dialkylamino-)sulfonyl refers to the moiety S(O).sub.2NRR, in which R and R each independently represents H, alkyl, aryl or aralkyl, all as previously defined.

(31) The term alkylsulfonylamino refers to the moiety NHS(O).sub.2-alkyl, in which alkyl is as previously defined.

(32) The term hydroxysulfonyloxy refers to the moiety OS(O).sub.2OH.

(33) The term alkoyxsulfonyloxy refers to the moiety OS(O).sub.2O-alkyl, in which alkyl is as previously defined.

(34) The term alkylsulfonyloxy refers to the moiety OS(O).sub.2-alkyl, in which alkyl is as previously defined.

(35) The term hydroxysulfonyl refers to the moiety S(O).sub.2OH.

(36) The term alkoxysulfonyl refers to the moiety S(O).sub.2O-alkyl, wherein alkyl is as previously defined.

(37) The term alkylsulfonylalkyl refers to the moiety -alkyl-S(O).sub.2-alkyl, wherein alkyl (each instance) is as previously defined.

(38) The term amino(monoalkylamino-, dialkylamino-)sulfonylalkyl refers to the moieties -alkyl-S(O).sub.2NRR, wherein alkyl is as previously defined, and R and R each independently represents H, alkyl, aryl or aralkyl, all as previously defined.

(39) The term amino(monoalkylamino-, dialkylamino-)sulfinylalkyl refer to the moieties -alkyl-S(O)NRR, wherein alkyl is as previously defined, and R and R each independently represents H, alkyl, aryl or aralkyl, all as previously defined.

(40) Preferred are the compounds of Formula I, above, wherein phenyl rings A and/or B are mono- or di-substituted. When the A and/or B ring is mono-substituted, para-substitution is preferred. When the A and/or B ring is di-substituted, 3,4 di-substitution is preferred. Most preferred are compounds in which the A ring is para-substituted, e.g., N-phenylcarbamoyl-3-(p-methyl-benzyl)sydnominine, compounds in which the B ring is 3,4-di-substituted, e.g., N-(3,4-dichlorophenyl)carbamoyl3-phenethyl-sydnominine and compounds in which the A ring is para-substituted and the B ring is 3,4-di-substituted, e.g., N-(3,4-dinitro-phenyl)carbamoyl-3-(p-nitrophenethyl)-sydnominine.

(41) The term pharmaceutically acceptable salts as used herein refers to salts derived from non-toxic physiologically compatible acids and bases, which may be either inorganic or organic. Thus, when a compound of Formula I has an acid moiety, e.g., 3-(p-carboxylbenzyl), sydnominine-N-phenylcarbamoyl, useful salts may be formed from physiologically compatible organic and inorganic bases, including, without limitation, alkali and alkaline earth metal salts, e.g., Na, Li, K, Ca, Mg, as well as ammonium salts, and salts of organic amines, e.g., ammonium, trimethylammonium, diethylammonium, and tris-(hydroxymethyl)methylammonium salts. The compounds of the invention also form salts with organic and inorganic acids, including, without limitation, acetic, ascorbic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, salicyclic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methane sulfonic, naphthalene sulfonic, benzene sulfonic, toluene sulfonic and similar known, physiologically compatible acids. In addition, when a compound of Formula I contains both a basic moiety and an acidic moiety, zwitterions (inner salts) may be formed and are included within the term salt(s) as used herein.

(42) The sydnominine derivatives described herein, including the pharmaceutically acceptable salts thereof, can be conveniently prepared by those having ordinary skill and experience in organic synthesis, using known starting materials, and following the general synthetic scheme shown below, in which the radicals R.sub.1-R.sub.6 are as previously defined:

(43) ##STR00002##
Chemical reactions for the preparation of specific sydnominine derivatives which may be used in the practice of this invention are described in further detail hereinbelow. The starting materials for these reactions are available from commercial sources. See also, U.S. Pat. No. 3,277,108.

(44) In vitro studies have been performed that demonstrate the specific DAT inhibition activity of the compounds of the invention. DAT inhibition activity was tested according to the procedure described by J. Javitz et al., Mol. Pharmacol., 26: 35-44 (1984). The test results for a number of representative compounds of the invention are reported hereinbelow.

(45) As used herein, the expression method of treating disease alleviated by inhibiting DAT refers to a treatment using one or more of the compounds described above, which provides relief either by freeing the recipient of a disease or condition mediated by DAT or easing the symptoms or effects of such disease or condition. The method of the invention is intended for treating, preventing, managing and/or delaying the progression of the following: pulmonary conditions such as lung edema; ischemia-reperfusion injury; cardiac conditions, such as acute decompensated heart failure and the cardiorenal syndrome; hyperprolactinaemia (BrE), hyperprolactinemia (AmE) and microprolactinoma; pain including chronic or neuropathic pain; catatonic, dyskinesia, restles legs syndrome and related movement disorders; stress, chronic posttraumatic stress disorder, anxiety disorders, obsessive-compulsive disorders, postpartum depression; schizophrenia, manic, bipolar, and affective disorder; executive function disorders, such as ADHD, Tourette syndrome and autism; cocaine, amphetamine, alcohol dependency, and addictive behavior, such as pathological gambling; neuroendocrinal regulatory disorders; inflammatory conditions, autoimmune diseases and rheumatism; neoplastic disorders, such as pituitary carcinomas, macroprolactinomas; visual sensory disorders, color deficiency; and ejaculatory and related sexual dysfunction. The diseases and conditions enumerated above are given by way of example and not by way of limitation.

(46) In general, the compounds of the invention can be administered to achieve specific dopamine reuptake inhibition by using any acceptable means known in the art, either alone or in combination with one or more other therapeutic agent. Thus, the active agent(s) can be administered enterally, parenterally, such as by intravenous infusion, intramuscular, intraperitoneal or subcutaneous injection, by liposome-mediated delivery, vaginally, by inhalation or insufflation, transdermally or by otic delivery.

(47) Normally, a daily dose of the compound of the invention in the range from about 0.01 mg to about 200 mg/kg of body weight can be administered. A daily dose of from 0.1 to 100, and preferably from 1 to 30 mg/kg per day in one or more applications per day should be effective to produce the desired result. By way of example, a suitable dose for oral administration would be in the range of 1-30 mg/kg of body weight per day, whereas a typical dose for intravenous administration would be in the range of 1-10 mg/kg of body weight per day. Of course, as those skilled in the art will appreciate, the dosage actually administered will depend upon the condition being treated, the gender, age, health and weight of the recipient, the type of concurrent treatment, if any, and the frequency of treatment. Moreover, the effective dosage amount may be determined by one skilled in the art on the basis of routine empirical activity testing to measure the bioactivity of the compound(s) in a bioassay, and thus establish the appropriate dosage to be administered.

(48) The compounds of the invention will typically be administered from 1-4 times a day, so as to deliver the above-mentioned daily dosage. However, the exact regimen for administration of the compounds and compositions described herein will necessarily be dependent on the needs of the individual subject being treated, the type of treatment administered and the judgment of the attending medical specialist. As used herein, the term subject includes both humans and animals.

(49) The compounds of the invention may be administered as such, or in a form from which the active agent can be derived, such as a prodrug. A prodrug is a derivative of a compound described herein, the pharmacologic action of which results from the conversion by chemical or metabolic processes in vivo to the active compound. Prodrugs include, without limitation, ester derivatives of the compounds of formula I, above. Other prodrugs may be prepared according to procedures well known in the field of medicinal chemistry and pharmaceutical formulation science. See, e.g., Lombaert et al., J. Med. Chem., 37: 498-511 (1994); and Vepsalainen, Tet. Letters, 40: 8491-8493 (1999).

(50) The DAT specific compounds described herein and the pharmaceutically acceptable salts thereof are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The expression unit dosage form as used herein refers to a physically discrete unit of the active agent appropriate for the subject to be treated. Each dose should contain the quantity of active ingredient calculated to produce the desired therapeutic effect, either as such, or in association with the selected pharmaceutical carrier medium and/or supplemental active agent(s), if any. Typically, the DAT inhibitory compounds of the invention will be administered in dosage form containing from about 0.01 mg to about 200 mg of the active ingredient, with a range of about 30 mg to about 100 mg being preferred.

(51) The orally administered dosage unit may be in the form of tablets, caplets, dragees, pills, semisolids, soft or hard gelatin capsules, aqueous or oily solutions, emulsions, suspensions or syrups. Suitable dosage forms for parenteral administration include injectable solutions or suspensions, suppositories, powder formulations, such as microcrystals or aerosol spray. The active agent may also be incorporated into a conventional transdermal delivery system.

(52) A pharmaceutical composition in accordance with the present invention comprises one or more of the compounds of Formula I, above, in combination or admixture with a pharmaceutically acceptable carrier medium. The composition may, if desired, be administered in conjunction with one or more supplemental active agents. For example, the DAT inhibiting agent may be used in combination with L-dopa for the treatment of Parkinson's disease; or in combination with a selective serotonin reuptake inhibitor (SSRI) for the treatment of depression and or cocaine abuse and addiction; or in combination with dopamine D2 antagonist for the treatment of schizophrenia; or in combination with cholinergic modulators for the treatment of Alzheimer disease or other diseases or conditions in which patients have a cognitive deficit. The compound(s) of the invention may be administered either simultaneously (e.g., in the same formulation or not) or sequentially with the supplemental therapeutic agent(s).

(53) As used herein, the expression pharmaceutically acceptable carrier medium includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface agent agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, fillers and the like as suited for the particular dosage form desired. Remington: The Science and Practice of Pharmacy, 20.sup.th edition, A. R. Genaro et al., Part 5, Pharmaceutical Manufacturing, pp. 669-1015 (Lippincott Williams & Wilkins, Baltimore, Md./Philadelphia, Pa. (2000)) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional pharmaceutical carrier medium is incompatible with the DAT inhibitor compounds of the present invention, such as by producing an undesirable biological effect or otherwise interacting in an deleterious manner with any other component(s) of a formulation comprising such compounds, its use is contemplated to be within the scope of this invention.

(54) For the production of solid dosage forms, including hard and soft capsules, the therapeutic agent may be mixed with pharmaceutically inert, inorganic or organic excipients, such as lactose, sucrose, glucose, gelatine, malt, silica gel, starch or derivatives thereof, talc, stearic acid or its salts, dried skim milk, vegetable, petroleum, animal or synthetic oils, wax, fat, polyols, and the like. For the production of liquid solutions, emulsions or suspensions or syrups one may use excipients such as water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerine, lipids, phospholipids, cyclodextrins, vegetable, petroleum, animal or synthetic oils. For suppositories one may use excipients, such as vegetable, petroleum, animal or synthetic oils, wax, fat and polyols. For aerosol formulations, one may use compressed gases suitable for this purpose, such as oxygen, nitrogen and carbon dioxide. The pharmaceutical composition or formulation may also contain one or more additives including, without limitation, preservatives, stabilizers, e.g., UV stabilizers, emulsifiers, sweeteners, salts to adjust the osmotic pressure, buffers, coating materials and antioxidants.

(55) The present invention further provides controlled-release or sustained-release therapeutic dosage forms for the pharmaceutical composition, in which the composition is incorporated into a delivery system. This dosage form controls release of the active agent(s) in such a manner that an effective concentration of the active agent(s) in the bloodstream can be maintained over an extended period of time, with the concentration in the blood remaining relatively constant, to improve therapeutic results and/or minimize side effects. Additionally, a controlled-release system would provide minimum peak to trough fluctuations in blood plasma levels of the active agent.

(56) In the pharmaceutical compositions of the invention, the active agent(s) may be present in an amount of at least 0.5 and generally not more than 95% by weight, based on the total weight of the composition, including carrier medium and/or supplemental active agent(s), if any. Preferably, the proportion of active agent(s) varies between 30-90% by weight of the composition.

(57) While not wishing to be confined to any particular theory as to the biochemical mechanism of action of the compounds described herein, considering that these compounds have shown 1) highly potent and specific interaction with dopamine reuptake proteins, 2) fast onsite absorption and distribution (e.g. brain), and 3) little interaction with other proteins including the most prevalent metabolic enzymes, it is believed that these compounds may be used 1) alone to ameliorate disease conditions, central and or peripheral, when endogenous dopaminergic functions are augmented by the inhibition of dopamine reuptake; 2) in combination with dopamine (or dopaminergic agonists) to provide synergistic effects of augmented endogenous dopaminergic functions and drug effects; and 3) in combination with other than dopaminergic mechanism medications, when the treatment of disease requires the consideration of complex and multifaceted disease etiology.

(58) The following examples are provided to describe the invention in further detail. These examples are provided for illustrative purposes only and are not intended to limit the invention in any way.

EXAMPLE 1

Preparation of N-phenylcarbamoyl-3-(benzyl)-sydonimine

(59) 1.2 ml 7.5N aq. HCl was stirred (at 0 C.) into a mixture of 0.94 g benzyl amine and 0.58 g of potassium cyanide in 2 ml of water. 0.7 g formaldehyde was then added dropwise into the mixture. The resulting mixture was stirred at room temperature for 2 hours and then cooled to 0 C. A solution of 0.62 g sodium nitrite in 1 ml water was added slowly dropwise to the mixture followed by the addition of 1.2 m17.5N HCl aq. solution while cooling. The mixture was stirred at room temperature for 1 hour. Ether was used to extract the resulting mixture three times. The combined ether solution was dried with anhydrous sodium sulfate. The solvent was removed under reduced pressure. N-nitroso-benzylaminoacetonitrile (see reaction scheme) was obtained as a yellow oil. The nitroso intermediate was then treated with 500 ml HCl ethereal solution (2.0 M) and stirred for 30 minutes at room temperature. White precipitate was obtained and recrystallized with 2-propanol, resulting in a white crystal. 2.84 g of the 3-benzyl-sydnonimine hydrochloride thus produced was dissolved in 25 ml of water. To the solution was added 1.34 g sodium bicarbonate at 0 C. 2.45 ml of phenyl isocyanate was then added dropwise to the mixture and stirred at 0 C. for 4 hours to the resulting mixture was added 10 ml of ether to help the yellow crystal precipitate. Methanol was used as solvent for recrystallization. The desired product was obtained as yellow crystal.

EXAMPLES 2-7

(60) The compounds N-phenylcarbamoyl-3-(p-carboxylbenzyl-syndonimine (2); N-phenylcarbamoyl-3-(p-methyl-benzyl)-sydnonimine (3); N-(3,4-dichlorophenyl) carbamoyl-3-phenethyl sydnonimine (4); N-(3,4-dinitrophenyl)carbamoyl-3-p-nitrophenethyl sydnonimine (5); N-phenylcarbamoyl-3-(phenylpropyl)-sydnonimine (6); and N-phenylcarbamoyl-3-(p-fluoro-benzyl)-sydnonimine (7) were synthesized following essentially the same procedure as described in Example 1, above, with appropriate substitution of different starting materials in equivalent amounts.

EXAMPLE 8

(61) I. Dopamine Transporter Binding Assay [3H]WIN 35,428

(62) This assay was carried out according to the procedure described by Javitch, J. J. et al., Mol. Pharmacol. 26: 35-44; 1984

(63) A. Tissue Preparation (Prepare all Solutions on Ice) 1. Thaw frozen brains from male Guinea Pigs (in assay buffer) and place in 50 mM TRIS-HCl (pH 7.4 at 25 C. with 120 mM NaCl). Isolate striatum. 2. Use a Polytron to homogenize tissue in 20 vols. (w/v) of 50 mM Tris-HCl (pH 7.4 at 25 C. with 120 mM NaCl). 3. Centrifuge homogenate at 48,400g for approximately 10 minutes at 4 C. Discard supernatant. 4. Wash pellet an additional time as described in steps 2 and 3. 5. Store pellet on ice until needed for binding assay. 6. Using a Polytron (setting 5; approximately 10 seconds) resuspend pellet in 50 mM Tris-HCl (pH 7.4 at 25 C. with 120 mM NaCl) to an initial concentration of 10 mg/ml (original wet weight), such that the final concentration is 8 mg/ml or 4.0 mg tissue/tube.

(64) B. Binding Reaction 1. Each tube receives the following components: 50 ul drug or vehicle 50 ul [3H]-WIN 35,428 400 ul tissue suspension. 2. Initiate binding reaction with the addition of tissue, and incubate for 120 minutes at 0 C. (on ice). 3. Terminate binding reaction by rapid filtration of tube contents onto untreated Whatman GF/C filters. 4. Rinse the assay tubes once with ice cold 50 mM TRIS HCl (pH 7.4 at 25 C. with 120 mM NaCl, BSA), then rapidly rinse the filters with 61 mls/tube of the same wash buffer. 5. Radioactivity trapped onto the filters is assessed using LSM machine. Let sit for 3 hours in scint cocktail.

(65) C. Dopamine Tranasporter Binding Assay

(66) Materials and Reagents 1. [3H]-WIN 35-428 is diluted to a concentration of 60 nM in 50 mM TRIS HCl (pH 7.4 at 25 C. with 120 mM NaCl). Thus, the final ligand concentration is 6 nM. 2. Non-specific binding is defined as that remaining in the presence of 110-6 M GBR12909 (room temperature cabinet). GBR12909 MW=523.5 g/mol. Will solubilize in 4% DMSO (sonicate 15 minutes). Aliquots can be used (1E-3). 3. The reference compound for the assay is GBR12909 and is diluted in 4% DMSO and is then run at the following final concentrations: 110.sup.10, 510.sup.10, 210.sup.9, 510.sup.9, 110.sup.8, 210.sup.8, 510.sup.8, 110.sup.7, 210.sup.7, 510.sup.7, 110.sup.6, 510.sup.6. 4. The positive control GBR12909 and is run at the final concentrations of: 210-8, 110.sup.7, 510.sup.7M. 5. The K.sub.d for the receptor is 28.0 nM.
Buffers

(67) TABLE-US-00001 Assay Buffer 1 L 2 L 3 L 4 L 50 mM Tris- 6.06 g 12.12 g 18.18 g 24.24 HC1 pH 7.4 120 mM NaC1 7.01 g 14.02 g 21.03 g 28.04 g
Wash Buffer (Add 1 g BSA/1L Assay Buffer)

(68) TABLE-US-00002 50 mM Tris-HCl pH 7.4 24.2 g/4 L 120 mM NaCl 28.0 g/4 L BSA 4 g/4 L Ki: 2.25-22.5 e9
II: Norepinephrine Transporter (Human Recombinant) Binding Assay This assay was carried out in accordance with the procedure described by Raisman, R, et al. Eur. Jrnl. Pharmacol. 78: 345-351 (1982) with modifications. See also, Langer, S., et al., Eur. Jrnl. Pharmacol, 72: 423-4 (1981).
Tissue Preparation 1. hrNET (Receptor Biology; RBNET) is diluted in assay buffer to 2.5 microunits/ml, so that each tube receives 0.5 microunits, or 2 microunits/ml. NOTE: 100 microassays, add 20 mis of buffer.
Binding Reaction 1. Each tube receives the following components: 25 ul drug or vehicle 25 ul [.sup.3H]-Nisoxetine 200 ul tissue suspension. 2. Initiate binding reaction with the addition of tissue, and incubate at room temperature for 1 hour. 3. Terminate binding reaction by rapid filtration of tube contents onto 0.1% PEI treated GF/C filters (TopCount). 4. Rinse the assay tubes once with ice cold 50 mM NaCl, then rapidly rinse the filters with 61 ml/tube of the same wash buffer. 5. Radioactivity trapped onto the filters is assessed using liquid scintillation spectrophotometer after soaking the filters for at least 1 hour in scintillation cocktail.
Materials and Reagents 1. The receptor source is human recombinant/CHO. 2. [.sup.3H]-Nisoxetine, diluted to a concentration of 10 nM in assay buffer, is used as the radioligand. Thus, the final ligand concentration is 1 nM. 3. Non specific binding is defined as that remaining in the presence of 110.sup.6M desipramine (MW=302.8) (Make fresh: in bag with dessicating rocks 1, dissolves in water 1E-3). 4. The reference compound for the assay is desipramine. Desimpramine is run at following final concentrations: 110.sup.10, 210.sup.10, 510.sup.10, 110.sup.9, 210.sup.9, 510.sup.9, 110.sup.8, 210.sup.8, 510.sup.8, 110.sup.7, 210.sup.7, 510.sup.7M 5. The positive control is any of the above run at the final concentrations of: 110.sup.9, 510.sup.9, 210.sup.8 M. 6. The K.sub.d for the receptor is 3 nM.
Buffers

(69) TABLE-US-00003 MW (g/mole) g/1 L Incubation Buffer: 50 mM Tris, pH 7.4 121 6.06 5 mM KCl 74.6 0.38 120 mM NaCl 58.4 7.02 Wash Buffer: 50 mM NaCl 58.4 3.0

(70) The results of the DAT and NET binding assays are set forth in the following table:

(71) TABLE-US-00004 INH INH at 10 at 10 Ex. M M Ki No. Name Structure DAT NET DAT 1 3-(benzyl)- syndonimine-N- phenylcarbamoyl 90% 0% 36 nM 2 3-(p-carboxylbenzyl)- syndonimine N- phenylcarbamoyl 92% 11% 2.7 nM 3 3-(p-methyl-benzyl)- syndonimine-N- phenylcarbamoyl 94% 14% 0.19 nM 4 3-phenethyl- syndonimine-N- (3,4-dichlorophenyl) carbamoyl 99% 2% 40 nM 5 3-(p-nitrophenethyl) syndonimine-N- (3,4-dinitrophenyl) carbamoyl 85% 4% 100 nM 6 3-(phenylpropyl)- syndonimine-N- phenylcarbamoyl 96% 1.44% 25 nM 7 3-(p-fluorobenzyl)- syndonimine-N- phenylcarbamoyl 99% 15% 0.46 nM

(72) Preferred compounds of the invention have a DAT binding inhibition (at 10 m) of greater than or equal to 90% and a NET binding inhibition (at 10 M) of less than or equal to 15%; or a ratio of DAT:NET binding inhibition (at 10 M) of at least 9:1.

EXAMPLE 9

(73) The following materials and methods are provided to facilitate the practice of Example 9, in which drug concentration in certain biological samples were assessed using different routes of administration.

(74) Parameters Used in Dosing and Drug Administration of the Compound of Example 3:

(75) TABLE-US-00005 Dose route Dose range Dose volume (Use a 1-ml syringe with appropriate oral feeding needle.) i.v. 10 mg/kg 5-10 bodyweight (5-10 ml/kg, i.e. a 25 g mouse receives 125-250 ul) i.p 10 mg/kg 10 bodyweight Oral 10, 30 mg/kg 10 bodyweight
Mouse Strain

(76) Adult C57BL/6 mice (preferred 16 weeks old).

(77) Time Point

(78) Pre-dose, 5, 15, 30, 60, 120, 240 and 480 mins for IV.

(79) Pre-dose, 15, 30, 60, 120, 240, 480 and 1,440 mins for IP and oral.

(80) Three mice per time point. If all three routes are conducted in same day, one group of pre-dose is efficient.

(81) Dosing Formula

(82) 10% EtOH

(83) 0.05-0.08% 7.5N HCL

(84) 30% Captisol in Saline

(85) Final PH at 2.5-3

EXAMPLE 10

Spontaneous Locomotor Activity (Open Field Study)

(86) A dose response study was conducted of locomotor depression induced by the compound of Example 3, according to NIDA's Medication Development Division (MDD) locomoter activity studies standard protocol. The study was conducted using 40 Digiscan locomotor activity testing chambers (40.540.530.5 cm) housed in sets of two, within sound attenuating chambers. A panel of infrared beams (16 beams) and corresponding photodetectors were located in the horizontal direction along the sides of each activity chamber. A 7.5 W incandescent light above each chamber provided dim illumination. Fans provided an 80 dB ambient noise level within the chamber. Separate groups of 8 non-habituated male Swiss Webster mice (Hsd:ND4, aged 2-3 months) were injected via the intraperitoneal (IP) route with either vehicle (2% methyl cellulose) or the compound of Example 3, above, (3, 10, 30 or 100 mg/kg) twenty minutes prior to locomotor activity testing. Just piro to placement in the apparatus, all mice received a saline injection IP. In all studies, horizontal activity (interruption of photocell beams) was measured for 1 hour within 10 minute periods. Testing was conducted with one mouse per activity chamber.

(87) The same protocol was followed in a study of locomotor depression induced by the compound of Example 6.

EXAMPLE 11

Time-Course (8 Hr.) Mouse Locomotor Activity Testing

(88) A time course/dose response study of the locomotor depression inducing effect of the compound of Example 3 was conducted, according to the same MDD locomotor activity studies time course protocol described immediately above, except that groups of 8 mice were injected with either vehicle (2% methylcellulose) or the compound of Example 3 (3, 10, 30 or 100 mg/kg), immediately prior to locomotor activity testing. Behavioral observations were recorded on each mouse at 30, 120 and 480 minutes following 100 mg/kg of the test compound. The vehicle used in this study was 2% methylcellulose.

(89) A separate time course/dose response study of the locomotor depression inducing effect of the compound of Example 6 was also conducted, under the same conditions described above for testing of the compound of Example 3. Additional groups of 8 mice were injected with either vehicle (2% methylcellulose) or the test compound (3, 10, 30 or 100 mg/kg), immediately prior to locomotor activity testing. Behavioral observations were recorded on each mouse at 30, 120 and 480 minutes following 100 mg/kg of the test compound. The vehicle used in this study was 2% methylcellulose.

Results

(90) The inventors have identified DAT selective inhibitors for use in the treatment and prevention of cocaine abuse and other disorders associated with aberrant dopamine reuptake. Two such inhibitors, namely, the compounds of Examples 3 and 6, above, are well tolerated up to at least 200 mg/kg. Observable changes in behavior occurred in minutes and lasted for about 2-3 hours. Notably subjects treated with the compound of Example 3 became lethargic at dosages above 125 kg/mg, whereas this effect was reduced at higher concentrations of the compound of Example 6.

(91) In the studies using adult C57BL/6 mice (preferably approximately 16 weeks old), dosing and drug administration parameters for the compound of Example 3 were explored. Table 2 summarizes the results of these studies. Clearly, the compound is suitable for oral administration and lasts in the plasma up to 2-3 hours. Moreover, the compound of Example 3 reaches the brain whether administered orally or via intravenous administration. See Table 3.

(92) TABLE-US-00006 TABLE 2 Plasma Concentration (ng/mL) by Different Routes of Drug Administration Time 10 mg/kg (IV) 10 mg/kg (IP) 10 mg/kg (PO) 30 mg/kg (PO) 0 18.33 21.00 10.60 8.93 5 2091.03 973.00 218.50 837.87 15 2110.87 315.97 115.45 669.80 30 915.00 544.25 59.90 374.53 60 416.63 115.40 21.30 113.87 120 139.27 82.47 17.57 41.87 240 47.43 34.03 15.80 48.53 480 94.63 31.20 14.30 13.40

(93) TABLE-US-00007 TABLE 3 Brain Drug Concentration (ng/mL) by Different Routes of Drug Administration Time 10 mg/kg (IV) 10 mg/kg (IP) 10 mg/kg (PO) 30 mg/kg (PO) 0 95.10 0.00 107.55 123.97 5 2313.10 1456.65 417.80 868.87 15 1723.27 374.43 297.60 844.87 30 1139.30 242.00 149.93 626.63 60 464.40 189.93 158.00 217.60 120 78.70 70.20 165.13 203.07 240 177.87 143.13 97.47 183.03 480 48.95 122.95 163.00 168.35
Additional pharmacokinetic studies were performed, as shown in FIGS. 2A-2D. It appears that the compound of Example 6 exhibits slightly better bioavailability in the plasma and brain when compared to the compound of Example 3 at the same dosage. The compound of Example 6 also exhibits relatively higher oral bioavailability when administered at different dosages. See FIG. 3.

(94) The spontaneous locomotor activity assessments were performed following administration of the DAT inhibitors of the present invention according to the procedure described in Tella et al. (1996) Pharmacol Biochem. Behav. 54:343-54; Elmer et al. (1996) Pharmacol Biochem Behav 53:911-918. The results of these assessments appear in FIGS. 4 and 5. FIGS. 4A and 4B are graphs that show that the compound of Example 3 demonstrates a dose dependent suppression of spontaneous locomotor activity. The compound of Example 6 showed a similar dose dependent response, as can be seen in FIGS. 5A and 5B.

(95) The results of the above-described time-course mouse locomotor activity experiment using the compound of Example 3 are set forth in FIG. 6A, which shows average horizontal activity counts/10 min as a function of time (0-8 hr) and dose of the compound (top to bottom panels). Treatment with this compound resulted in time- and dose-dependent depression of locomotor activity following 30 and 100 mg/kg. Depressant effects of 30 and 100 mg/kg occurred within 10 minutes following injection and lasted 140 to 160 minutes. The period 20-50 min was selected for analysis of dose-response data because this was the time period in which maximal suppression first appeared as a function of dose. The mean average horizontal activity counts/10 min for this 30-min period were fit to a linear function of log.sub.10 dose of the descending portion of the dose-effect curve (3 to 100 mg/kg dose range). The ID.sub.50 (dose producing maximal depressant activity, where maximal depression=0 counts/30 mint) was estimated to be 24.0 mg/kg.

(96) A two-way analysis of variance conducted on horizontal activity counts/10 min indicated a significant effect for Treatment F(4,35)=5.35, p=0.002, 10-Minute Periods F(47,1645)=42.63, p<0.001, and for the interaction of Periods and Treatment F(188,1645)=2.26, p<0.001. A one-way analysis of variance conducted on log.sub.10 horizontal activity counts for the 20-50 min time period (maximal depressant effect) indicated a significant effect of Treatment F(4,35)-27.59, p<0.001, and planned comparisons (a priori contrast) against the vehicle group showed a significant depressant effect for 30 and 100 mg/kg (ps<0.05 denoted on FIG. 6A with an asterisk).

(97) The results of the time-course mouse locomotor activity experiment using the compound of Example 6, is provided in FIG. 6B, which shows average horizontal activity counts/10 min as a function of time (0-8 hr) and dose of the test compound (top to bottom panels). Treatment with this compound resulted in time-dependent depression of locomotor activity following 100 mg/kg. Depressant effects of 100 mg/kg occurred within 10 minutes following injection and lasted 160 minutes. The period 50-80 min was selected for analysis of dose-response data because this was the time period in which maximal suppression first appeared as a function of dose. The mean average horizontal activity counts/10 min for this 30-min period were fit to a linear function of log.sub.10 dose of the descending portion of the dose-effect curve (10 to 100 mg/kg dose range). The ID.sub.50 (dose producing maximal depressant activity, where maximal depression=0 counts/30 min) was estimated to be 30.2 mg/kg.

(98) A two-way analysis of variance conducted on horizontal activity counts/10 min indicated a significant effect of Treatment F(4,35)=6.84, p<0.001, 10-Minute Periods F(47,1645)=48.92, p<0.001, and the interaction of Periods and Treatment F(188,1645)=1.64, p<0.001. A one-way analysis of variance conducted on log 10 horizontal activity counts for the 50-80 min time period (maximal depressant effect) indicated a significant effect of Treatment F(4,35)=6.01, p=0.001, and planned comparisons (a priori contrast) against the vehicle group showed a significant depressant effect for 100 mg/kg (ps<0.05 denoted on FIG. 6B with an asterisk).

(99) To further characterize the pharmacological effects of these compounds, Irwin behavioral battery testing was performed.

(100) Testing occurred towards the end of the light cycle between 8-11 a.m. (lights go off at 11:00 a.m.). The mice were acclimated to the testing room for at least 30 minutes. The Irwin test equipment (timer, viewing jar and support, open arena, grid, ruler, sound box, wood stocks, kimwipes box) was placed under a laminar flow hood. The mouse was first placed in the viewing jar for five (5) minutes and the following parameters were scored:

(101) 1. General appearancecoat quality, whiskers

(102) 2. Body position

(103) 3. Spontaneous activity

(104) 4. Respiration rate

(105) 5. Tremors

(106) 6. Twitches

(107) 7. Bizarre behaviorcomments

(108) 8. Convulsions

(109) 9. Defecationnumber of feces at the end of the session

(110) At the end of the 5-minute period, the viewing jar with the muose in it was transferred to the arena, where the jar was disassembled and the mouse released into the arena. The mouse was not handled by the experimenter. The following parameters were scored in the arena in the following order:

(111) 1. Transfer arousalduring the 10 first seconds

(112) 2. Locomotor activitynumber of squares entered by all four feet in 30 seconds

(113) 3. Palpebral closure

(114) 4. Piloerection

(115) 5. Startle response90 dB sound from clickbox 30 cm above arena

(116) 6. Gait

(117) 7. Pelvic elevation

(118) 8. Tail elevationduring forward motion

(119) 9. Touch escapefinger stroke from above

(120) 10. Tail pinchwith forceps 3 cm from the base of tail

(121) 11. Body temperaturehypo- or hyperthermia

(122) 12. Positional passivitystruggle response to sequential handling by tail, neck, hind legs, or held supine

(123) Then the mouse was picked up by its tail and kept above the arena to score the following set of parameters:

(124) 1. Trunk curl

(125) 2. Limb grasping

(126) 3. Visual placingextension of forelimbs when animal is lowered by base of tail from a height of 15 cm above a wire grid

(127) 4. Grip strengthmouse is lowered and allowed to grip the grid; gentle horizontal backwards pull is applied

(128) 5. Body tonesides of mouse are compressed between thumb and index finger

(129) 6. Pinna reflexmouse is gently restrained on grid and the proximal part of the inner cathus is touched lightly with the tip of fine wire probe; ear retraction is observed

(130) 7. Corneal reflexmouse is gently restrained no grid and the cornea is touched lightly with the side of fine wire probe; eye-blink response is observed

(131) 8. Toe pinchthe mid digit of hind foot is gently compressed laterally with fine forceps. The hind limbs are lifted clear of the grid

(132) 9. Wire Maneuvermouse is held above the wire by tail suspension and lowered to allow the forelimbs to grip the horizontal wire; the mouse is held in extension and rotated around to the horizontal and released

(133) The mouse was then placed in supine restraint to score the below parameters in the following order:

(134) 1. Body lengthfrom tip of nose to base of tail (mm)

(135) 2. Skin colorplantar surface and digits of forelimbs

(136) 3. Heart ratefelt by palpation below sternum

(137) 4. Limb toneresistance to gentle finger tip pressure on plantar surface of left/right hind paw

(138) 6. Lacrimation

(139) 7. Pupil reflex

(140) 8. Salivation

(141) 9. Provoked bitingdowel rod gently inserted between the teeth at the side of the animal's mouth

(142) 10. Irritability

(143) The final three parameters were scored with the mouse above or in the arena:

(144) 1. Righting reflexmouse is held by the tail and flicked backgrounds through the air such that it performs a backward somersault when released; the landing position is observed

(145) 2. Contact righting reflexmouse is placed into a plastic tube and turned upside down

(146) 3. Negative geotaxismouse is placed no horizontal grid; the grid is raised to vertical with mouse facing the floor; behavior is observed for 30 seconds

(147) ##STR00010##
The test results presented in FIGS. 7A-7C demonstrate that the compound of Example 3 induced significant behavioral effects in the Irwin Behavioral Batter. FIG. 7A shows the results on spontaneous activity. FIG. 7B shows the results on grip strength. FIG. 7C shows the effects of the compound administration on limb tone.

(148) Open Field testing was also performed. In carrying out this testing, individually housed male and female mice aged 8-12 weeks were used. The mice were maintained on a reverse L:D/12 p.m.: 12 a.m. cycle in a barrier facility. Food and water were available ad libitum. Testing occurred towards the end of the light cycle between 6-12 a.m. Mice were acclimated to the testing room for at least 30 minutes. The assay was performed in a custom-made Open Field Apparatus. Each chamber was a 50 by 50 cm square. The experiment was recorded and tracked by a tracking system obtained from Viewpoint.

(149) The time and the path length in the center of the open field were determined. The center of the open field is defined as a 13.513.5 cm square in the geometric center of the arena. The percent of path in the center is calculated as

(150) $\frac{\mathrm{Path}\mathrm{length}\mathrm{in}\mathrm{the}\mathrm{center}}{\mathrm{Total}\mathrm{path}\mathrm{length}}100\%$
For each mouse, the total path length and path length for 60 minutes at 5 minute intervals was determined as a measure of locomotor activity. In addition, feces produced by each experimental animal during the test was counted. Each chamber was cleaned between individual mouse testing.

(151) Path length for 60 minutes at 5 minute intervals was analyzed by repeat measures using SPSS software. All other parameters were compared using unpaired t-test (GraphPad Prism).

(152) ##STR00011##
The data presented in FIG. 8 demonstrates that the compound of Example 3 is effective to reduce locomotor activity in open field testing. In yet another assay, the same compound produced demonstrable anxiolytic effects. See FIGS. 9A-9D.

(153) Novel environment-induced feeding suppression (NEIFS) testing was also performed. Individually housed male mice aged 8-12 weeks are used for this testing. Mice were maintained no a reverse L:D/12 p.m.:12 a.m. cycle in a barrier facility with food and water available ad libitum. Testing occurred towards the end of the light cycle between 6-12 a.m. The mice were acclimated to the testing room for at least 30 minutes.

(154) Days 1, 2, 3 and 5 (Home cage): A Petri dish containing crushed Graham crackers was placed in the farthest corner from each mouse in its own home cage. The time to approach (defined as nose of the mouse directed at or within 1 cm of the dish) and consume (eat, not pick up) the crackers was recorded immediately following the placement of the dish into home cage.

(155) Day 4 (Novel environment): The procedure followed was the same as described for home cage; however, on day 4 the mouse was placed in a new cage with bedding (novel environment) for the duration of the experiment. The mice were subsequently returned to their respective home cages for day 5 testing. The remaining crackers and Petri dish were disposed of at the end of the experiment for each mouse.

(156) In a novel environment induced feeding suppression assay, the compound of Example 3 induced measurable anxiolytic effects. See FIG. 10. Notably, the compound had no effect on the number of fecal boli in open field testing.

(157) Rotarod assays were also performed using the experimental protocol and design set forth below. Individually housed male mice aged 8-12 weeks were used for this experiment. Mice were maintained on a reverse L:D/12 p.m.: 12 a.m. cycle in a barrier facility with food and water available ad libitum. Testing occurred towards the end of the light cycle between 6-12 a.m. Mice were acclimated to the testing room for at least 30 minutes.

(158) The assay was carried out using four EzRod test chambers kept in a laminar hood for testing. For the accelerating rotarod paradigm, mice were given 10 trials with the maximum duration of 3 minute and a 30-second intertrial interral (ITI). Each mouse was placed on the EZRod machines and the latency to fall was recorded for all trials. If the mouse fell or 3 minutes elapsed, the mouse was left in the bottom of the EzRod test chamber for 30 seconds before starting the next trial.

(159) The latency to fall was compared between the two groups by analysis of variance (ANOVA) with repeated measures.

(160) ##STR00012##

(161) The data presented in FIG. 11 reveal that the compound of Example 3 did not affect rotarod performance.

(162) The foregoing specification includes citations to certain patent and literature references, which are provided to indicate the state of the art to which this invention pertains. The entire disclosure of each of the cited references is incorporated by reference herein.

(163) While certain embodiments of the present invention have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. The present invention is, therefore, not limited to the particular embodiments described and/or exemplified, but is capable of considerable variation and modification without departure from the scope of the appended claims.