WO1998017636A1 - Phenylalaninol derivatives for the treatment of central nervous system disorders - Google Patents

Abstract

This invention relates to a series of phenylalaninol derivatives of formula (I) where X, R, R1, R2, Y, q, n, and p defined herein. In addition the invention relates to pharmaceutical compositions containing them and intermediates used in their manufacture. The compounds of the invention are modulators of the NPY1 receptor and display anxiolytic animal models.

Description

PHENYLALANINOL DERIVATIVES FOR THE TREATMENT OF CENTRAL NERVOUS SYSTEM DISORDERS This invention relates to a series of phenylalaninol derivatives, pharmaceutical compositions containing them and intermediates used in their manufacture. The compounds of the invention are ligands for the neuropeptide Y1 (NPY1 ) receptor, a receptor which is associated with a number of central nervous system disorders In addition, select compounds of the invention exhibit anxiolytic activity in an animal model.

BACKGROUND OF THE INVENTION Regulation and function of the mammalian central nervous system is governed by a series of interdependent receptors, neurons, neurotransmitters, and proteins. The neurons play a vital role in this system, for when externally or internally stimulated, they react by releasing neurotransmitters that bind to specific proteins. Common examples of endogenous small molecule neurotransmitters such as acetylcholine, adrenaline, norepinephrine, dopamine, serotonin, glutamate, and gamma-aminobutyric acid are as well known, as the specific receptors that recognize these compounds as ligands ("The Biochemical Basis of Neuropharmacology", Sixth Edition, Cooper, J. R.; Bloom, F. E.; Roth, R. H. Eds., Oxford University Press, New York, NY 1991 ). There are a number of diseases that are associated with this interdependent system such as anxiety, depression, pain and schizophrenia. These diseases have been treated with small molecules and peptides which modulate neuronal responses to endogenous neurotransmitters. However, in addition to the endogenous small molecule neurotransmitters, there is increasing evidence that neuropeptides play an integral role in neuronal operations. Neuropeptides are now believed to be co-localized with perhaps more than one-half of the 100 billion neurons of the human central nervous system. Aside from humans, neuropeptides have been discovered in a number of animal species. In some instances the composition of these peptides is remarkably homogenous between species. This finding suggests that the function of neuropeptides is vital and has been impervious to evolutionary changes. Therefore, agents which affect the binding of neuropeptides to their respective receptors or affect responses which are mediated by neuropeptides are potential therapies for diseases associated with neuropeptides.

Neuropeptides, unlike small molecule neurotransmitters, are typically synthesized by the neuronal ribosome. In some cases, the active neuropeptides are produced as part of a larger protein which is enzymatically cleaved to yield the active substance. Compared to small molecule neurotransmitters, neuropeptide- based strategies may offer novel therapies for CNS diseases and disorders.

One example of the class of neuropeptides, is neuropeptide Y (NPY). NPY was first isolated from brain and was shown to be structurally similar to other members of the pancreatic polypeptide (PP) family such as peptide YY (Tatemoto, K. et al. Nature 1982, 296, 659). Neuropeptide Y is a single peptide protein that consists of thirty-six amino acids containing an amidated C-terminus. NPY sequences from a number of animal species have been elucidated and all show a high degree of amino acid homology to the human protein (>94% in rat, dog, rabbit, pig, cow, sheep) (see Larhammar, D. in "The Biology of Neuropeptide Y and Related Peptides", Colmers, W. F. and Wahlestedt, C. Eds., Humana Press, Totowa, NJ 1993).

NPY is often co-localized with norepinephπne (NE) rich neurons and NPY can regulate classic NE actions such as constriction of peripheral vascular beds, stimulation of sensory nerves as well as control of certain primary CNS functions (pituitary hormone release, behavior, central autonomic control).

Endogenous receptor proteins that bind NPY and related peptides as ligands have been identified and distinguished, and several such proteins have been cloned and expressed. Six different receptor subtypes (Y1 , Y2, Y3, Y4(PP1 ), Y5, PYY) are recognized today based upon binding profile, pharmacology and / or composition if identify is known (Lundberg, J. M. et. al. Trends in Pharmaceutical Sciences 1996, 17, 301 ; Gerald, C. et. al. Nature 1996, 382, 168; Weinberg, D. H. et. al. Journal of Biological Chemistry 1996, 271, 16435; Gehlert, D. et. al. Current Pharmaceutical Design 1995, 1, 295)). Most and perhaps all NPY receptor proteins belong to the family of so-called G-protein coupled receptors (GPCRs). The neuropeptide Y1 receptor, a known GPCR, is negatively coupled to cellular cyclic adenosine monophosphate (cAMP) levels via the action of adenylate cyclase (Westlind- Danielsson, A. et. al. Neuroscience Letter 1987, 74, 237; McDermott, B. J. et. al. Cardiovascular Research 1993, 27, 893). For example, NPY inhibits forskolin- induced cAMP production / levels in SK-N-MC neuroblastoma cell line. A Y1 ligand that mimics NPY in this fashion is an agonist whereas one that competitively reverses the NPY inhibition of forskolin-induced cAMP production is an antagonist. Neuropeptide Y itself is the archetypal substrate for the Y1 receptor and its binding can elicit a variety of pharmacological and biological effects in vitro and in vivo. When administered to the brain of live animals (intraventricularly (icv) or into the amygdala), NPY produces anxiolytic effects in established animal models of anxiety (e.g., the elevated plus-maze, Vogel punished drinking and Geller-Seifter's bar-pressing conflict paradigms) (Heilig, M. et. al. Psychopharmacology 1989, 98, 524; Heilig, M. et. al. Reg. Peptides 1992, 41, 61 ; Heilig, M. et. al. Neuropsycho- pharmacology 1993, 8, 357.). Thus compounds that mimic NPY are postulated to be useful for the treatment of anxiolytic disorders associated with the Y1 receptor. The immunoreactivity of neuropeptide Y is notably decreased in the cerebrospinal fluid of patients with major depression and those of suicide victims (Widdowson, P. S. et. al. Journal of Neurochemistry SI, 59, 73), but rats treated with tricyclic antidepressants display significant increases of NPY relative to a control group (Heilig, M. et. al. European Journal of Pharmacology 1988, 147, 465). These findings suggest that an inadequate NPY response may play a role in some depressive illnesses, and that compounds that regulate the NPY-ergic system may be useful for the treatment of depression.

Neuropeptide Y improves memory and performance scores in animal models of learning (Flood, J. F. et. al. Brain Research 1987, 421, 280) and therefore may serve as a cognition enhancer for the treatment of neurodegenerative diseases such as Alzheimer's Disease (AD) and AIDS-related and senile dementia.

Elevated plasma levels of NPY are present in animals and humans experiencing episodes of high sympathetic nerve activity such as surgery, newborn delivery and hemorrhage (Morris, M. J. et. al. Journal of Autonomic Nervous System 1986, 17, 143). Thus chemical substances that alter the NPY-ergic system may be useful in the treatment of stress. The binding of NPY to the Y1 receptor also mediates endocrine functions such as the release of luteinizing hormone (LH) in rodents (Kalra, S. P. et. al. Frontiers in Neuroendrocrinology 1992, 13, 1 ). Since LH is vital for mammalian ovulation, a compound that mimics the action of NPY could be useful for the treatment of infertility, particularly in women with so-called luteal phase defects. NPY is orexigenic in rodents (Clark, J. T. et. al. Endocrinology 1984, 115, 427; Levine, A. S. et. al. Peptides 1984, 5, 1025) , but not anxiogenic when given intracerebro-ventricularly. Thus antagonism of the neuropeptide Y1 receptor may be useful for the treatment of eating disorders such as those associated with obesity and anorexia.

In the periphery, NPY inhibits pre-synaptic release of substance P from sensory neurons and such an effect can cause diminution of pain (analgesia) (Hua, X. Y. et. al. Journal of Pharmacological Experimental Therapy 1991 , 258, 243);. In animals, NPY produces dose-related antinociception (hot plate test) not associated with opioid (naloxone competition) or adrengeric (idazoxan) activities (Broqua, P. et. al. Brain Research 1996, 724, 25). Thus NPY receptor ligands may be useful for the treatment and relief of pain.

SUMMARY OF THE INVENTION The present invention is related to compounds of Formula I

wherein:

R is selected from the group consisting of hydrogen, d-βalkyl, C^cycloalkyl, Ci-βalkoxy, (RaJm- C_j- ^* where m is 1-5, R₃ is selected from one or more or the group consisting of Cι_-5alkyl, substituted Cι.₅alkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), Cι.₅alkoxy, Cι.₅alkylthio, nitro, amino, Cι_-5alkylamino, cyano, carboxylic acid Cι-5alkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, and halogen, and o

⁴ ^ whe ^■re R₄ is aminoCι_-9alkyl or amino;

Ri is selected form the group consisting of hydrogen, Cι_-5aikyl, substituted Ci.salkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), Cι_-5alkoxy, Cι_-5alkylthio, nitro, amino, C_1-5alkylamino, cyano, carboxylic acid Cι_-5alkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, or halogen;

R₂ is selected form the group consisting of hydrogen, C_1-5alkyl, substituted

Ci.salkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), Ci-saikoxy, Ci.salkylthio, nitro, amino, Cι.₅alkylamino, cyano, carboxylic acid Cι_-5alkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, or halogen;

n is 1-5 X is O or NH; Y is NH or CH_2; and q is O or l ; or pharmaceutically acceptable salts thereof.

As modulators of the NPY1 receptor, the compounds of Formula 1 are useful for treating central nervous system disorders such as anxiety, depression, cognition impairment, pain, and alcohol abuses. The compounds compete with the natural ligands, NPY1 and PYY and bind to the NPY1 receptor. In addition, the compounds demonstrate agonist activity by mimicking the activity of NPY1 in a cyclic adenosine monophosphate assay which uses human ceils. In addition, the compounds exhibit anxiolytic activity in the rat although not in the monkey assay. The compounds appear to be selective for anxiolytic activity in the rat, for they do not exhibit anticonvulsant activity or general skeletal CNS activity in a comparable rodent model.

The compounds are ligands of the NPY receptor, but are not necessarily limited solely in their pharmacological or biological action due to binding to this or any neuropeptide, neurotransmitter or G-protein coupled receptor. For example, the described compounds may also undergo binding to adrengeric receptors. In addition, the compounds described herein are potentially useful in the regulation of endocrine functions, particularly those associated with the pituitary and hypothalamic glands, and may be useful for the treatment of inovulation/infertility due to insufficient release of luteinizing hormone (LH).

The present invention comprises pharmaceutical compositions containing one or more of the compounds of Formula I. In addition, the present invention comprises intermediates used in their manufacture of compounds of Formula I

DETAILED DESCRIPTION OF THE INVENTION

As used herein unless otherwise noted the terms "alkyl" and "alkoxy" whether used alone or as part of a substituent group, include straight and branched chains. For example, alkyl radicals include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, 2-methyl-3-butyl, 1- methylbutyl, 2-methylbutyl, neopentyl, hexyl, 1-methylpentyl, 3-methylpentyl. Alkoxy radicals are oxygen ethers formed from the previously described straight or branched chain alkyl groups. With reference to substituents, the term "independently" means that when more than one of such substituent is possible, such substituents may be the same or different from each other. When compounds contain a basic moiety, acid addition salts may be prepared and may be chosen from hydrochloric, hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic, cinnamic, mandelic, methanesuifonic, p-toluenesulfonic, cyclohexanesulfamic, salicylic, 2-phenoxybenzoic, 2-acetoxybenzoic, or saccharin, and the like. Such salts can are made by reacting the free base of compounds of formula I with the acid and isolating the salt.

The compounds of formula I are prepared using the synthetic strategies depicted in Scheme I; more details are provided in subsequent schemes. This chemistry allows for the production of carbamate, urea and amide derivatives. Each approaches offer distinct advantages. For example, when X is an oxygen atom, the phenylalaninol reacts efficiently with electron poor phenyl isocyanates; however a copper I salt is needed to catalyze reaction with some electron rich isocyanates. Alternatively, activation of the phenylalaninol as a chloroformate allows for simple amines to be introduced, especially those difficult to activate as an isocyanate. As discussed below, the methodology to construct urea, ether and carbonate derivatives has also been successfully developed.

Scheme I

(i)

More specifically, a N-protected phenylalaninol a is reacted with a electron- poor phenylisocyanate b in an appropriate solvent. The reaction can be carried out at ambient temperature or with heating; an amine base can be added but is not necessary. If the phenylisocyanate does not possess at least one (but preferably two or more) electron-withdrawing groups, then to the reaction is added copper (I) sulfate (not shown). With either method, the resultant carbamate adduct c is then subjected to removal of the nitrogen protecting group using conditions and methods known in the art (Scheme 2) to afford final product d

Scheme 2

removal of protecting group

P = protecting group

An alternate technology that utilizes the identical chemistry described above is that of solid-support-assisted organic synthesis (Scheme 3). The phenyalaninol can be attached to a resin through the nitrogen atom and then reacted with a phenylisocyanate or mixtures of phenylisocyanates. Cleavage from the resin affords either pure compound d (R₂= same) or mixtures of compound d (R₂ = different) referred to as libraries. In this strategy, attachment of the substrate to the resin serves to mask the reactivity of the nitrogen center and therefore mimics conventional protecting groups. A variety of resins are commercially available as are different apparatus for performing such manipulations; these are known to those skilled in the art. The choice of resin includes, but is not limited to, the Wang resin and the chlorotrityl and trityl resins. In the first case, the nitrogen center is joined via a carbamate linkage formed upon reaction of the phenylalaninol with the para- nitrophenyi carbonate derivative of the Wang resin (Scheme 3). In the case of the trityl resins, reaction with the phenylalaninol results in displacement of chloride; the resultant tritylated amine is reluctant to undergo further reaction at the nitrogen center due to steric hinderance. In all cases, the alcohol oxygen center is unperturbed and free to undergo reaction with isocyanates as described above. A known advantage of solid-support-assisted organic synthesis is that large excesses of reagents can be used to improve yields and purity; the unused materials, as well as impurities are typically removed by washing the resin. Cleavage from the resin typically involves treatment with trifluoroacetic acid in an inert solvent. These operations are well known to those skilled in the art.

Scheme 3

The replacement of the carbamate linkage with a urea linkage a (X = NH) is carried out by converting the alcohol a to an amine f prior to reaction with the isocyanate b This is accomplished by activating the corresponding carbon center towards nucleophilic attack through the use of a leaving group (LG) e; this strategy is well known to those skilled in the art. For example, reaction of the phenylalaninol precursor a with methanesulfonyl chloride in the presence of a base, or alternatively, reaction of the same precursor with carbon tetrahalide in the presence of triphenylphosphine produces the mesylate or chloride or bromide respectively (Scheme 4). The amine center is then introduced via displacement of the leaving group with an amine such as ammonia or with azide followed by reduction to the amine f.

Scheme 4

This chemistry can also be carried out using solid-support-assisted techniques. Urea formation g^ occurs upon treatment with phenylisocyanates and the final products t are obtained upon removal of the protecting group or cleavage from the resin. It is advantageous to employ protecting groups or resin linking groups that decrease the reactivity of the origin nitrogen center to avoid side reactions. The Cbz (benzyloxycarbonyl) group is one such protecting group and the carbamate linkage is a suitable method of attachment to the Wang resin (see Scheme 5). Alternatively, the leaving group can be displaced by sodium cyanate to produce the isocyanate j, and this intermediate can then be reacted with an amine such as an aniline j to produce the desired compounds (Scheme 6). Once again, this chemistry is amenable to solid-support techniques.

Scheme 5

NaNCO

LG = OMs, Cl, Br

removal of protecting group

P = protecting group or resin-linked

Isocyanates i can also be reacted with benzylic carbanions k to afford amide derivatives I (X = NH; Y = CH₂) (Scheme 6). Grignard reagents and alkyllithiums species are suitable for this approach.

Scheme 6

removal of protecting group

P = protecting group M = MaBr. Li or resin-linked

I

Alternatively, amine f can be coupled to phenylacetic acids using conventional peptide chemistry (DCC / HOBt, BOP, etc.) (Scheme 7). There are many reagents and conditions that carry out this condensation and these are well known to those who practice chemical synthesis.

Scheme 7

To prepare pharmaceutical compositions of any of the aforementioned inventions, one or more compounds or salts thereof, as the active ingredient, is intimately mixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending upon the desired route of administration (e.g., oral, parenteral). Thus for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, stabilizers, coloring agents and the like; for solid oral preparations, such as powders, capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Solid oral preparations may also be coated with substances such as sugars or be enteric-coated so as to modulate major site of absorption. For parenteral administration, the carrier will usually consist of sterile water and other ingredients may be added to increase solubility or preservation. Injectable suspensions or solutions made also be prepared utilizing aqueous carriers along with appropriate additives. For the treatment of disorders of the central nervous system, the pharmaceutical compositions described herein will typically contain from 1 to about 1000 mg of the active ingredient per dosage; one or more doses per day may be administered. Determination of optimum doses and frequency of dosing for a particular disease state or disorder is within the experimental capabilities of those knowledgeable in the treatment of central nervous system disorders. The preferred dose range is 50-100 mg/kg.

Biological Assays

Select compounds of the invention were evaluated for their ability to competitively bind to the neuropeptide receptor, Y1 (NPY1 ), where the assay was based on scintillation proximity technology (SPA). A human neuroblastoma cell line,

SK-N-MC, expresses NPY1 and provides the receptor source. The membranes were first trapped by wheat germ aggiutinin (WGA) coated SPA beads. Compounds were evaluated in terms of their ability to inhibit binding of the 125|_h_uman neuropeptide Y ligand. In addition to this ligand, the compounds were evaluated for their ability to inhibit the binding of porcine PYY. Reagents and Materials

1. SK-N-MC membranes (freshly prepared on the day of assay);

2. ¹²5|-human neuropeptide Y (Amersham, cat #IM170, 2000Ci/mmol); or porcine PYY (Amersham, cat. #IM 259)

3. Human neuropeptide Y peptide (Palomar Organics, diluted to afford a 300 uM stock solution);

4. Control compound RWJ53797, 1 mM stock solution in DMSO;

5. Bacitracin (Sigma, 10% stock solution); 6. Leupeptin (Sigma, 10 mg/ml);

7. Homogenization buffer: 10 mM HEPES, pH 7.4 containing 0.1 % bacitracin, 2 mM CaCl2, 2 mM MgCl2 and 0.3 M sucrose;

Wash buffer: 10 mM HEPES, pH 7.4 containing 2 mM CaCl2, 2 mM MgCl2 and 0.1% bacitracin;

8. Assay buffer (1X): 10mM HEPES, pH 7.4 containing 0.1 % bacitracin, 0.1%

BSA, 2 mM CaCl2 and 2 mM MgCl2;

9. Packard Microlite or Dynatech Optiplate 96-well plate; 10. WGA-SPA beads (Amersham, 40mg/ml assay buffer);

11. Protein assay kit (BioRad); 12 Sucrose solution.

Reagent Preparation a) Cell culture The human neuroblastoma cell line, SK-N-MC, that expresses the NPY1 receptor, was obtained from ATCC. Cells were maintained in DMEM medium supplemented with 10%FCS, glutamate and antibiotics in 5%CO2 at 37°C. Cells were allowed to go to confluence before harvesting. They were either used immediately or frozen at -80 °C. b) Cell membrane preparation P2 membrane preparations from SK-N-MC cells were used. Protein concentration was determined using the BioRad Protein assay performed in a microtitre plate with 0.1 % BSA as standard. Membranes were diluted to 5ug/ul in wash buffer. Each well would require 50ug of membranes.

Working Reagents

1 ) WGA-SPA beads

40 mg/ml in assay buffer. Each well required 25 ul beads.

2) 125l-neuropeptide Y ' Lyophilized powder (50 uCi) was dissolved in 1 ml of assay buffer. The ligand solution was stored in 100 ul portions at -80°C and thawed only before use. Each well requires 0.5 ul of the stock ligand solution diluted into 76.25 ul in assay buffer.

3) Test compounds

Test compounds were dispensed into 96-well plate (3.75 ul of 1 mM).

Test Procedure a) Adsorption of membranes onto beads The amount of beads required for 5 plates (25 ul x 5 x 100 wells = 12,500 ul or 12.5 ml) was dispensed into a 50 ml sterile Falcon tube. The amount of membranes required for 5 plates was then added (50 ug x 5 plates x 100 well, e.g. if membrane concentration was 5 ug/ul, use 10 ul per well, or 10 x 5 x 100 = 5ml). The tube was then placed on a nutator and mixed for 1 hr at room temperature. Non- specific sites blocking agent (2 ml) was added and the mixture was further incubated for at least 30 min. At the end of the incubation, 7.5 ml of sucrose and 9.5 ml of assay buffer was added). Each well receives 70 ul of this membrane+beads mixture. b) Test plate To the control column, 3.75 ul of 300 uM NPY peptide were added to rows C and D (cold peptide, non-specific binding control); 3.75ul 1 mM BIBP 3226 methyl ether to rows G and H (control compound); and 3.75 ul of 30% DMSO HEPES buffer to rows A, B, E and F (total binding). Ligand was then added to each well (76.25 ul).

c) Plate handling The plate was sealed with adhesive paper, wrapped in lead foil and put on a shaker for 14-16 hr. The plates were then left for at least 4 hours at room temperature before reading in a Topcount. Data is saved onto a floppy diskette.

Data Analysis Total binding (T) = Mean cpm in rows E,F of column 12

Non-specific binding (NSB) = mean cpm in rows C,D of column 12

Total specific binding (TS) = T - N cpm in each well = R

Net cpm in each well (N) = R-NSB % Inhibition = 100% - { N_x 100%}

TS

Putative hits are identified as compounds that give >50% inhibition at 25uM in the described assay, and confirmed by repeating the assay at least 2 times.

Rating Scale

The inhibitory activity of the candidate compounds are scored as follows:

Rating Percent Inhibition Description

1 <20% INACTIVE

2 21-40% MARGINALLY ACTIVE

3 41-80% ACTIVE

4 81-95% VERY ACTIVE >95% EXTREMELY ACTIVE

Compounds scored as Active, Very Active or Extremely Active are subsequently re-assayed at various concentrations so as to obtain Inhibition Concentration values, specifically IC50 values are obtained by standard methods. Data for select compounds of the invention is listed in Tables A & B. IC∞s are listed and if that number was not determined, the percentage of inhibition at a listed concentration is given. If a "#" is listed, that indicates that no data was obtained. Compoounds where "^*" is indicated are the S-isomers, compounds where "**" is indicated are the R-isomers, compounds where "^***" is indicated are the racemic mixtures..

Table A

Cpd. # R R, R, NPY PYY

11 NH₂(CH₂)₇C(0)- H 3,5-(CF₃)₂ # 88 @ 25 μM

12 H phenyl 3,5-(CF₃)₂ # 33 @ 30 μM

4 4-NH₂CH₂phenyl-C(0) H 3,5-(CF₃)₂ # 81 @ 30 μM^*

13 4-NH₂CH₂phenyl-C(0) H 3,5-(CF₃)₂ # 80 @ 30 μM~

2 H H 3,5-(CF₃)₂ 1.0 μM 30.0 μM**

14 4-NH₂CH₂phenyl-C(0) H 3,4-(CI)₂ # 27 @ 30 μM*

15 4-NH₂CH₂phenyl-C(0) H 4-OCH₃ # 18 @ 30 μM

5 H H 3,4-(CI)₂ # 20 μM

16 H H 4-OCH₃ # 18 @ 30 μM

10 NH₂CH₂phenylC(0) H 3,5-(CF₃)₂ 3 μM*^**

17 H H 2,6-(F)₂ 0 @ 30 μM

18 H H 3-N0₂ # 5 @ 30 μM

19 H H 2-CI-5 -CF₃ 20 μM 0 H H 4-phenyl 0 @ 30 μM 1 H H 6-OCH₃ 20 @ 20 μM 2 H H 3,5-diN0₂ 26 @ μM

H H 3,5-(CF₃)₂ 0.5 μM** # 6 CH₃C(0) H 3,5-(CF₃)₂ 0 @ 30 μM

Table B

Cod. # R Rι R? NPY PYY

23 H H 2-CI # 21 @ 30.0 μM*

24 H H 3-CI # 20 @ 30 μM**

25 H H 4-C(CH₃)₃ 200nM 14 @ 25 μM

In addition to the binding assay, select compounds of the invention were evaluated in an cyclic adenosine monophosphate assay. This assay determines whether the compounds are agonists of NPY. The known Y1 antagonist that was used in this test was BIBP-3226.

Cell culture Human neuroblastoma cells, SK-N-MC (American Tissue Culture Center), was maintained in DMEM containing 10% FCS and antibiotics under standard cell culture conditions. Approximately, the same number of cells was seeded to each well in a 12-well plate and the cells were allowed to grow to 70% confluence.

Forskolin-induced cAMP extraction

Wells were rinsed twice with Hank's solution (Gibco BRL, lot 9P6360) and the cells were incubated for 10 minutes at 37°C in 1 mM 3-isobutyl-1-methyl-xanthine (IBMX) (Sigma, lot 85H1331 ) dissolved in Hank's solution. Varying concentrations of test compounds or human neuropeptide Y (17 nM), or both, (Palomar Research Organics, lot 0496) were added to the appropriate wells and incubated for 1 minute at room temperature. Control wells received the same volume of DMSO in place of drugs.

Forskolin (Sigma, lot 102H78131 ) dissolved in DMSO was added to each well to a final concentration of 10uM. The cells were incubated at 37°C for 20 minutes. The supernatant in the wells was removed and rinsed once with Hank's solution. Ice cold 65% ethanol (0.5ml) was added to each well and incubated for 5 minutes at 0°C to extract cellular cAMP.

The ethanol extract was transferred into separate 1.8 mi microfuge tube and cleared by brief centrifugation for 30 seconds in a microfuge. The supernatant was transferred to a fresh microfuge tube.

cAMP measurements

The cAMP content in separate extracts were determined using a cAMP Scintillation Proximity Assay System kit (Amersham) and cAMP standards according to the supplier's instructions. Effects on forskolin induced cAMP Ivels in SK-N-MCcells

1 0 0 2 0 0 3 0 0

Mean of 3 separate experiments

CAMP [pM] Interpretation of Data and Conclusions

Legend Treatment

+/- cells + IBMX

Result: endogeneous low cAMP background. IBMX is ATPase inhibitor; allows for accumulation of intracellular cAMP.

+/+ cells + IBMX + forskolin

Result: forskolin increases intracellular level of cAMP via adenylate cyclase.

0.1 uM NPY cells with forskolin and IBMX, treated with NPY

Result: NPY lowers forskoiin-induced cAMP production.

Legend Treatment

1uMa cells + IBMX + forskolin + known Y1 antagonist (a) 1uM 10uMa cells + IBMX + forskolin + known Y1 antagonist (a) @ 10 uM

Result: cAMP levels return to forskolin-treated control group. 10uMb cells + IBMX + forskolin + O-(N-3,5-bis(thfluoromethyl) phenyl)carbamoyl-N-D-phenyialaninol

Result: Claimed compound (@ 10 uM) mimics NPY by reducing forskolin-enhanced cAMP levels.

1u a + 1uMb Varying concentrations (0.1 uM, 1 uM, 10 uM) of

1uMa + 10 uMb claimed compound (b): Cpd. 1[O-(N-3,5- bis(trifluoromethyl) 10 uMa + 1 uMb phenyl)carbamoyl-N-D-phenyialaninol]

0.1 uMa + 10uMb and known Y1 antagonist (a) [BIBP-3226]

Conclusion: Claimed compound is confirmed agonist of NPY1 receptor since it behaves like NPY ligand by lowering forskolin-enhanced cAMP levels but is competitively inhibited by known Y1 antagonist.

The anxiolytic activity of selected compounds of the invention was assessed by determining their ability to encourage behavior that had been suppressed by punishment (Vogel, J.R. et al. Psychopharmacology 1971 , 21, 1 ). Male rats were deprived of water for 48 hours and were deprived of food for 24 hours prior to testing. After the first 24 hours of water deprivation, they were placed in the conflict chamber for a training period; wherein, they were allowed 200 unpunished licks from a bottle containing tap water. The experiment was run the next day. At the expected time of peak activity, the animals were placed in the chamber and allowed access to tap water. If they failed to drink, the experiment was terminated in 5 min, and animals were evaluated for signs of CNS depression. Their first lick initiates a 3-min test session. Subsequently, every 20th lick was punished by a 0.2-s shock delivered via the stainless-steel d nking-tube. Vehicle-treated control animals generally were willing to accept a median number of 3 to 8 shocks per test session. Animals treated with an active anxiolytic drug tolerated significantly more shocks than control animals. The Wilcoxon rank-sum test (Mann-Whitney U-test) was used to test for an increase (p<0.05, 1 -tailed) in the median number of shocks in drug-treated groups, compared to a concurrently run vehicle-treated group. The biological assay is considered to be valid if the effects of a known anxiolytic (positive control) are detected, within the same experiment. A compound was considered active if there is a significant difference in the median number of shocks tolerated between the drug-treated group and the control group. The minimum effective doses (MED) for Cpd. 2 is 10 mg/kg where the median number of shocks tolerated was 4.5 in one experiment and 6 in another. The critera for activity is 5.5 shocks. The MED was defined as the minimum dose of the drug-treatment as analyzed using the Wilcoxon rank-sum test (SAS; Statistical Analysis System, version 5.16). If the MED value is greater than 10, an active dose of the compound being tested had not been determined. When tested at 30 mg/kg, the median number of shocks tolerated over 3 experiments was 6.5, 3, and 8 shocks.

A similar test was conducted on adult monkeys using a single compound. However, Cpd. 2 showed no anxiolytic activity in the monkey assay. Selected compounds of the invention were tested for their ability to reduce metrazol-induced convulsions in mice (Swinyard, E.A. J. Am. Pharm Assoc. 1949, 38, 201 ). Male CDi mice, were fasted at least 16 hours, were divided into equal groups and test compounds or vehicle were administered parenterally. Water was not withheld except during the period of observations. At the time of suspected peak activity, anti-pentylenetetrazol (anti-metrazol) activity was evaluated by the subcutaneous administration of the CDgo dose of metrazol (the dose of metrazol was determined from the dose-response curve producing clonic convulsions in 90% of animals that received the corresponding vehicle for this experiment). Metrazol was dissolved in 0.9% sodium chloride solution, and its dose volume was 10 ml/kg. Animals were housed individually for observation of clonic convulsions, tonic convulsions and death for a period of 30 min. Test compounds that blocked the clonic seizure component of the convulsion in at least 50% of the animals were considered active. The biological assay was considered to be valid if the effects of a known anticonvulsant (positive control) were detected, within the same experiment. Activity was reported as percent reduction of clonic convulsions from the vehicle group. The ED50 values of active compounds were calculated by the method of probits (Finney, D.J. 1971. Probit Analysis. London: Cambridge University Press) and are listed in Tables 1. An ED50 value of greater than 30 indicates that an active dose for the compound being tested had not been determined. Compounds active in this screen are considered active anticonvulsant / antiepileptic agents. Cpd. 2 was tested and was inactive in this assay.

A compound of the invention was tested for its ability to alleviate the anxiety of rat in a behavioral model of trait anxiety. This behavioral assay is qualitatively unique in that it is based on the innate behavior of the animal and may model human anxiety traits (Pellow, S. et al. J. Neurosci. Methods 1985, 14, 149). It is thought to be a model for short term anxiolytic events where the Vogel model is thought to represent chronic anxiety. Adult male Long-Evans hooded rates (Charles River Laboratories were used. Animals had unlimited accss to food and water except during the experiment but were deprived of food but not wateer for 18 hours before use. Test compounds were evaluated by the oral route of administration. Each black plastic maze had two open arms and two arms with 40 cm high walls (enclosed arms), of equal length (50 cm), extending rom the center at right angles, suh that arms of similar type were opposite each other. Each plus-maze was elevated approximately 60 cm above the floor. Infrared photo-beams that crossed the entrance of each arm and the center of the maze detected the exploratory activity of an animal in the maze. At one hour after treatment, animals were placed on an open arm of the plus-maze facing the center. The 10-min test was initiated when the animal entered the center of the apparatus. Data collection was automated and was obtained while the investigator was outside of the laboratory. The percent of total time spent (%Time = 11x[time in open arms divided by 600 sec]),, and statistical significance was etermined using ht eMann Whitney test. Active treatments increased %Tm and/or %Entries compared to vehicle- treated animals (p<0.05, one-tail). Compound 2 was inactive in this screen. Some of the compounds of the invention were tested for their ability to act as general CNS agents and particularly as skeletal muscle reiaxants and hypnotics/sedatives (Coughenour, L.L. et al. Pharm. Biochem. Behav. 1977, 6, 351 ). Male CDi mice, fasted for at least 16 hours but allowed access to water except during the period of observation, were placed on a horizontally- held screen (mesh size 1/4", wire diameter approximately 1.0 mm). The screen was inverted and mice which successfully climb to the top side of the screen within one minute were selected for testing. Selected mice were weighed and divided into equal groups. Test compounds or vehicle were administered to those mice parenterally. At a pre-determined interval (or intervals) after administration, the animals were tested for their ability to climb to the top side of the inverted screen (pass the test). Activity is reported as the percent reduction in the number of animals that pass the test in each treatment group relative to the corresponding vehicle-treated group. Percent Reduction = 100 X ([Percent Pass in Vehicle Group] - [Percent Pass in Test Group]/Percent Pass in Vehicle Group). Test compounds which produce a 50% or greater reduction in the number passing the test were considered active. Cpd. 2 was tested and had no affect upon the six tested mice.

Even though all compounds of the invention are useful in the treatment of neuropeptide related disorders, some compounds are more active than others and are either preferred or particularly preferred. Examples of preferred compounds of Formula I include compounds where R are hydrogen or 4-aminomethylbenzoyl; R-i is hydrogen; n is 0; R₂ is trifluoromethyl or chloro; p is 2; X is O; Y is NH; q is 1.

Examples of particularly preferred compounds of formula I include: O-(N-3,5-bis(trifluoromethyl)phenyl)carbamoyl-D-phenylalaninol

N-(4-aminomethyl)benzoyl-O-(N-3,5-bis(thfluoromethyl)phenyl)carbamoyl-D- phenylalaninol O-(N-3,4-dichlorophenyl)carbamoyl-D-phenylalaninol

(R)-Λ/-(2-Amino-3-phenylpropyl)-Λ/'-(3,5-bis(trifluoromethyl)ρhenyl)urea.

(R)-(Λ/-(2-Amino-3-phenylpropyl))-(3,5-bis(trifluoromethyl)phenyl)acetamide

(R)-(Λ/-(2-Amino-3-phenylpropyl))- 3,5-bis(trifluoromethyl)benzamide

EXAMPLES The following examples describe the invention in greater detail and are intended to illustrate the invention, but not to limit it. All compounds were identified by a variety of methods including nuclear magentic resonance spectroscopy, mass spectrometry and in some cases, infrared spectroscopy and elemental analysis. Nuclear magentic resonance (200 MHz NMR) data is reported in parts per million downfield from tetramethylsilane. Mass spectra data is reported in mass/charge (m/z) units. Unless otherwise noted, the materials use in the examples were obtained from readily available commercial sources or synthesized by standard methods known to those skilled in the art.

O-(N-3,5-bis(trifluoromethyl)phenyl)carbamoyl-N-(f-Butoxycarbonyl)-D- phenylalaninol Cpd. 1 N-(f-Butoxycarbonyl)-D-phenylalaninol (2.51 g, 10 mmol) was dissolved in dichloroethane (50 mL) at ambient temperature. To this solution was added 3,5- bis(trifluoromethyl)phenyl isocyanate (1.90 mL, 11 mmol) and the mixture was stirred overnight. A white solid formed, most of the solvent was removed via rotary evaporation and hexanes (60 mL) was added. The white solid was filtered off and washed with fresh hexanes to afford the desired product (5.5 g, >100%) of approximately 85-90% purity. This material could be carried forward as described below or purified in the following manner. The solid was dissolved in a minimum amount of hot toluene (approx. 30 mL) and let cool to form a viscous white sludge. Hexanes (approx. 80 mL) were added and the resultant white solid was filtered off and washed with fresh hexanes to afford product (3.4 g, 70%); NMR (DMSO-d₆): 10.4 (bs, 1 H, carbamate NH), 8.12 (s, 2H, carbamate aromatic C-H), 7.70 (s, 1 H, carbamate aromatic C-H), 7.35-7.18 (m, 5H, phenyl), 6.96 (d, 1 H, NH-BOC), 4.10 (dd, 2H, CH₂-O), 3.95 (m, 1 H, N-CH-), 2.78 (m, 2H, CH₂-Ph), 1.31 (s, 9H, t-Bu); MS: 507 MH⁺, 407 (M-tBu-O-C(O)).

0-(N-3,5-bis(trifluoromethyl)phenyl)carbamoyl-N-D-phenylalanino trifluroacetate

Cpd. 2 0-(N-3,5-bis(trifluoromethyl)phenyl)carbamoyl-N-(f-Butoxycarbonyl)-D- phenylalaninol, prepared as described above (5.5 g (85%-90% purity), was dissolved in dichloroethane (75 mL) at ambient temperature. Trifluoroacetic acid (25 mL) was added and the reaction was stirred overnight. The solvents were removed via rotary evaporation and a viscous oil resulted. With stirring, a minimum amount of ethyl ether was added (in order to free oil from sides of vessel) and the mixture was again subjected to rotary evaporation. The procedure was repeated two more times and a white solid resulted. Hexanes was added over the solid and the solid was crushed, filtered and washed with fresh hexanes. The resultant desired product was isolated as a mono-trifluoroacetate salt (4.4 g, >90%); NMR (DMSO-ds): 10.5 (bs, 1 H, carbamate NH), 8.4 (bs, 3H, amine N-H), 8.15 (s, 2H, carbamate aromatic C-H), 7.73 (s, 1 H, carbamate aromatic C-H), 7.39-7.21 (m, 5H, phenyl), 4.20 (ABX dd, 2H, CH₂-O), 3.71 (m, 1 H, N-CH-), 2.97 (m, 2H, CH₂-Ph); MS: 407 MH⁺.

Example 3

O-(N-3,5-bis(trifluoromethyl)phenyl)carbamoyl-N-(4-(t- butoxycarbonyl)aminomethyl)benzoyl-D-phenylalaninol

Cpd. 3 O-(N-3,5-bis(thfluoromethyl)phenyl)carbamoyl-N-D-phenylalaninol trifluoroacetate salt (2.0 g, 3.85 mmol) was dissolved in anhydrous N,N-dimethylformamide (25 mL) under a dry nitrogen atmosphere. Diisopropylethylamine (2.0 mL, 11.5 mmol) was added followed by the addition of 4-(t-butoxycarbonyl)aminomethylbenzoic acid (1.45 g, 5.78 mmol). Castro's reagent (benzotriazoie-l-yl-oxy-tris-(dimethylamino)- phosphonium hexafiuorophosphate (5.1 g, 11.5 mmol) was added to the homogeneous reaction. Lastly, 4-dimethylaminopyridine (0.5 g) was added and the mixture was stirred at ambient temperature overnight. The resultant brownish solution was diluted with ethyl acetate (150 mL) and transferred to a separatory funnel and washed with 1 M hydrochloric acid (3 x 25 mL) and then with brine (1 x 25 mL). The organics were dried (MgSO₄), filtered and the solvent removed by rotary evaporation to afford a brownish oil. The crude product was dissolved in a mimimum amount of chloroform and purified via a silica gel plug (chloroform as eluent); dark materials remained on the silica gel. Removal of chloroform gave a viscous oil to which hexanes were added slowly to produce a fine off-white solid. This solid was filtered and washed with hexanes to obtain the desired product (2.4 g, 97%); NMR (DMSO-d₆): 10.4 (bs, 1 H), 8.31 (d, 2H), 8.12 (s, 2H), 7.70 (s, 1 H), .61 (d, 2H), 7.35-7.18 (m, 5H), 6.66 (d, 1 H), 4.44 (m, 1 H), 4.20 (dd, 2H), 4.10 (d, H), 2.96 (m, 2H), 1.48, (s, 9H); MS: 640 MH⁺.

Example 4

O-(N-3,5-bis(thfluoromethyl)phenyl)carbamoyl-(N-(4-aminomethyl) benzoyl-D- phenylalaninol Cpd. 4 O-(N-3,5-bis(trifluoromethyl)phenyl)carbamoyl-N-(4-(t-butoxycarbonyl) aminomethyl)benzoyl-D-phenylalaninol (2.4 g, 3.75 mmol) was dissolved in dichloroethane (45 mL). Trifluoroacetic acid (15 mL) was added and the homogeneous mixture was stirred at ambient temperature overnight. The solvents were removed via rotary evaporation. Ethyl ether was added to the resultant oil and then removed by rotary evaporation. Fresh ether was added (mimium amount to free oil from vessel walls) and again removed by evaporation. The resultant white solid, the TFA salt of the desired product, was neutralized via dissolution into a minimum amount of 90:10:1 CH₂CI₂/CH₃OH/NH₄OH and passed through a short silica gel column. The eluent was collected and the solvents removed by rotary evaporation to afford the desired product as a white solid (1.4 g, 70%); NMR (DMSO-d₆): 8.38 (d, 1 H), 8.12 (s, 2H), 7.73 (s, 1 H), 7.71 (d, 1 H), 7.39 (d, 2H), 7.32- 7.15 (m, 5H), 4.51 (m, 1 H), 4.26 (dd, 2H), 3.71 (s, 2H), 2.95 (m, 2H); MS: 540 MH⁺.

Example 5

O-(N-3,4-dichlorophenyl)carbamoyl-N-D-phenylalaninol Cpd. 5 & Examples of SolidSupport Syntheses (Scheme 3) 4-Nitrophenyl chloroformate (16.8 g, 83.2 mmol) was added in portions to a suspension of p-benzyloxybenzyl alcohol resin (Wang resin) (0.52 mmol/g, 40.0g, 20.8 mmol) in a solution of 4-methylmorpholine (10.3 mL, 93.6 mmol) in dichloromethane (DCM, 350 mL) at 20 to 26 °C. The resultant mixture was stirred with a low speed mechanical stirrer at ambient temperature for 18 h. This activated resin (see Scheme?), capped as a 4-nitrophenylcarbonate, was collected by filtration and washed successively with DCM (3x), DMF (3x), THF (3x) and DCM (3x). The resin was dried under a flow of nitrogen. This material was then taken up in DMF (350 mL) and treated with (R)-(+)-2- amino-3-phenyl-1-propanol (14.1 g, 94 mmol). The resulting mixture was stirred at ambient temperature for 2.5 days. The resin was collected, washed and dried as above to give 43.6 g. This resin was shown to have incorporated the phenylalaninol substrate via the displacement of 4-nitrophenol (which could be used to monitor the progress of the reaction). The bulk of this resin (referred to as resin-bound phenylalaninol) was then used in subsequent reactions as described below. In each step, an aliquot of the derivatized resin was removed to confirm the attachment of the desired substrate onto the resin and to quantify the substrate to resin ratio (mmol substrate / g resin). In all cases, each product was readily obtained via cleavage from the resin by treating the resin for 10 to 20 min with TFA/water (95:5). The resultant solution was filtered and evaporated. The residue was analyzed by HPLC and MS and shown to be desired product. Specific examples follow and illustrate the usefulness of this methodology.

To the resin-bound phenylalaninol (2 g, 0.5 mm/g) was added, as 1M solutions in DMF, 3,4-dichlorophenyl isocyanate (40 mL) followed by CuCI (20 mL). The mixture was mixed via gentle aspiration with nitrogen gas for 1 hour at room temperature. The resultant resin was successively washed with 10% Acetic acid-H2θ (1 L), THF (500 mL, 2X), acetone (500 mL, 2X), and CH2CI2 (500 mL, 3X). The product was cleaved from the resin by treatment with TFA:CH2Cl2 (80:20, 100mL); the organics were removed in vacuo to isolate the desired phenylalaninol-derived carbamate trifluoroacetate salt. The phenylalaninol-derived carbamate could be also be obtained as the corresponding free base or as the hydrochloride salt as described here. To the carbamate TFA salt, was added aqueous NaHCO3 and the aqueous layer was extracted thoroughly with CH2CI2. The organic extract was subsequently washed with H2O, dried over MgS04, and the solvent removed via rotary evaporation to afford the free base of the desired product, as a clear oil. This oil was dissolved in diethyl ether and ethereal HCI was added which resulted in precipitation of the hydrochloride salt as a white solid; NMR (DMSO-dβ): 10.2 (bs, 1 H), 8.45 (bs, 2H), 7.8 (d, 1 H), 7.6 (d, 1 H), 7.4- 7.35 (m, 5H), 7.5-7.45 (dd, 1 H), 4.28-4.25 (dd, 1 H), 4.10-4.05 (dd, 1 H), 3.75-3.62 (m, 1 H), 3.13-3.08 (dd, 1 H), 2.98-2.85 (m, 1 H); MS: 339 MH⁺.

(R)-Λ/-(2-Amino-3-phenylpropyl)-Λ/'-(3,5-bis(trifluoromethyl)phenyl)urea.

Cpd. 6 A solution of methanesulfonyl chloride (1.75 mL, 22.6 mmol) in DCM (20 mL) was added dropwise to a suspension of the resin-bound phenylalaninol (10 g, 5 mmol) in a solution of /V,Λ/-diisopropylethylamine (8 mL, 45.9 mmol) in DCM (100 mL) at a rate in which the temperature never exceeds 25 °C. The resultant suspension is stirred at ambient temperature for 18 h. The solution was filtered from the resin, and the resin was washed successively with DCM (3x), DMF (3x), MeOH (3x) and THF (3x). This resin which contained the appropriate mesylated phenylalaninol was dried at room temperature under a flow of nitrogen. This resin-bound mesylate (9 g, 4.5 mmol) was suspended in DMF (100 mL), treated with lithium azide (8 g, 0.163 mol) and heated to 70 °C for 18 h. The resultant resin-bound azide was collected by filtration washed successively with DMF (3x), water (3x), MeOH (3x), THF (3x) and DCM (3x) and dried under a flow of nitrogen.

This material (approx. 4.5 mmol) was suspended in THF (90 mL), treated with triphenylphosphine (8.26 g, 31.5 mmol) and heated to 60 °C for 2 h. The solution was filtered from the resin, and the resin was washed with three portions of THF. This material was suspended in THF (100 mL), treated with water (10 mL) and heated to 60 °C for 2 h. The resin was collected by filtration and washed successively with THF (3x), MeOH (3x) and DCM (3x) and dried under a flow of nitrogen. At this point the resin, independently shown to contain the corresponding phenylalaninol-derived amine, was divided into portions for derivatization into the corresponding ureas, amides etc. as described below.

The above resin (1.0 g, 0.54 mmol) was suspended in DCM (10 mL) and treated with 3,5-bis(thfiuoromethyl)phenylisocyanate (1.0 mL, 5.79 mmol). The mixture was shaken at room temperature for 2.5 h. The resin was collected by filtration, and washed successively with DCM (3x), DMF (3x), MeOH (3x), THF (3x) and DCM (3x). The resin was treated twice with TFA/water (95/5) for 15 min and 5 min, filtered and washed with DCM. The solutions were combined, and the solvent was evaporated at 60 °C, under a flow of nitrogen. The residue was dried in vacuo at rt to give the TFA salt of the product as an oil 0.24 g (85%); NMR (DMSO-d₆): 9.87 (s, 1 H, exchanges with D₂O), 8.14 (s, 2H), 7.97 (br s, 3H, exchanges with D₂0), 7.57 (s, 1 H), 7.25-7.38 (m, 5H), 7.24 (t, 1 H, exchanges with D₂O), 3.16-3.53 (m, 3H), 2.73-2.97 (m, 2H); MS: 406 MH⁺.

(R)-(/V-(2-Amino-3-phenylpropyl))-(3,5-bis(trifluoromethyl)phenyl) acetamide Cpd. 7 1 ,3-Diisopropylcarbodiimide (67 mL, 0.429 mmol) was added to a mixture of the resin of Example 6 (0.191 g, 0.096 mmol), 3,5-bis(trifluoromethyl)phenylacetic acid (0.130 g, 0.478 mmol) and 1-hydroxybenzotriazole hydrate (26 mg, 0.191 mmol) in DMF (3 mL), and the resultant mixture was shaken at rt for 3 h. The resin was collected by filtration, and washed successively with DMF (3x), MeOH (3x), THF (3x) and DCM (3x). The resin was treated twice with TFA/water (95/5) for 10 and 5 min, then washed with DCM. The combined solutions were evaporated at 60 °C under a flow of nitrogen, and the residue dried in vacuo to give the TFA salt of the product as a sticky solid, 41 mg (82%): NMR (DMSO-d₆): 8.39 (t, 1 H), 8.00 (s, 2 H), 7.97 (br s, 3 H), 7.23 (m, 6 H), 3.75 (s, 2 H), 3.38-3.52 (m, 1 H), 3.19-3.28 (m, 2 H), 2.75-2.96 (m, 2 H); MS: 405 MH⁺.

Example 8

(R)-(Λ/-(2-Amino-3-phenylpropyl))-3,5-bis(trifluoromethyl) benzamide Cpd. 8 1-Hydroxybenzotriazole hydrate (1.0 M in DMF, 230 mL, 0.23 mmol) was added to a mixture of the resin (0.22 g, 0.144 mmol) and 3,5- bis(trifluoromethy)benzoic acid in DMF (3 mL) followed by 1 ,3- diisopropylcarbodiimide (81 mL, 0.515 mmol). The mixture was shaken at rt for 20 h. The resin was collected by filtration and washed with DMF (3x), MeOH (3x), THF (3x) and DCM (3x), then treated twice with TFA/water (95/5) for 10 and 5 min. The resin was washed with DCM and the combined solutions were evaporated under a flow of nitrogen at 60 °C. The residue was dried in vacuo to give the TFA salt of the product as an off white solid 37 mg (51 %): NMR (DMSO-d₆): 9.12 (m, 1 H, exchanges with D₂O), 8.45 (s, 1 H), 8.34 (s, 1 H), 8.06 (br s, 3 H, exchanges with D₂0), 7.22-7.40 (m, 5 H), 3.34-3.68 (m, 3 H), 2.81-3.07 (m, 2 H); MS: 391 MH⁺.

Claims

What is claimed is:

1. A compound having central nervous system activity of the Formuia I

wherein:

R is selected from the group consisting of hydrogen, d-βalkyl, C₃-βcycloalkyl, Ci-βalkoxy,

where m is 1 -5, R₃ is selected from one or more or the group consisting of Cι.₅alkyl, substituted Cι-₅alkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine),

Cι-₅alkoxy, Cι_-5alkylthio, nitro, amino, Cι-₅alkylamino, cyano, carboxylic acid Cι.₅alkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, and halogen, and

where R₄ is aminoCι.₉alkyl or amino;

Ri is selected form the group consisting of hydrogen, Ci-salkyl, substituted

Ci-salkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), Cι_-5alkoxy, Cι.₅alkylthio, nitro, amino, Cι-₅alkylamino, cyano, carboxylic acid Cι-₅alkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, or halogen;

R₂ is selected form the group consisting of hydrogen, Ci-salkyl, substituted

Cι-₅alkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), Cι_-5alkoxy, Cι.₅alkylthio, nitro, amino, Cι.₅alkylamino, cyano, carboxylic acid Cι.₅alkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, or halogen;

n is 1-5

X is O or NH; Y is NH or CH_2; and q is 0 or 1 ; or pharmaceutically acceptable salts thereof. 2. The compound of claim 1 wherein R is hydrogen or

(where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine). 3. The compound of claim 1 wherein X is oxygen. 4. The compound of Claim 1 wherein Y is NH.

5. The compound of Claim 1 wherein q is 0.

6. The compound of Claim 1 , selected from any of 0-(N-3,5-bis(trifluoromethyl)phenyl)carbamoyl-D-phenylalaninol,

N-(4-aminomethyl)benzoyl-O-(N-3,5-bis(trifluoromethyl)phenyl)carbamoyl-D- phenylalaninol,

0-(N-3,4-dichlorophenyl)carbamoyl-D-phenylalaninol,

(R)-Λ/-(2-Amino-3-phenylpropyl)-Λ/'-(3,5- bis(trifluoromethyl)phenyl)urea,

(R)-(/v-(2-Amino-3-phenylpropyl))-(3,5- bis(thfluoromethyl)phenyl)acetamide, and (R)-(Λ/-(2-Amino-3-phenylpropyl))- 3,5-bis(trifluoromethyl)benzamide.

7. A pharmaceutical composition comprising a compound of formula I:

wherein:

R is selected from the group consisting of hydrogen, d-βalkyl, C₃-₈cycloalkyl, Ci-βalkoxy,

where m is 1 -5, R₃ is selected from one or more or the group consisting of Ci-salkyl, substituted Ci-salkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), Ci-salkoxy, Cι_-5alkylthio, nitro, amino, Ci-salkylamino, cyano, carboxylic acid Cι-₅alkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, and halogen, and o

⁴ ^ where R₄ is aminoCι.₉alkyl or amino;

Ri is selected form the group consisting of hydrogen, Cι.₅alkyl, substituted

Cι.₅alkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), d.₅alkoxy, Ci-salkylthio, nitro, amino, Ci-salkylamino, cyano, carboxylic acid Ci.salkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, or halogen; R₂ is selected form the group consisting of hydrogen, Ci-salkyl, substituted

Ci-salkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), Cι.₅alkoxy, Ci-salkylthio, nitro, amino, Ci-salkylamino, cyano, carboxylic acid Cι-_saikoxycarbonyl, carboxaldehyde, carboxamide, phenyl, or halogen;

n is 1-5

X is 0 or NH;

Y is NH or CH_2: and q is O or l; or pharmaceutically acceptable salts thereof in an amount effective for treating disorders of the central nervous system and a pharmaceutically acceptable carrier or diluent.

8. The composition of Claim 7 wherein the amount of a compound of Formula 1 is 50 to 500 mg/kg.

3. A method for treating disorders of the central nervous system consisting administering a compound of the Formula I:

wherein:

R is selected from the group consisting of hydrogen, d-βalkyl, C^cycloalkyl, Ci-βalkoxy,

where m is 1 -5, R₃ is selected from one or more or the group consisting of Ci-salkyl, substituted Cι.₅alkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine),

Ci-salkoxy, Ci-salkylthio, nitro, amino, Ci-salkylamino, cyano, carboxylic acid Ci-salkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, and halogen, and

where R is aminoCι-₉alkyl or amino;

Ri is selected form the group consisting of hydrogen, Ci-salkyl, substituted

Ci-salkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), d-₅alkoxy, Cι-₅alkylthio, nitro, amino, Cι.₅alkylamino, cyano, carboxylic acid d.₅alkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, or halogen;

R₂ is selected form the group consisting of hydrogen, Cι-₅alkyl, substituted

Ci-salkyl (where the alkyl substituents are one or more of chlorine, bromine, iodine or fluorine), d.₅alkoxy, Cι.₅alkylthio, nitro, amino, Cι.₅alkylamino, cyano, carboxylic acid Ci-salkoxycarbonyl, carboxaldehyde, carboxamide, phenyl, or halogen;

n is 1-5 X is O or NH; Y is NH or CH_2: and q is 0 or 1 ; or pharmaceutically acceptable salts thereof to a mammal affiliated with a disorder of the central nervous system in an amount effective for treating such disorder.

10. The method of claim 9, wherein the effective amount is of from about 50 to 250 mg/kg per day.

12. The method of claim 9, wherein the disorder is anxiety.

13. The method of claim 9 wherein the disorder is convulsions.

14. The method of claim 9 wherein the disorder is sleeplessness.

15. The method of claim 9 wherein the disorder is muscle spasm.

16. The method of claim 9 wherein the disorder is benzodiazepine drug overdose.