Selective potentiation of serotonin receptor subtypes

The selective potentiation and/or inhibition of the 5-HT2A and/or 5-HT1A response to serotonin (5-HT) is achieved using analogs of oleamide. Selective potentiation and/or inhibition of the 5-HT2A and/or 5-HT1A leads to a modulation of serotonergic signal transduction of cells having various receptor subtypes. A subset of analogs is identified that inhibits rather than potentiates the 5-HT2A, but not the 5-HT1A, receptor response. These analogs enable the selective modulation of serotonin receptor subtypes and even have opposing effects on the different subtypes. An analysis of the activity of the oleamide analogs discloses that the structural features required for activity are highly selective. In particular, the presence, position, and stereochemistry of the 9-cis double bond is required and even subtle structural variations reduce or eliminate activity. Secondary or tertiary amides may replace the primary amide but follow a well-defined relationship requiring small amide substituents suggesting that the carboxamide serves as a hydrogen bond acceptor but not donor. Alternative modifications at the carboxamide as well as modifications of the methyl terminus or the hydrocarbon region spanning the carboxamide and double bond typically eliminate activity.

TECHNICAL FIELD

The invention relates to the potentiation of serotonin receptors and serotonergic activities. More particularly, the invention related to the selective potentiation of specific serotonin receptor subtypes with oleamide based analogs.

BACKGROUND

Fatty acid amide hydrolase (FAAH) is an integral membrane protein that degrades 1 to oleic acid and potent inhibitors of the enzyme have been detailed (Koutek et al. (1994)J. Biol. Chem.269, 22937-22940; Petrocellis et al. (1997)Biochem. Biophys. Rsch. Commun.231, 82-88; Deutsch et al. (1997)Biochem. Pharmacol.53, 255-260). The characterization and neuronal distribution of FAAH have been disclosed and the enzyme was found to possess the ability to hydrolyze a range of fatty acid amides including anandamide which serves as an endogenous ligand for the cannabinoid receptor (Devane et al. (1992)Science258, 1946-1949; Di Marzo et al. (1995)Prostaglandins, Leukot. Essent. Fatty Acids53, 1-11). Unlike anandamide, an appealing feature of this new class of biological signaling agents is the primary amide suggesting that their storage and release may be controlled in a manner analogous to that of peptide hormones terminating in a primary amide.

Recent studies have shown the oleamide modulates serotonergic neurotransmission (Huidobro-Toro et al. (1996)Proc. Natl. Acad. Sci. USA93, 8078-8082; Thomas et al. (1997)Proc. Natl. Acad. Sci. USA94, 14115-14119). In the first disclosure of such effects, oleamide was shown to potentiate 5-HT2Cand 5-HT2Areceptor-mediated chloride currents in transfected frog oocytes, but not those elicited by the 5-HT3ion-gated channel receptor or other G protein coupled receptors. This potentiation was greatest for the 5-HT2Creceptor subtype where the effect was observed at concentrations as low as 1 nM and was maximal at 100 nM oleamide. Oleamide did not alter the serotonin (5-HT) EC50but instead increased receptor efficacy.

Similarly, oleamide has been reported to potentiate phosphoinositide hydrolysis in rat pituitary P11 cells expressing the 5-HT2receptor but to inhibit 5-HT7receptor-mediated stimulation of cAMP levels in HeLa cells transfected with the receptor. In these efforts, oleamide was shown to act as a weak agonist at the 5-HT7receptor but to behave as an unsurmountable antagonist in the presence of serotonin illustrating that it may act at an allosteric site (Huidobro-Toro et al. (1996)Proc. Natl. Acad. Sci. USA93, 8078-8082; Thomas et al. (1997)Proc. Natl. Acad. Sci. USA94, 14115-14119). Thus, oleamide has been shown to enhance (5-HT2A, 5-HT2C), disrupt (5-HT7), or have no effect (5-HT3) on serotonergic signal transduction at various receptor subtypes. Serotonin receptors have been implicated in anxiety, depression, appetite, thermoregulation as well as sleep and mood regulation and strong links between 5-HT1, 5-HT2, and 5-HT7and the regulation of sleep have been disclosed (Leonard et al. (1996)Psychother. Psychosom.65, 66-75; Lovenberg et al. (1993)Neuron11, 449-458).

What is needed are analogs which possess inhanced activity and selectivity over that of oleamide for the potentiation of serotonergic signal transduction at various receptor subtypes.

SUMMARY OF THE INVENTION

The invention is directed to the use of analogs which potentiate serotonin receptor subtypes. Many of the analogs are more potent agonists than oleamide and some of the analogs possess dual agonist/antagonist activity. Such analogs may permit selective modulation of or even have opposing effects on different serotonin receptor subtypes.

One aspect of the invention is directed to a method for selectively potentiating a cell having a serotonin receptor subtype 5-HT1A. The method employs the step of contacting the cell having the serotonin receptor subtype 5-HT1Awith a serotonergic agent possessing 5-HT1Aagonist activity. Preferred serotonergic agents include compounds represented by the structure:
In the above structure, X is a diradical selected from the group represented by the following structures:
In the above structures, Z is a radical selected from the group consisting of: —CH2and O; Y is a diradical selected from the group consisting of: —CH2—, —CH(CH3)—, —C(CH3)2—, —O—, —NH—, —CH(SH)—, —CHSAc)—, —CH(OH)—, —CHCl—, —C(═O)—, —C(═O)CH2—, —CH2NHC(═O)—, and —CH2N(CH3)C(═O)—; R1is a radical selected from the group consisting of: hydrogen, —NH2, OH, MeNH—, Me2N—, EtNH—, Et2N—, CH2═CHCH2NH—, n-propyl-NH—, i-propyl-NH—, cyclopropyl-NH—, i-propyl-NMe-, butyl-NH—, pyrrolidine-, phenyl-NH—, phenyl(CH2)3NH—, HONH—, MeONMe-, NH2NH—, CH3O—, CH3CH2O—, CH3(CH2)2O—, Me2CHCH2O—, H—, CF3-, BrCH2—, ClCH2—, N2CH—, HOCH2CH2NH—, (HOCH2CH2)2N—, HOCH2CH2CH2NH— and HOCH2CH(OAc)CH2O—; and R2is a radical selected from the group consisting of: —CH3, —(CH2)2CH3, —(CH2)4CH3, —(CH2)6CH3, —CH2OCH3, —CH2OH, —CONH2and —CO2H. In each instance, n is an integer from 0 to 15; m is an integer from 0 to 15 with the requirement that the sum of n+m is an integer from 11 to 15. However, if Y is CH2, 4≦n≦9, 4≦n≦7, and R2is CH3, then R1cannot be NH2and OH.

Another aspect of the invention is directed to a method for selectively enhancing a serotonergic signal transduction response of a cell having serotonin receptor subtype 5-HT1A. Enhancement is achieved by contacting the cell, in the presence of serotonin, with the above serotonergic agent possessing 5-HT1Aagonist activity.

Another aspect of the invention is directed to a method for selectively potentiating a cell having a serotonin receptor subtype 5-HT2A. The method employs the step of contacting the cell having the serotonin receptor subtype 5-HT1Awith a serotonergic agent possessing 5-HT2Aagonist activity. Preferred serotonergic agents include compounds represented by the structure:
In the above structure, X is a diradical selected from the group represented by the following structures:
In the above structures, Z is a radical selected from the group consisting of: —CH2and O; Y is a diradical selected from the group consisting of: —CH2—, —CH(CH3)—, —C(CH3)2—, O—, —NH—, —CH(SH)—, —CHSAc)—, —CH(OH)—, —CHCl—, —C(═O)—, —C(═O)CH2—, —CH2NHC(═O)—, and —CH2N(CH3)C(═O)—; R1is a radical selected from the group consisting of: hydrogen, —NH2, OH, MeNH—, Me2N—, EtNH—, Et2N—, CH2═CHCH2NH—, n-propyl-NH—, i-propyl-NH—, cyclopropyl-NH—, i-propyl-NMe-, butyl-NH—, pyrrolidine-, phenyl-NH—, phenyl (CH2)3NH—, HONH—, MeONMe-, NH2NH—, CH3O—, CH3CH2O—, CH3(CH2)2O—, Me2CHCH2O—, H—, CF3-, BrCH2—, ClCH2—, N2CH—, HOCH2CH2NH—, (HOCH2CH2)2N—, HOCH2CH2CH2NH— and HOCH2CH(OAc)CH2O—; and R2is a radical selected from the group consisting of: —CH3, —(CH2)2CH3, —(CH2)4CH3, —(CH2)6CH3, —CH2OCH3, —CH2OH, —CONH2and —CO2H. In each instance, n is an integer from 0 to 15; m is an integer from 0 to 15 with the requirement that the sum of n+m is an integer from 11 to 15. However, if Y is CH2, 4≦n≦9, 4≦n≦7, and R2is CH3, then R1cannot be NH2and OH.

Another aspect of the invention is directed to a method for selectively enhancing a serotonergic signal transduction response of a cell having serotonin receptor subtype 5-HT2A. Enhancement is achieved by contacting the cell, in the presence of serotonin, with the above serotonergic agent possessing 5-HT2Aagonist activity.

Another aspect of the invention is directed to a method for selectively inhibiting a serotonergic signal transduction response of a cell having serotonin receptor subtype 5-HT1A. Inhibition is achieved by contacting the cell with an inhibitory concentration of a serotonergic agent possessing S-HT1Aantagonist activity. Preferred serotonergic agents having antagonist activity include compounds represented by the structure:
In the above structure, X is an ethene diradical; Y is a methylene diradical; R1is a radical selected from the group consisting of —NH2and cyclopropyl-NH—; and R2is a radical selected from the group consisting of —CH3, —CH2OH and —CONH2. “n” is an integer from 0 to 15; and “m” is an integer from 0 to 15 with the requirement that the sum of n+m is an integer from 11 to 15. However, there are two provisos, viz., if R1is —NH2, then R2is not —CH3; and if R2is —CH3, then R1is not —NH2.

Another aspect of the invention is directed to a method for selectively inhibiting a serotonergic signal transduction response of a cell having serotonin receptor subtype 5-HT2A. Inhibition is achieved by contacting the cell with an inhibitory concentration of a serotonergic agent possessing 5-HT2Aantagonist activity. Preferred serotonergic agents having antagonist activity include compounds represented by the structure:
In the above structure, X is an ethene diradical; Y is a diradical selected from the group consisting of: —CH2—, —C(CH3)2—, —NH—, —CH(SH)—, —CH(SAc)—, —CH2NHC(═O)—, and —CH2N(CH3)C(═O)—; R1is a radical selected from the group consisting of: —NH2, —OH, cyclopropyl-NH—, butyl-NH—, phenyl-NH—, CH3O—, CH3CH2O—, N2CH—, and HOCH2CH(OAc)CH2—; and R2is a radical selected from the group consisting of: —CH3, —(CH2)2CH3, —(CH2)4CH3, and —(CH2)6CH3. “n” is an integer from 0 to 15; and “m” is an integer from 0 to 15 with the requirement that the sum of n+m is an integer from 11 to 15. However, there are two provisos, viz., if R1is —NH2, then Y is not —CH2—; and if Y is —CH2—, then R1is not —NH2.

DETAILED DESCRIPTION OF THE INVENTION

The following examples disclose a set of analogs that potentiate both 5-HT2A, and 5-HT1A, receptor responses. Some of the analogs inhibit rather than potentiate the 5-HT2A, but not 5-HT1A, receptor response suggesting such agents may permit selective modulation of serotonin receptor subtypes or even have opposing effects on the different subtypes. These analogs provide information which defines features of oleamide required for potentiation of the5-HT2Areceptor response and report the analogous but more tolerant potentiation of the 5-HT1Areceptor which has not been previously examined.

Oleamide is an endogenous fatty acid primary amide which possesses sleep-inducing properties in animals and has been shown to effect serotonergic receptor responses and block gap junction communication. Herein, the potentiation of the 5-HT1Areceptor response is disclosed and a study of the structural features of oleamide required for potentiation of the 5-HT2Aand 5-HT1Aresponse to serotonin (5-HT) is described. Of the naturally occurring fatty acids, the primary amide of oleic acid (oleamide) is the most effective at potentiating the 5-HT2Areceptor response. The structural features required for activity were found to be highly selective. The presence, position, and stereochemistry of the9cis double bond is required and even subtle structural variations reduce or eliminate activity. Secondary or tertiary amides may replace the primary amide but follow a well-defined relationship requiring small amide substituents suggesting that the carboxamide serves as a hydrogen bond acceptor but not donor. Alternative modifications at the carboxamide as well as modifications of the methyl terminus or the hydrocarbon region spanning the carboxamide and double bond typically eliminate activity.

A less extensive study of the 5-HT1Apotentiation revealed that it is more tolerant and accommodates a wider range of structural modifications. An interesting set of analogs was identified that inhibits rather than potentiates the 5-HT2A, but not the 5-HT1A, receptor response further suggesting that such analogs may permit the selective modulation of serotonin receptor subtypes and even have opposing effects on the different subtypes.

The effects of oleamide and its analogs on rat 5-HT2Aand human 5-HT1Areceptors were examined using R-SAT transfected cellular assays linked to a colorimetric β-galactosidase assay (Messier et al. (1995)Pharmacol. Toxicol.76, 308-311; Brann et al. (1996)J. Biomol. Screening1, 43-45; Brauner-Osborne et al. (1996)Eur. J. Pharm.295, 78-102) which provide results identical to those derived from second messenger assays. Activation of the 5-HT2Aor 5-HT1Areceptors in 5-HT dependent cell lines results in cell proliferation measured by the levels of β-galactosidase produced. Analogous to the findings of Huidobro-Toro and Harris (Huidobro-Toro et al. (1996)Proc. Natl. Acad. Sci. USA93, 8078-8082.), treatment with oleamide alone had no effect, but its coadministration with 5-HT provided a significant potentiation of the effect of 5-HT administration alone.

A maximal response with the rat 5-HT2Areceptor was observed at 100 nM oleamide when assayed at 100 nM or 1 μM 5-HT, the concentrations at which the potentiation response (165 and 170%, respectively) was greatest. At higher concentrations of oleamide (1 μM), no additional potentiation was observed with the rat 5-HT2Areceptor (FIG.11A). Similarly, the maximal potentiation for human 5-HT1Awas observed at 100 nM 5-HT (FIG. 2) and concentrations as low as 1-10 mM oleamide produced a measurable effect (FIG.11B). The maximum effect was observed at concentrations of 100 nM 5-HT and oleamide treatment provided a 370% (100 nM oleamide) or 560% (1 μM oleamide) potentiation approximating the magnitude observed with the 5-HT2Creceptor.

An extensive series of agents was tested at 500 nM for their ability to potentiate the rat 5-HT2Aor human 5-HT1Areceptor response to 100 nM 5-HT. This spans the concentration range (100 nM-1 μM) in which oleamide exhibits its greatest potentiation and employs a 5-HT concentration at which the effect was found to be largest. The concentrations employed in the examples below are well within physiologically relevant concentrations. Serotonin levels in human CSF (3.3 ng/ml), plasma (3.4 ng/ml), and platelets (748 ng/109platelets) translate into 10-20 nM concentrations (Kumar et al. (1990)Life Sciences47, 1751-1759) which could be much higher at the synapse, and oleamide levels in human plasma (31.7 μg/mL, 110 μM) (Arafat et al. (1989)Life Sci.45, 1679-1687) and mouse neuroblastoma N18TG2cells (1.5 μg/109cells, ca. 100× the concentration of anandamide) typically exceed those examined (Bisogno et al. (1997)Biochem. Biophys. Res. Commun.239, 473-979). There was no additional effect when the FAAH enzyme inhibitor PMSF was included in the assay suggesting that the agents susceptible to protease degradation are stable in the assay. The analog screening was conducted with the rat 5-HT2Aassay and a subset of analogs was also examined in the human 5-HT1Aassay. The results reported are normalized to a 100% potentiation by oleamide for the ease of direct comparisons.

Potentiation with Fatty Acid Primary Amide Analogs

The first series examined was the primary amides (Wakamatsu et al. (1990)Biochem. Biophys. Res. Commun.168, 423-429; Jain et al. (1992)J. Med. Chem.35, 3584-3586) of the naturally occurring fatty acids and related synthetic analogs (FIGS.3-4). From these studies, important trends in structural requirements of the endogenous agent emerge. The most effective primary amide of the naturally occurring fatty acids was oleamide. In the 5-HT2Aassay and for agents that contain one double bond, the presence, position, and stereochemistry of the olefin as well as the chain length were found to have a pronounced effect.

Removal of the9double bond (18:0) or its replacement with a trans double bond (18:19-trans) resulted in no observable potentiation. Shortening the chain length to 14 or 16 carbons resulted in the loss of activity providing weak inhibitors while lengthening the chain substantially diminished the effectiveness. A9cis olefin exhibited the strongest effect and the potency sharply declined as its position was moved in either direction. This is especially clear in the oleamide series (18:1) where the potency sharply declined as the distance from the 9 position increased. Although this is central to oleamide's structure and potentially represents a relationship with either the carboxamide or methyl terminus, the inactivity of the primary amides of 20:19, 22:19, and 24:19and the modest activity of 20:113, 22:113, and 24:115suggest that it may be the9relationship with the methyl terminus that may be most important. Moreover, while extending the distance between the carboxamide and the double bond resulted in reduced or inactive compounds, shortening the length typically provided agents that displayed progressively more potent inhibition versus potentiation. Although the significance of this is not yet clear, it suggests the possibility that tightly regulated endogenous agents may serve to both potentiate or inhibit a serotonin receptor response (FIGS.3-4).

With the polyunsaturated fatty acid primary amides, those containing two cis double bonds exhibited modest activity and those containing 3-6 double bonds were typically less active. The exception to this generalization is -linolenamide (18:36,9,12) which proved to be a more effective but still less potent agent. Arachidonamide containing four cis double bonds was ineffective.

The behavior of 18:0, 18:19-trans, 18:18, 18:112and 18:29,12proved analogous to the observations detailed by Huidobro-Toro and Harris et al. (1996)Proc. Natl. Acad. Sci. USA93, 8078-8082 in studies with the 5-HT2Creceptor. These similar observations not only indicate that the R-SAT assay for assessment of the 5-HT2Areceptor potentiation provides observations analogous to second messenger assays, but also implies that the oleamide structural features required for activity may be well conserved throughout the 5-HT2receptor subtypes.

In contrast, the 5-HT1Areceptor was found to be more tolerant of structural changes. Like the effects at the 5-HT2Areceptor, the saturated or trans fatty acid primary amides 18:0 and 18:19-transas well as 18:18were less effective than oleamide, albeit not inactive, on the 5-HT1Areceptor. Similarly, 14:19, 16:19, and linoleamide (18:29,12) were more effective on the 5-HT1Areceptor than 5-HT2Awith the latter two approaching the potency of oleamide.

Potentiation with Carboxamide Terminus Analogs

A study of the carboxamide terminus revealed well-defined structural requirements. Not only was the primary amide of oleic acid capable of potentiating the 5-HT response, but secondary and tertiary amides also provided a comparable potentiation provided the amide substituents were small (FIGS.4-5). The activity smoothly progresses through the series with the maximum effect observed with the NMe2tertiary amide. As the amide substituents further increased in size, the effect diminished and ultimately provided inactive derivatives. Thus, the primary carboxamide is not required although its efficacy approximates that of the most potent amide. This suggests that the carboxamide may serve as a H-bond acceptor but need not serve as a H-bond donor. In addition, the cyclopropyl amide was uniquely effective at inhibiting the response to serotonin at the 5-HT2Areceptor. Although endogenous agents that may act similarly have not been identified, such allosteric inhibitors at 5-HT2Amay prove to be useful biochemical tools and potentially interesting therapeutics.

In contrast to the well defined effects on the 5-HT2Areceptor, both the isopropyl and cyclopropyl amides as well as the methyl amide were found to be effective at potentiating 5-HT1Aeven though the first two were inactive or inhibitory on 5-HT2A. Such distinctions suggest that derivatives of oleamide may be developed that not only possess greater potentiation effects, but that can also selectively modulate the various serotonin receptor subtypes or even have opposing effects (i.e., cyclopropyl amide).

Alternative substitutions for the carboxamide including oleic acid itself, oleyl esters, alcohols, amines, aldehydes, acetals, and electrophilic ketones did not provide a comparable potentiation of 5-HT2Abut appear to be better tolerated with 5-HT1A(FIGS.4-5). Of particular interest are oleyl aldehyde and the trifluoromethyl ketone (FIG. 5, R=H and CF3). Both not only possess polarized carbonyls and can serve as H-bond acceptors, but both are potent inhibitors of FAAH which is responsible for the degradation of oleamide (Patterson et al. (1996)J. Am. Chem. Soc.118, 5938-5945). This dual activity suggests that they may not only potentiate the activity of oleamide by inhibiting its degradation, but that they may also serve as oleamide agonists at the 5-HT1A receptor.

Potentiation with Oleyl Ethanolamide, Anandamide and Related Analogs

An important subset of modified carboxamides is the ethanolamide derivatives (Bachur et al. (1965)J. Biol. Chem.240, 1019-1024; Ramachandran et al. (1992)Biochem. Arch.8, 369-377; Schmid et al. (1990)Prog. Lipid Res.29, 1-43; Hanus et al. (1993)J. Med. Chem.36, 3032-3034) which include anandamide. Consequently, the ethanolamides and bis-(ethanol)amides of oleic and arachidonic acid were examined (FIG.6). Both derivatives of oleic acid were inactive providing no effect on the 5-HT2Areceptor while those of arachidonic acid including anandamide were weakly inhibitory. Similarly, recent studies have implicated 2-arachidonyl glycerol as an endogenous ligand for the cannabinoid receptor (Mechoulam et al. (1995) Biochem. Pharmacol. 50, 83-90) and diacylglycerols including 1-oleyl-2-acetylglycerol have been reported as inhibitors of Chinese hamster V79 cell gap junctions (Aylsworth et al. (1986)Cancer Res.46, 4527-4533). This latter compound was examined and it did not potentiate, but rather weakly inhibited the 5-HT2Areceptor response.

Both oleyl ethanolamide and anandamide were found to potentiate the 5-HT1Areceptor response to 5-HT and the former was more potent. Although this might be interpreted to suggest a special significance for the ethanolamides, oleyl propanolamide was equally effective. As such, the results are more consistent with the simpler interpretation that the 5-HT1Areceptor potentiation is more tolerant of modifications in the carboxamide terminus and accommodates a wider range of secondary or tertiary amides (FIG.6).

Potentiation with Putative Precursors and Related Analogs

The potential that oleamide may be stored as a N-oleyl glycinamide derivative and released upon -hydroxylation of glycine by a peptidylglycine-amidating monoxygenase (Merkler et al. (1996)Arch. Biochem. Biophys.330, 430-434) led to the examination of a set of N-oleyl glycine derivatives (FIG.7). None of the derivatives potentiated the 5-HT2Aserotonin receptor response and most proved to be weak inhibitors. This is consistent with their behavior as large secondary or tertiary amide derivatives and their activity follows the prior trends (FIGS.4-6). In contrast, N-oleyl glycine was a weak potentiator of the 5-HT1Areceptor consistent with its more tolerant accommodation of modifications in the carboxamide terminus.

Potentiation with Methyl Terminus Analogs

The potentiation of 5-HT2Aor 5-HT1Awas especially sensitive to the structural characteristics at the methyl terminus (FIG.8). Extending the length of the methyl terminus chain and the incorporation of polar functional groups resulted in a loss of activity.

Potentiation with Analogs which Have Modifications in the Double Bond

The agents 2-10 were examined to define the role of the olefin (FIG.9). Analogous to the observations made with octadecanamide (18:0) and the trans-9-octadecenamide (18:19-trans) which were ineffective at potentiating 5-HT2A, nearly all agents were ineffective. These include 7-10 for which a benzene ring was incorporated into the structure at a location that mimics the9double bond as well as 5 and 6 which mimic a hairpin conformation. Although it is difficult to draw specific conclusions from their inactivity, it highlights that the effects of oleamide at the 5-HT2Areceptor are surprisingly selective for the endogenous lipid. The exceptions include 9-octadecynamide (2) which was nearly equipotent with oleamide and 4 which was approximately 50% as effective. The observation that the former is so effective suggests that the appropriate presentation of a -system in addition to the conformational effects of the cis double bond may be important. Consistent with this, 4 versus 3 proved surprisingly effective and may benefit from the partial characteristics of the cyclopropane which would allow it to mimic both and conformational characteristics of the cis double bond.

Similar observations with the 5-HT1Areceptor were made with 2 and 4. In contrast to the 5-HT2Aresults, both 9 and 10, but not 8, were effective at potentiating the 5-HT1Areceptor response and imply that an extended versus hairpin conformation of oleamide might be important at the 5-HT1Areceptor.

Potentiation with Analogs which Have Modifications in the Linking Chain

Modifications in the seven carbon chain linking the olefin and carboxamide were examined and found to have a detrimental effect with 5-HT2A(FIG.10). Substitution of the -carbon or its replacement with a heteroatom resulted in a loss of activity or provided agents that inhibited the 5-HT2Aresponse. Most notable are 2,2-dimethyloleamide as well as the urethane (X=NH) which proved to be potent inhibitors.

In contrast, most of the linking chain modifications did not adversely affect the 5-HT1Apotentiation and this is significant in several respects. It is consistent with the greater tolerance for carboxamide modifications observed at the 5-HT1Areceptor and highlights again that many oleamide analogs may have distinguishing effects on the serotonin receptor subtypes. Moreover, the first seven entries inFIG. 10constitute analogs that are more resistant to hydrolytic FAAH degradation and suggest an improved duration of effect would accompany their enhanced efficacy in vivo. In addition, the two-keto amide (X=CO, COCH2) inFIG. 10are potent inhibitors of FAAH illustrating that they may serve to potentiate the effects of oleamide by inhibiting its hydrolysis and serve as oleamide agonists in their own right at the 5-HT1Areceptor.

General Synthetic Conditions for the Preparation of Oleamide Based Analogs

The fatty acid primary amides (FIG. 3) were prepared by treating the acid chlorides, generated from the corresponding carboxylic acid and oxalyl chloride, with aqueous NH4OH according the procedures in Cravatt et al. (1996)J. Am. Chem. Soc.118, 580-590; Patterson et al. (1996)J. Am. Chem. Soc.118, 5938-5945). Many of the fatty acids were commercially available (Sigma, Aldrich, Fluka), and the remainder were synthesized as detailed vida infra.

The ethanolamides (FIG. 6) were similarly prepared or purchased (Pfaltz & Bauer). The synthesis of the 18-hydroxyoleamide was described in Cravatt et al. (1996)J. Am. Chem. Soc.118, 580-590 and standard transformations following protocols detailed therein were used to prepare the remaining agents in FIG.8.

N-Oleoyl glycine (FIG. 7) was prepared by coupling glycine ethyl ester to oleic acid with EDCI (Cravatt et al. (1996)J. Am. Chem. Soc.118, 580-590; Patterson et al. (1996)J. Am. Chem. Soc.118, 5938-5945) followed by sequential transformation to the carboxylic acid (LiOH) and glycinamide (EDCI, NH4OH;). N-Oleoyl sarcosine was purchased (Pfaltz and Bauer) and converted to its ethyl ester by coupling with EtOH (DCC).

The agents inFIG. 9were prepared as described in Cravatt et al. (1996)J. Am. Chem. Soc.118, 580-590; Simmons et al. (1959)J. Am. Chem. Soc.81, 4256-4264) or by a series of Wittig couplings to the appropriately substituted o, m, or p-cyanobenzaldehyde. Substitutions at the α-carbon (FIG. 10) were installed by treating the enolate of oleic acid or methyl oleate (generated by LDA) with an appropriate electrophile as previously detailed in Patterson et al. (1996)J. Am. Chem. Soc.118, 5938-5945). The primary carbamate and urethanes were prepared from the corresponding alcohols or amines (HCl, NaOCN).

The above examples illustrate that the structural features of oleamide required for potentiation of the 5-HT2Areceptor response are well-defined supporting a selective site of action. Of the naturally occurring fatty acids, oleamide is the most effective and other endogenous fatty acid amides including arachidonamide, anandamide, and oleyl ethanolamide were less active or ineffective. For oleamide, the presence, position, and stereochemistry of9cis double bond is required and even subtle structural variations reduce or eliminate activity. Secondary or tertiary amides but not acids, esters, aldehydes, alcohols, amines, acetals, or electrophilic or polarized ketones may replace the primary carboxamide. Even the amide substitutions follow a well-defined relationship limited to small amide substituents. Modifications of the methyl terminus or in the hydrocarbon chain linking the carboxamide and cis double bond typically eliminate the activity. In contrast, the 5-HT1Areceptor was more tolerant of structural modifications especially at the carboxamide terminus.

The well-defined structural features of oleamide required for potentiation of the 5-HT2Aor 5-HT1Areceptor response in the presence of serotonin provides the opportunity to correlate the properties with physiological states including sleep (Lerner, R. A. (1997)Proc. Natl. Acad. Sci. USA94, 13375-13377). Such studies will clarify whether the serotonergic effects of oleamide and related agents may be responsible. Many of the well-defined structural features of oleamide are tightly conserved among the 5-HT2A, 5-HT1A, and 5-HT2Creceptors suggesting they may be well conserved throughout the 5-HT1and 5-HT2receptor subtypes. However, distinguishing structural effects were observed where the 5-HT1Areceptor was more tolerant of structural modifications in the carboxamide terminus of oleamide.

In addition, several agents including a small set of naturally occurring fatty acid primary amides were identified that inhibited rather than potentiated the 5-HT2Abut not 5-HT1Areceptor response. Although the significance of these observations is not yet clear, it not only suggests the possibility that tightly regulated endogenous agents may serve to both potentiate or inhibit a serotonin response, but that analogs of oleamide may permit the selective modulation of serotonin receptor subtypes and, in selected instances, even have opposing effects on the different receptor subtypes.

The studies to date have demonstrated that at concentrations of 100 nM serotonin, 100 nM oleamide potentiates 5-HT2C(365%, 11), 5-HT1A(370%, results herein), and 5-HT2Areceptors (165% for results herein, 228% (Thomas et al. (1997)Proc. Natl. Acad. Sci. USA94, 14115-14119) and 260% (Huidobro-Toro et al. (1996)Proc. Natl. Acad. Sci. USA93, 8078-8082), inhibits 5-HT7receptors (−50%, Thomas et al. (1997)Proc. Natl. Acad. Sci. USA94, 14115-14119), and has no effect on the ion gated channel 5-HT3receptor. The identification of such agents provide new biochemical tools for the study of serotonin receptors and may lead to therapeutic applications involving selective modulation of the serotonin response at the receptor subtypes.

While a preferred form of the invention has been shown in the drawings and described, since variations in the preferred form will be apparent to those skilled in the art, the invention should not be construed as limited to the specific form shown and described, but instead is as set forth in the following claims.

EXPERIMENTAL PROTOCOLS

General

1H and13C nmr spectra were recorded either on a Bruker AM-250, a Bruker AMX-400 or a Bruker AMX-500 spectrometer. Residual protic solvent CHCl3(δH=7.26 ppm, δC=77.0), d4-methanol (δH=3.30 ppm, δC=49.0) and D2O (δH=4.80 ppm, δC(ofCH3CN)=1.7 ppm) or TMS (δH=0.00 ppm) were used as internal reference. Coupling constants were measured in Hertz (Hz). HRMS were recorded using FAB method in a m-nitrobenzylalcohol (NBA) matrix doped with NaI or CsI. Infra-red spectra were recorded on a Perkin-Elmer FTIR 1620 spectrometer. Enantiomeric excess was determined by HPLC using a Daicel Chemical Industries CHIRALPAK AD column. Optical rotations were measured with an Optical Activity AA-1000 polarimeter. Melting points were taken on a Thomas Hoover capillary melting point apparatus and are uncorrected. Column chromatography was performed on Merck Kieselgel 60 (230-400 mesh). Analytical thin layer chromatography was performed using pre-coated glass-backed plates (Merck Kieselgel F254) and visualized by cerium molybdophosphate or ninhydrin. Diethyl ether, tetrahydrofuran (THF) and toluene (PhCH3) were distilled from sodium-benzophenone ketyl, dichloromethane (DCM) and acetonitrile from calcium hydride. Other solvents and reagents were purified by standard procedures if necessary.

The assays were conducted with R-SAT kits (Receptor Technologies Inc, Winooski, Vt.) containing NIH 3T3 cells expressing the rat 5-HT2Areceptor (Suter et al. (1987)Fundam. Appl. Toxicol.9, 785-794; Pritchett et al. (1988)EMBO J7, 4135-4140) or RAT-1 cells expressing the human 5-HT1Areceptor (Lam et al. (1996)Biochem. Biophys. Res. Commun.219, 853-858; Kobilka et al. (1987)Nature329, 75-79) cotransfected with the -galactosidase gene and were performed according to the procedures provided (Messier et al. (1995)Pharmacol. Toxicol.76, 308-311; Brann et al. (1996)J. Biomol. Screening1, 43-45). The cells in Dulbecco modified Eagle's medium containing serotonin (100 nM) and the analogs (500 nM) were incubated in a humidified 5% CO2incubator at 37° C. for 4 or 5 days for the 5-HT2Aand 5-HT1Atransfected cells, respectively. Levels of galactosidase were measured after incubation with the chromogenic substrate o-nitrophenyl- -D-galactopyranoside (20) at 30° C. in a humidified incubator for a recommended period of time and the absorbance was measured at 405 nm. The results were normalized to 100% for oleamide (rel. % potentiation) for the ease of comparison and are the average of 2-8 determinations.

18:0 (stearamide) as illustrated in FIG.3: purchased from Aldrich and recrystallized once before use.

General procedure for the preparation of fatty amides (compounds disclosed in FIG.3):

The fatty acid (1 equiv) was dissolved in dry CH2Cl2(0.2 M) and cooled to 0° C. under a N2atmosphere. Oxalyl chloride (2M in CH2Cl2, 3 equiv) was added slowly. The solution was warmed to 25° C. and allowed to stir for 3 h in the dark. The solvent was removed in vacuo and the flask cooled to 0° C. Excess concentrated NH4OH was added slowly and the crude product was purified by chromatography on SiO2using EtOAc/hexanes as an eluent. Fatty acids were purchased from Sigma unless otherwise indicated.

Synthesized exactly as found in the general procedure vida supra; all starting reagents were purchased from Aldrich, Acros, or Sigma and resulting white solid was purified by chromatography or prepared as previously described exactly (JACS, 1996, 118, 580-590).

18:19(Oleamide) as illustrated in FIG.3:

18:19trans(Elaidamide) as illustrated in FIG.3:

18:111(Vaccenamide) as illustrated in FIG.3:

18:29,12(Linoleamide) as illustrated in FIG.3:

20:45,9,11,14as illustrated in FIG.3: purchased from Sigma.

General procedure for the preparation of oleic amide derivatives varying about carboxamide (unless otherwise described) as illustrated in FIG.4:

One equivalent of oleic acid was dissolved in dry CH2Cl2(0.2 M) and cooled to 0° C. under a N2atmosphere. Oxalyl chloride (2M in CH2Cl2, 3 equiv) was added slowly. The solution was warmed to 25° C. and allowed to stir for 3 hours in the dark. The solvent was then removed in vacuo and the flask cooled to 0° C. Excess free amine (amines that were available as the hydrochloride salts were extracted into EtOAc from a 50% NaOH solution before use) or alcohols were added slowly. The crude product was purified by chromatography on SiO2using EtOAc/hexanes as an eluent.

Agent purchased from Aldrich.

Synthesized exactly as found in the general procedure for derivatives varying about carboxamide vida supra using i-Propyl-N-methyl-amine; all starting reagents were purchased from Aldrich, Acros, or Sigma.

Synthesized exactly as found in the general procedure for derivatives varying about carboxamide vida supra using the cyclopropyl amine; all starting reagents were purchased from Aldrich, Acros, or Sigma.

Synthesized exactly as found in the general procedure for derivatives varying about carboxamide vida supra using the commercially available Ph(CH2)3NH amine; all starting reagents were purchased from Aldrich, Acros, or Sigma.

NHOH derivative as illustrated in FIG.4: prepared as previously described (JACS, 1996, 118, 5938-5945).

NH2NH derivative as illustrated in FIG.4: prepared as previously described (JACS, 1996, 118, 5938-5945).

H(oleyl aldehyde) as illustrated in FIG.5: prepared as described in the literature (JOC 1978, 43,2480-2482).

N2CH as illustrated in FIG.5: prepared as previously described (JACS, 1996, 118, 5938-5945).

Oleyl alcohol as illustrated in FIG.5: prepared as described in the literature (JOC 1978, 43,2480-2482).

Oleyl acetate: purchased from Sigma

Oleyl amine: purchased from Pfaltz and Bauer

Oleyl aldehyde dimethyl acetal as illustrated in FIG.5: prepared as described in the literature (J Med Chem, 1989, 32, 1319-1322).

CoA-SCO derivative as illustrated in FIG.5: purchased from Sigma

HOCH2CH2NH derivative as illustrated in FIG.6:

(HOCH2CH2)2NH derivative as illustrated in FIG.6:

Purchased from Pfaltz and Bauer.

HOCH2CH(OAc)CH2O derivative as illustrated in FIG.6:

Purchased from Sigma

HOCH2CH2NH derivative as illustrated in FIG.6:

Purchased from Sigma

OH derivative as illustrated in FIG.7:

OEt derivative as illustrated in FIG.7:

OH/R1=Me derivative as illustrated in FIG.7: purchased from Sigma.

CH2OCH3derivative as illustrated in FIG.8.

CH2OH derivative as illustrated inFIG. 8is commercially available from Aldrich.

CO2H derivative as illustrated in FIG.8:

3 derivative as illustrated in FIG.9:

4 derivative as illustrated in FIG.9:

Prepared as described in the literature (JACS, 1959, 81, 4256).

6 derivative as illustrated in FIG.9:

8 derivative as illustrated in FIG.9.

CH(CH3) derivative as illustrated in FIG.10.

C(CH3)2derivative as illustrated in FIG.9:

“O” derivative as illustrated in FIG.9and scheme shown on FIG.21:

Commercially available from Aldrich.

“NH” derivative as illustrated in FIG.9and scheme shown on FIG.21:

Commercially available from Aldrich.

CH(SH) derivative as illustrated in FIG.9and scheme shown in FIG.22:

CH(SAc): derivative as illustrated in FIG.9:

CH(OH)derivative as illustrated in FIG.9: prepared as previously described (JACS, 1996, 118, 5938-5945).

CHCl derivative as illustrated in FIG.9: prepared as previously described (JACS, 1996, 118, 5938-5945).

C(═O) derivative as illustrated in FIG.9: prepared as previously described (JACS, 1996, 118, 5938-5945).

C(═O)CH2derivative as illustrated in FIG.9: prepared as previously described (JACS, 1996, 118, 5938-5945)