Inhibitors of the anandamide transporter

Abstract

Disclosed are compounds that are anandamide transport inhibitors and their pharmacological use.

Description

BACKGROUND OF THE INVENTION

The marijuana derived cannabinoid Δ⁹-tetrahydrocannabinol, Δ⁹THC, is known to bind to CB1 receptors in the brain and CB2 receptors in the spleen. Compounds which stimulate those receptors have been shown to induce analgesia and sedation, to cause mood elevation including euphoria and dream states, to control nausea and appetite and to lower intraocular pressure. Cannabinoids have also been shown to suppress the immune system. Thus, compounds which stimulate the receptors, directly or indirectly, are potentially useful in treating glaucoma, preventing tissue rejection in organ transplant patients, controlling nausea in patients undergoing chemotherapy, controlling pain and enhancing the appetite and controlling pain in individuals with AIDS Wasting Syndrome.

In addition to acting at the receptors, cannabinoids also affect cellular membranes, thereby producing undesirable side effects such as drowsiness, impairment of monoamine oxidase function and impairment of non-receptor mediated brain function. The addictive and psychotropic properties of cannabinoids also limit their therapeutic value.

Arachidonyl ethanolamide (anandamide) is an endogenous lipid that binds to and activates cannabinoid receptors and mimics the pharmacological activity of Δ⁹THC. In general, anandamide has been found to be somewhat less potent than Δ⁹THC. Despite having a rapid onset of action, the magnitude and duration of action of anandamide is relatively short, presumably because of a rapid inactivation process consisting of carrier-mediated transport into cells followed by intra-cellular hydrolysis by a membrane-bound amidohydrolase, anandamide amidase. Thus, inhibitors of anandamide amidase have the effect of indirectly stimulating the receptors by increasing in vivo levels of anandamide. In this connection, attention is directed to Makriyannis et al U.S. Pat. Nos. 5,688,825 and 5,874,459, the disclosures of which are incorporated herein by reference.

Anandamide released by depolarized neurons is believed to be subject to rapid cellular uptake followed by enzymatic degradation. Indeed, rat brain neurons and astrocytes in primary culture avidly take up radioactively labeled anandamide through a mechanism that meets four key criteria of a carrier-mediated transport; temperature dependence, high affinity, substrate selectivity, and saturation. In that other lipids including polyunsaturated fatty acids and prostaglandin E₂ (PGE₂) enter cells by carrier-mediated transport, it is possible that anandamide uses a similar mechanism. This accumulation may result from the activity of a transmembrane carrier or transporter, which may thus participate in termination of the biological actions of anandamide. This carrier or anandamide transporter is believed to be involved in the inactivation of anandamide. Thus, anandamide released from neurons on depolarization may be rapidly transported back into the cells and subsequently hydrolyzed by an amidase thereby terminating its biological actions. Consequently, the anandamide transporter is a potential therapeutic target for the development of useful medications.

There is considerable interest in understanding the mechanism of anandamide transport and in developing pharmacological agents that selectively interfere with it. Anandamide transport inhibitors may be used as experimental tools to reveal the possible physiological functions of this biologically active lipid. Many of these functions are still elusive despite a growing body of evidence suggesting that the endocannabinoid system is intrinsically active not only in brain and spinal cord, but also in peripheral tissue. Furthermore, anandamide transport inhibitors may offer a rational therapeutic approach to a variety of disease states, including pain, psychomotor disorders, and multiple sclerosis, in which elevation of native anandamide levels may bring about a more favorable response and fewer side effects than direct activation of CB1 receptors by agonist drugs.

SUMMARY OF THE INVENTION

It has now been found that certain analogs of anandamide are potent inhibitors of transport of anandamide across cell membranes. The inventive analogs do not activate the cannabinoid receptors or inhibit anandamide hydrolysis per se but instead prevent anandamide reuptake thereby prolonging the level of the undegraded anandamide. Previously, cannabinoid drugs were targeted toward cannabinoid receptors and amidase enzymes. The anandamide transport inhibitor of the present invention targets activity of the anandamide transporter.

The inhibitors are analogs of anandamide and exhibit the tail, central and head pharmacophore portions represented by Structural Formula IX—Y-Z.  (I)

The tail portion X is a fatty acid chain remnant, or an aliphatic hydrocarbon as defined later, or a biphenyl group with an alkyl chain.

The central portion Y is a member selected from the group consisting of —NH—C(O)—, —NH—, —NH—C(O)—NH—, —NH—C(O)—O—, —O—C(O)—NH—, —C(O)—C(O)—NH, —NH—C(O)—C(O)—, —O—C(O)—O—, —C(O)—NH, —O—C(O)—, —O—, —S— and —H. It should be noted that the X and Z portions may be connected to the Y portion at either of the Y portion connecting atoms. Thus, for example, the Y portion —C(O)—NH— will lead to analogs X—C(O)—NH-Z and Z-C(O)—NH—X.

The head portion Z is selected from the group consisting of hydrogen, aryl, substituted aryl, alkyl, alkyl aryl, halogen substituted alkyl aryl, cyclic glycerols and substituted cyclic glycerols, COCF₃, C(O)-alcohol, —(CH₂)_m—(C(CH₃)₂)_p—(CH₂)_n-T₂-T₃, —(CH₂)_m—(CH(CH₃))_q—(CH₂)_n-T₂-T₃ (where m and n are each independently selected from 0 to 6 integer, p and q are each independently 0 or 1, T₂ is optionally present and comprises aryl, a cyclic ring, a bicyclic ring, a tricyclic ring, a heterocyclic ring, a heterobicyclic ring, a heterotricyclic ring, a heteroaromatic ring, 1- or 2-glycerol, 1- or 2-cyclic glycerol, alkyl, alkenyl, alkynyl, T₃ comprises H, OH, SH, halogen, C(halogen)₃, CH(halogen)₂, O-alkyl, N₃, CN, NCS, NH₂, alkylamino, dialkylamino or a substituent group as defined later).

In variations of the invention the following provisos apply.

When Y is —C(O)—N(H)— and X is the tail remnant of arachidonyl acid, Z excludes 4-hydroxyphenyl.

When Y is —O—C(O)—NH— and X is the tail remnant of arachidonyl acid, Z excludes ethyl, iso-propyl and propyl.

When Y is —NH—C(O)—NH— and X is the tail remnant of arachidonyl acid, Z excludes methyl, iso-propyl, propyl, iso-butyl, CH₂CH₂F, CH₂CH₂OH, and CH2CH2OCH3.

When Y is —NH—C(O)—O— and X is the tail remnant of arachidonyl acid, Z excludes ethyl, iso-propyl and CH₂CH₂F.

When Y is —NH—C(S)—NH— and X is the tail remnant of arachidonyl acid, Z excludes 4-methyl-2-methoxy-phenol, and 4-methyl-2-chloro-phenol.

Some of the inventive analogs described herein, and physiologically acceptable salts thereof, have high potential when administered in therapeutically effective amounts for providing a physiological effect useful to treat pain; peripheral pain; glaucoma; epilepsy; nausea such as associated with cancer chemotherapy; AIDS Wasting Syndrome; cancer; neurodegeneration; neurodegenerative diseases including Multiple Sclerosis, Parkinson's Disease, Huntington's Chorea and Alzheimer's Disease; to enhance appetite; to reduce fertility; to prevent or reduce diseases associated with motor function such as Tourette's syndrome; to provide neuro-protection; to produce peripheral vasodilation and to suppress memory. Thus, another aspect of the invention is the administration of a therapeutically effective amount of an inventive compound, or a physiologically acceptable salt thereof, to an individual or animal to provide a physiological effect.

Some of the novel inhibitors of the present invention, when tested in vitro, inhibit accumulation of anandamide in rat cortical neurons and astrocytes and enhance various effects of anandamide administration both in vitro and in vivo. The vasodepressor responses are significantly potentiated and prolonged by the transport inhibitors. Thus, the inhibitors are believed to be effective drugs for the treatment of cardiovascular diseases and blood pressure disorders.

Beneficially, the inventive compounds would typically not be expected to have the undesirable membrane-related side-effects associated with cannabinoids. In addition, the compounds disclosed herein may be immunosuppressive and can therefore be used to prevent organ rejection in an individual undergoing an organ transplant. The compounds could also be used to treat psychomotor disorders and peripheral hypertension. In all of the above conditions, inhibition of the anandamide transporter and subsequent decrease in anandamide hydrolysis may bring about a more favorable response and fewer side effects than direct activation of CB1 and CB2 receptors by agonist drugs.

The novel inhibitors of anandamide transport disclosed herein also have research uses. For example, they can be used to maintain the level of anandamide in vivo to study the effect of anandamide on individuals and animals. The anandamide transport inhibitors disclosed herein can also be used as an aid in drug design, for example as a control in assays for testing other compounds for their ability to inhibit anandamide transport and to determine the structural and activity requirements of such inhibitors. These results, together with data from initial experiments on the selectivity of radioactively labeled [³H]anandamide uptake by rat brain astrocytes, suggest that the interactions of anandamide with its putative transporter protein are governed by strict structural requirements. These results delineate the broad molecular requisites for this process, thus providing a basis for the design of more potent and selective inhibitors with potential applications to medicine.

Anandamide uptake in neurons and astrocytes has been found to be mediated by a high-affinity, Na⁺-independent transporter that is selectively inhibited by the inhibitors of the present invention. The structural determinants governing recognition and translocation of substrates by the anandamide transporter have been determined. The secondary amido group interacts favorably with the transporter, but may be replaced with other radicals, suggesting that it may serve as hydrogen acceptor. Putative endogenous cannabinoid esters also serve as a substrate for the transporter. Substrate recognition and translocation require the presence of at least one cis double bond situated at the middle region of the fatty acid hydrocarbon chain or a biphenyl group with an aliphatic chain, indicating a preference for ligands whose hydrophobic tail can adopt a bent U-shaped or hair-pin configuration. Uptake experiments with radioactively labeled substrates favor two or more and preferably four cis nonconjugated double bonds for optimal translocation across the cell membrane, suggesting that substrates are transported in a folded hairpin conformation.

The inventive compounds include any and all possible isomers, stereoisomers and enantiomers. In general, the compositions of the invention may be alternately formulated to comprise, consist of, or consist essentially of, any appropriate components herein disclosed. The compositions of the invention may additionally, or alternatively, be formulated so as to be devoid, or substantially free, of any components, materials, ingredients, adjuvants or species used in the prior art compositions or that are otherwise not necessary to the achievement of the function and/or objectives of the present invention.

A better understanding of the invention will be obtained from the following detailed description of the presently preferred, albeit illustrative, embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the translocation of substrate inhibitors of the present invention at different concentration levels.

FIG. 2 is a graph similar to FIG. 1 for two different substrate inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is directed to the discovery of a putative anandamide transporter system which has been characterized biochemically and pharmacologically and which can be used as a target for the discovery of novel medications. These would include all compounds that can inhibit the function of this transporter. The invention further includes the pharmacological formula containing an effective amount of the inhibitor while another embodiment is directed to a method of inhibiting anandamide transport in an individual or animal by administering a therapeutically effective amount of the inhibitor and/or physiologically acceptable salts thereof. The inhibition results in increased levels of anandamide in the individual or animal, thereby causing prolonged stimulation of cannabinoid receptors in the individual or animal, e.g., the CB1 receptor in the brain and the CB2 receptor in the spleen. Thus, the present invention involves not only the inhibitor itself but also a method of reducing anandamide transporter activity in an individual or animal. It is to be understood that the present invention may also be used to reduce the activity of transporters not yet discovered for which anandamide and/or a cannabinoid act as an agonist.

In some embodiments the anandamide transport inhibitors of the present invention include amide, reverse amide or carbonyl amine, urea, carbamate and ester analogs of anandamide having the three pharmacophores of the Structural Formula I wherein the tail portion X is a fatty acid hydrophobic carbon chain having one or more nonconjugated cis double bonds in the middle portion of the aliphatic hydrocarbon chain or a biphenyl group having an alkyl or branched alkyl distal moiety of about 1 to about 10 carbon atoms. The biphenyl group is substituted with 1-6 substituents including OH, CH₃, halogen, SCH₃, NH₂, NHCOR, SO₂NHR, NO₂, COCF₃, or a substituent group as defined later. The fatty acid chain may contain four to thirty carbon atoms but preferably the chain length is about 10 to 28 carbon atoms and more preferably contains from about 17 to about 22 carbon atoms. The aliphatic hydrocarbon chain may terminate with an aryl or substituted aryl group. By contrast, analogs with fully saturated chains or with a trans or terminal double bond fail to compete successfully with [³H]anandamide for transport and thus are ineffective as inhibitors. The central pharmacophore Y is selected from the group set forth hereinbefore. However, compounds containing a free carboxylic acid, carboxyethyl and carboxymethyl groups, or a primary alcohol are inactive. The head portion Z is selected from the group set forth hereinbefore.

Exploration of the Y and Z pharmacophores shows that compounds containing primary, secondary and tertiary amido groups as well as hydroxymethyl ester or glycerol ester moieties are capable of competing with [³H]anandamide, but exhibit a wide range of potencies. Structural variations of the head group Z leads to analogs with diverse selectivities for the anandamide transporter. Thus substitution of the terminal hydroxyl with a hydrogen causes a substantial decrease in potency, whereas replacement of the entire hydroxyalkyl moiety with hydrogen yields compounds that are as potent as anandamide. Introduction of a methyl group alpha to the amido nitrogen also leads to active compounds. Some chiral molecules display considerable enantioselective inhibition of [³H]anandamide transport. The (S) enantiomer is approximately four times more potent than its (R) isomer.

One striking structure-activity correlation was observed with analogs having hydroxyphenyl radicals at the head group. Use of the hydroxyphenyl group leads to relatively potent uptake inhibitors, with the 4-hydroxyphenyl analog being distinctly the most successful.

Unless otherwise specifically defined, “acyl” refers to the general formula —C(O)alkyl.

Unless otherwise specifically defined, “acyloxy” refers to the general formula —O-acyl.

Unless otherwise specifically defined, “alcohol” refers to the general formula alkyl-OH and includes primary, secondary and tertiary variations.

Unless otherwise specifically defined, “alkyl” or “lower alkyl” refers to a linear, branched or cyclic alkyl group having from 1 to about 16 carbon atoms, and advantageously about 1 to about 6 carbon atoms, including, for example, methyl, ethyl, propyl, butyl, hexyl, octyl, isopropyl, isobutyl, tert-butyl, cyclopropyl, cyclohexyl, cyclooctyl, vinyl and allyl. The alkyl group can be saturated or unsaturated. Unless otherwise specifically limited, an alkyl group can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position. Unless otherwise specifically limited, a cyclic alkyl group includes monocyclic, bicyclic, tricyclic and polycyclic rings, for example norbornyl, adamantyl and related terpenes.

Unless otherwise specifically defined an aliphatic hydrocarbon includes, unless otherwise stated, one or more polyalkylene groups connected by one or more cis-alkenyl linkages such that the total number of methylene carbon atoms is within the ranges set forth herein. The structure of preferred aliphatic hydrocarbons comprising the tail portion X have the formula IIC(R₃)₃—(C(R₂R₁))_a-(cis-CH═CHC(R₁)₂)_b—(C(R₀R₂))_c—  (II)wherein each of R₀ to R₃ is independently selected from the groups consisting of hydrogen and lower alkyl groups, however the chain's terminal C(R₃)₃ may include phenyl and biphenyl groups that are unsubstituted or substituted with a member selected from the group consisting of hydroxyl, halogen, —NO₂, —NH₂, —SCH₃, —CH₃, —COCF₃, and —OCH₃ or a substituent group as defined later, and a and c are each independently selected integers 0 to 10 and b is an integer from 1 to 6. Specific examples include CH₃—(CH₂)₄-(cis-CH═CHCH₂—)₄—(CH₂)₂—, CH₃—(CH₂)₄-(cis-CH═CHCH₂)₃—(CH₂)₅, —CH₃—(CH₂)₆-(cis-CH═CHCH₂)₂—(CH₂)₆—, CH₃—(CH₂)₆-(cis-CH═CHCH₂)₂—(CH₂)₅—CH₃—(CH₂)₇-cis CH═CH—(CH₂)₉, CH₃—(CH₂)₇-cis-CH═CH—(CH₂)₇— and CH₃—(CH₂)₄—(CH═CHCH₂)₄—CH₂—C(CH₃)₂—.

The aliphatic hydrocarbon also includes long chain hydrocarbons possessing 8 to 22 carbons and having zero to six of double bonds and optionally substituted in any possible position with lower-alkyl, di-lower-alkyl, cycloalkyl or heterocycloalkyl groups; of which the multiple double bonds can be conjugated or unconjugated. The aliphatic hydrocarbon backbone chain may be interrupted with, or otherwise include, methylene, (CH₃)₂C, O, NH, N-alkyl, cyclic alkyl groups, heterocyclic groups and aryl groups.

Unless otherwise specifically defined, “alkoxy” refers to the general formula —O-alkyl.

Unless otherwise specifically defined, “alkylmercapto” refers to the general formula —S-alkyl.

Unless otherwise specifically defined, “alkylamino” refers to the general formula —(NH)-alkyl.

Unless otherwise specifically defined, “di-alkylamino” refers to the general formula —N(alkyl)₂. Unless otherwise specifically limited di-alkylamino includes cyclic amine compounds such as piperidine and morpholine.

Unless otherwise specifically defined, an aromatic ring is an unsaturated ring structure having about 5 to about 7 ring members and including only carbon as ring atoms. The aromatic ring structure can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position.

Unless otherwise specifically defined, “aryl” refers to an aromatic ring system that includes only carbon as ring atoms, for example phenyl, biphenyl, 1-naphthyl or 2-naphthyl. The aryl group can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position.

Unless otherwise specifically defined, “aroyl” refers to the general formula —C(═O)-aryl.

Unless otherwise specifically defined, a bicyclic ring structure comprises 2 fused or bridged rings that include only carbon as ring atoms. The bicyclic ring structure can be saturated or unsaturated. The bicyclic ring structure can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position. The individual rings may or may not be of the same type. Examples of bicyclic ring structures include, Dimethyl-bicyclo[3,1,1]heptane, bicyclo[2,2,1]heptadiene, decahydro-naphthalene and bicyclooctane.

Unless otherwise specifically defined, a carbocyclic ring is a non-aromatic ring structure, saturated or unsaturated, having about 3 to about 8 ring members that includes only carbon as ring atoms, for example, cyclohexadiene or cyclohexane. The carbocyclic ring can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position.

Unless otherwise specifically defined, a cyclic glycerol includes members wherein 2 of the 3 hydroxy groups are tied to form a 5 to 8 member ring and the third hydroxyl group is substituted, for example in the form of an ester or an ether. The cyclic glycerol ring will typically, but not always, be saturated. The cyclic glycerol may be substituted in any possible position by one or more substituent groups. Examples of cyclic glycerols include

wherein n is an integer selected from 1 to 3.

Unless otherwise specifically defined, “halogen” refers to an atom selected from fluorine, chlorine, bromine and iodine.

Unless otherwise specifically defined, a heteroaromatic ring is an unsaturated ring structure having about 5 to about 8 ring members that has carbon atoms and one or more heteroatoms, including oxygen, nitrogen and/or sulfur, as ring atoms, for example, pyridine, furan, quinoline, and their derivatives. The heteroaromatic ring can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position.

Unless otherwise specifically defined, a heterobicyclic ring structure comprises 2 fused or bridged rings that include carbon and one or more heteroatoms, including oxygen, nitrogen and/or sulfur, as ring atoms. The heterobicyclic ring structure is saturated or unsaturated. The heterobicyclic ring can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position. The individual rings may or may not be of the same type. Examples of heterobicyclic ring structures include tropane, quinuclidine and tetrahydro-benzofuran.

Unless otherwise specifically defined, a heterocyclic ring is a saturated or unsaturated ring structure having about 3 to about 8 ring members that has carbon atoms and one or more heteroatoms, including oxygen, nitrogen and/or sulfur, as ring atoms, for example, piperidine, morpholine, piperazine, pyrrolidine, thiomorpholine, tetrahydropyridine, and their derivatives. The heterocyclic ring can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position.

Unless otherwise specifically defined, a heterotricyclic ring structure comprises 3 rings that may be fused, bridged or both fused and bridged, and that include carbon and one or more heteroatoms, including oxygen, nitrogen and/or sulfur, as ring atoms. The heterotricyclic ring structure can be saturated or unsaturated. The heterotricyclic ring structure can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position. The individual rings may or may not be of the same type. Examples of heterotricyclic ring structures include 2, 4, 10-trioxaadamantane, tetradecahydro-phenanthroline.

Unless otherwise specifically defined, a heteropolycyclic ring structure comprises more than 3 rings that may be fused, bridged or both fused and that include carbon and one or more heteroatoms, including oxygen, nitrogen and/or sulfur, as ring atoms. The heteropolycyclic ring structure can be saturated or unsaturated. The heteropolycyclic ring structure can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position. The individual rings may or may not be of the same type. Examples of heteropolycyclic ring structures include azaadamantine, 5-norbornene-2,3-dicarboximide.

Unless otherwise specifically defined, the term “phenacyl” refers to the general formula -phenyl-acyl.

Unless otherwise specifically defined, a polycyclic ring structure comprises more than 3 rings that may be fused, bridged or both fused and bridged, and that includes carbon as ring atoms. The polycyclic ring structure can be saturated or unsaturated.

The polycyclic ring structure can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position. The individual rings may or may not be of the same type. Examples of polycyclic ring structures include adamantine, bicyclooctane, norbornane and bicyclononanes.

Unless otherwise specifically defined, a spirocycle refers to a ring system wherein a single atom is the only common member of two rings. A spirocycle can comprise a saturated carbocyclic ring comprising about 3 to about 8 ring members, a heterocyclic ring comprising about 3 to about 8 ring atoms wherein up to about 3 ring atoms may be N, S, or O or a combination thereof.

Unless otherwise specifically defined, a tricyclic ring structure comprises 3 rings that may be fused, bridged or both fused and bridged, and that includes carbon as ring atoms. The tricyclic ring structure can be saturated or unsaturated. The tricyclic ring structure can be unsubstituted, singly substituted or, if possible, multiply substituted, with substituent groups in any possible position and may be substituted or unsubstituted. The individual rings may or may not be of the same type. Examples of tricyclic ring structures include fluorene and anthracene.

Unless otherwise specifically limited the term substituted means substituted by a below-described substituent group in any possible position. Substituent groups for the above moieties useful in the invention are those groups that do not significantly diminish the biological activity of the inventive compound. Unless otherwise specifically limited a substituent group or a substituent group that does not significantly diminish the biological activity of the inventive compound includes, for example, H, halogen, N₃, NCS, CN, NO₂, NX₁X₂, OX₃, C(X₃)₃, OAc, O-acyl, O-aroyl, NH-acyl, NH-aroyl, NHCOalkyl, CHO, C(halogen)₃, COOX₃, COCF₃, SO₃H, PO₃H₂, SO₂NX₁X₂, CONX₁X₂, alkyl, alcohol, alkoxy, alkylmercapto, alkylamino, di-alkylamino, sulfonamide or thioalkoxy wherein X₁ and X₂ each independently comprise H or alkyl, or X₁ and X₂ together comprise part of a heterocyclic ring having about 4 to about 7 ring members and optionally one additional heteroatom selected from O, N or S, or X₁ and X2 together comprise part of an imide ring having about 5 to about 6 members and X₃ comprises H, alkyl, loweralkylhydroxy, or alkyl-NX₁X₂. Unless otherwise specifically limited, a substituent group may be in any possible position.

Procedures for Synthesizing these Materials Are as Follows:

Arachidonyl alcohol: To a magnetically stirred solution of 0.5 ml (0.5 mmol) of LiAlH₄ in Et₂O, 100 mg (0.314 mmol) of arachidonic acid methyl ester in 2 mL of Et₂O was added dropwise at 0° C. The reaction mixture was stirred for 1 h and then quenched by addition of 1 mL of EtOAC. 2 mL of saturated NH₄Cl solution was added and the organic layer was separated, dried with MgSO₄, filtered and evaporated. Chromatography on silica gel (eluents: CH₂Cl₂/petroleum ether up to 70% CH₂Cl₂), evaporation, followed by Millipore filtration of a CH₂Cl₂ solution of the product gave 99.3 mg (0.292 mmol, 93% yield) of arachidonyl alcohol as a colorless oil: TLC (CHCl₃) R_f 0.28; ¹H NMR (200 MHz, CDCl₃) δ 5.37 (m, 8 H ), 3.61 (t, 2 H, J=6 Hz), 2.79 (m, 6H), 2.08 (m, 4 H), 1.66-1.17 (m, 8 H), 0.92 (t, 3 H, J=7 Hz).

Arachidonyl azide: To a magnetically stirred solution of 50 mg (0.17 mmol) of arachidonyl alcohol in 1 mL of pyridine 29.2 mg. (0.255 mmol) of mesyl chloride was added at 0° C. After stirring for 5 h, the reaction mixture was poured into 2 mL of iced water and extracted with Et₂O (2×4 mL). The ethereal layers were combined and washed with 1 N H₂SO₄, NaHCO₃, and evaporated in vacuo to dryness. The mesylate was not purified and it was directly converted to the corresponding azide: it was dissolved first in 2 ml DMF and then 4 ml of solution of 6.5 mg. (0.85 mmol) NaN₃ in DMF was added at room temperature. The reaction mixture was heated at 90° C. for 24 h. After the reaction mixture was cooled down to room temperature the inorganic material was filtered off and the filtrated was poured into 1 mL of iced H₂O and then extracted with Et₂O (2×6 mL). The ethereal layers were combined, dried, filtered, and evaporated in vacuo to dryness. Silica gel chromatography (eluent: petroleum ether), evaporation, followed by Millipore filtration of a CH₂Cl₂ solution of the product gave 39 mg (0.12 mmol, 73% yield) of arachidonyl axide as a colorless oil: ¹H NMR (200 MHz, CDCl₃) δ 5.38 (m, 8 H), 3.27 (t, 2 H, J=6 Hz), 2.81 (m, 6H), 2.11-2.01 (m, 4), 162 m, 2 H), 1.48-1.25 (m, 6 H), 0.89 (t, 3 H, J=7 Hz).

Arachidonylamine: To a magnetically stirred solution of 132 mg (0.43 mmol) of arachidonyl azide in 3 mL of Et₂O, 4 mL of a 1.0 M LAH solution in THF (4.0 mmol) was added dropwise at room temperature. The reaction mixture was refluxed for 3 h and then it was cooled to ambient temperature. 210 mg (5 mmol) of NaF was added and the reaction was quenched with wet Et₂O. The white mixture was filtered and the solvent was evaporated to dryness. Silica gel chromatography (eluents: CH₂Cl₂/MeOP-up to 50% MeOH), evaporation of solvent, followed by Millipore filtration of a CH₂Cl₂ solution of the product then gave 78.9 mg (0.28 mmol, 64% yield) of arachidonyl-amine as a colorless oil. TLC (EtOAc/CH₂Cl₂ (20:80)) R_f 0.33; ¹H NMR (200 MHz, CDCl₃) δ 5.38 (m, 8 H), 2.82 (m, 6 H), 2.70 (t, 2 H, J=6.6 Hz), 2.08 (m, 4 H), 1.40 (m, 4 H), 1.26 (m, 6 H), 0.89 (t, 3 H, J=6.4 Hz).

Typical procedure for synthesis of AM 1177, AM 1172 and AM 1191 is as follow: To a suspension of arachidonyl amine or arachidonyl acid (1 mmol) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodimide hydrochloride (EDCl) (1.2 mmol) in 5 mL THF was added a solution of 1.1 mmol of corresponding acid, or alcohol (Stekar, J., et. al. Angew. Chem.; GE; 1995, 107: 195-197.) in 1 mL THF at room temperature followed by addition of a solution of dimethylaminopyridine (DMAP) (1.1 mmol) in 1 mL THF. The stirring was continued for another 6 h. The THF was removed in vacuo, and the residue was re-dissolved in Et₂O. The resulting solution was washed wash with water, 1N HCl and brine, respectively, dried over Na₂SO₄. The solvent was removed in vacuo, and residue was purified by column chromatograph on silica gel to afford pure AM 1177 AM 1172 and AM 1191 (0.6-0.8 mmol, 60-80% yield) as oil.

Arachidonylamine-3′-(hydroxy)-propionate: To a magnetically stirred solution of 48 mg (0.17 mmol) of arachidonyl-amine in 2 mL of CH₂Cl₂, 58 μl (0.17 mmol) of a 2.0 M solution of (CH₃)₃Al in of hexane were added at room temperature. The mixture was stirred for 20 min and then 12.24 mg (0.17 mmol) of β-propiolactone was added dropwise. The reaction mixture was refluxed for 6 h, quenched with 1N HCl and extracted with methyl chloride. The product was purified with silica gel column chromatography (eluents with CH₂Cl₂/EtOAc, up to 80% EtOAc. Evaporation of the solvent, followed by Millipore filtration of a CH₂Cl₂ solution of the product gave 51 mg (0.14 mmol, 83% yield) of arachidonyl-amine-3′-(hydroxy)-propionate as a colorless oil; TLC (EtOAc) R_f 0.26; ¹H NMR (200 MHz, CDCl₃) δ 5.35 (m, 8 H), 3.85 (q, 2 H, J=5.4 Hz), 3.25 (q, 2 H, J=5.4 Hz), 2.84 (m, 6 H), 2.66 (t, 2 H, J=6.8 Hz), 2.05 (m, 4 H), 1.57 (m, 2 H), 1.35 (m, 6 H) 0.89 (t, 3, H, J=6.5 Hz); Anal. C, H, N.

Arachidonyl-amine-trifluoroacetate: To a magnetically stirred solution of 69 mg (0.6 mmol) of trifluoroacetic acid, in 2 ml of dry methylene chloride, at 0° C., 0.046 ml (0.6 mmol) of dry DMF was added and then 0.3 ml (0.6 mmol) of 2.0 M solution of oxalyl chloride in methylene chloride, dropwise. The reaction mixture was stirred for 20 min and then a solution of 172 mg (0.6 mmol) of arachidonyl amine in 2 ml of methylene chloride was added and the reaction was stirred for 2 hrs at ambient temperature. The product was purified with silica gel column chromatography (eluents: petroleum ether/ethyl acetate, up to 50% ethyl acetate). Evaporation of the solvent, followed by Millipore filtration of a CH₂Cl₂ solution of the product gave 153 mg (0.4 mmol, 67% yield) of arachidonyl-amine-trifluoroacetate as a colorless oil.

Preparation of Compound 1-12:

• • a. CO(imidazole)₂, amines, THF, 70-90%; b. diphosgene, Et₃N, THF, amines, 85-90%; c. Zn(N₃)₂·2Py, DIAD, PPh₃, toluene, 0° C.-r.t., 85%; d. H₄LiAl, Et₂O, 84%Compound 1-8 and 12

Method A: To a suspension of CO(imidazole)₂ (1.5 mmol) in 3 mL of anhydrous THF was added a solution of arachidonyl alcohol or arachidonyl amine (1 mmol) in 1 mL THF at 0° C. Stirring was continued for another 2 h at ambient temperature. Then, the reaction mixture was cooled to 0° C. again, and the respective amine (2 mmol) was added and stirred for overnight. THF was removed in vacuo, and the residue was diluted with Et₂O. The heterogeneous mixture was filtered with a pad of Celite, and filtrate was washed with 2N HCl, saturated NaHCO₃ and brine, respectively, dried over Na₂SO₄. The solvent was removed in vacuo, and residue was chromatographed on silica gel to afford pure compounds 1-8 and 12 (0.7-0.9 mmol, 70-90% yield) as oil.

Compound 9-11:

Method B: To a suspension of arachidonyl alcohol (1 mmol) and activated carbon (0.1 mmol) in 3 mL of anhydrous THF was added diphosgene (0.75 mmol) at 0° C. Stirring was continued for another 2 h at 0° C. Then, a solution of respective amine (2 mmol) Et₃N (6 mmol) in 1 mL of THF was added to above reaction mixture and stirring was continued for overnight. It was diluted with Et₂O, and heterogeneous mixture was filtered with a pad of Celite. The filtrate was washed with water and brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was chromatographed to give pure compounds 9-11 (0.82-0.90 mmol, 82-90% yield) as oil.

Preparation of Compound 13-15:

Compound 13-15:

Preparation of 13-15 can be fulfilled from corresponding commercially available alcohols following the procedure of preparation of compounds 1-8.

Preparation of Compound 16:

Compound M1:

To a solution of (S)-1,2-propandiol (5 mmol) in 5 mL of anhydrous CH₂Cl₂ were added 2-methyl-1-butene (5.5 mmol) and BF₃ etherate (0.5 mmol) at room temperature. The stirring was continued for another 24 h. The solvent was removed by vacuum, and the residue was purified by chromatograph on silica gel to afford intermediate (4 mmol, 80% yield) as oil. Then, to a suspension of this intermediate and imidazole (6 mmol) in 6 mL of anhydrous THF was added 4.8 mL of TBDMSCl (1M in THF) at 0° C. The solvent was removed by vacuum, and the residue was re-dissolved in Et₂O. The heterogeneous mixture was filtered over a pad of Celite. The filtrate was washed with 1N HCl, water and brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was chromatographed to give pure M1 (0.34 mmol, 85% yield) as oil.

Compound M2:

To a solution of M1 (3 mmol) in 3 mL of anhydrous CH₂Cl₂ was added TBDMSOTf (0.3 mmol) at room temperature, and the resulting solution was stirred for 24 hours. Saturated NaHCO₃ was then added and the solution extracted with AcOEt. Combined organic layers were dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by chromatograph on silica gel to afford M2 (1.8 mmol, 60% yield) as oil.

Compound 16:

The 16 can be made in two steps. The first one is coupling, which can be fulfilled by following the similar procures of preparation of 1-8 in 83% yield. The second step is deprotection by TBAF. To a solution of coupling product (1 mmol) in 2 mL of THF was added 1.2 mL of TBAF (1M in THF) at room temperature. The stirring was continued for another 1.5 h. Then, the THF was removed in vacuum, and the resulting mixture was dissolved in Et₂O. The resulting solution was washed by water and brine, dried over Na₂SO₄ and concentrated in vacuum. The crude product was purified by chromatograph on silica gel to afford 16 (0.8 mmol, 80% yield) as oil.

Preparation of Compound 17-25

Typical procedure for synthesis of compounds 17-25 is as following:

To a magnetically stirred solution of arachidonic acid (1 mmol) and DMF (0.1 mmol) in 5 mL of methylene chloride in a 25-mL three-neck flask at 0° C. was added oxalyl chloride (2 mmol) in a dropwise manner. The reaction mixture was stirred further at 0° C. for an additional hour. Then a solution of corresponding amine (10 mmol) in 5 mL of THF was added. Stirring was continued further for 15 min. The reaction mixture was diluted with 30 mL of CH₂Cl₂ and washed successively with 30 mL of 10% of aqueous HCl, 30 mL of 10% aqueous NaOH, and water. The organic phase was separated, dried (MgSO₄), and evaporated in vacuo to give a yellowish oily residue. Chromatography on silica gel afforded final compounds 17-25 (0.6-0.9 mmol, 60-90% yield) as oil.

The Transporter.

In order to properly evaluate the effectiveness of inhibitors of anandamide transport, it was necessary to establish the identity and character of the carrier-mediated transporter. The accumulation of radioactively labeled exogenous [³H]anandamide by neurons and astrocytes fulfills several criteria of a carrier-mediated transport. It is a rapid process that reaches 50% of its maximum within about four minutes. Furthermore, [³H]anandamide accumulation is temperature dependent and saturable. Kinetic analyses reveals that accumulation in neurons can be represented by two components of differing affinities (lower affinity: Michaelis constant, K_m=1.2 μM, maximum accumulation rate, V_max=90.9 pmol/min per milligram of protein; higher affinity: K_m=0.032 μM, V_max=5.9 pmol/min per milligram of protein). The higher affinity component may reflect a binding site, however, as it is displaced by the cannabinoid receptor antagonist, SR-141716-A (100 nM). In astrocytes, [³H]anandamide accumulation is represented by a single high-affinity component (K_m=0.32 μM, V_max=171 pmol/min per milligram of protein). Such apparent K_m values are similar to those of known neurotransmitter uptake systems and are suggestive therefore of high-affinity carrier-mediated transport.

To characterize further this putative anandamide transporter, cortical astrocytes in culture were employed. As expected from a selective process, the temperature-sensitive component of [³H]anandamide accumulation was prevented by nonradioactive anandamide, but not by palmitoyl ethanolamide, arachidonate, prostanoids, or leukotrienes. Replacement of extracellular sodium ion with N-dimethylglocosamine or choline had no effect suggesting that accumulation is mediated by a Na⁺-independent mechanism which has been observed for other lipids. Moreover, inhibition of fatty acid amide hydrolase (FAAH) activity indicates that an anandamide hydrolysis does not provide the driving force for anandamide transport into astrocytes within the time frame of the experiment. Finally, the cannabinoid receptor agonist WIN-55212-2 (1 μM) and antagonist SR-141716-A (10 μM) also had no effect, suggesting that receptor internalization was not involved.

A primary criterion for defining carrier-mediated transport is pharmacological inhibition. To identify inhibitors of anandamide transport, examination was made of various components that prevent the cellular uptake of other lipids such as fatty acids, phospholipids or bromcresol green. Among the compounds tested, only bromcresol green interfered with inanimate transport, albeit with limited potency and partial efficacy, bromcresol green inhibited [³H]anandamide accumulation with an IC₅₀ (concentration needed to produce half-maximal inhibition) of 4 μM in neurons and 12 μM in astrocytes and acted noncompetitively. Moreover, bromcresol green had no significant effect on the binding of [³H]WIN-55212-2 to rat cerebral membranes, on FAAH activity in brain microsomes and on uptake of [³H]arachidonate or [³H]ethanolamine in astrocytes.

The bromcresol green, which blocks PGE₂ transport, raised the question of whether anandamide accumulation occurred by means of a PGE₂ carrier. That this is not the case was shown by the lack of [³H]PGE₂ accumulation in neurons or astrocytes and by the inability of PGE₂ to interfere with [³H]anandamide accumulation. Previous results indicating that expression of PGE₂ transporter mRNA in brain tissue is not detectable further support this conclusion.

[³H] Anandamide Competition Assay Using a High Throughput Method.

Human CCF-STTG1 astrocytoma cells (American Type Culture Collection) were grown in RPMI 1640 culture medium containing 10% FBS and 1 mM glutamine. Cells were seeded at a density of 2×10⁵/well=6×10⁵/cm² and used at confluence (5 days post seeding). For standard competition assays, confluent cells grown in 96-well view plates were rinsed and preincubated for 10 min. at 37° C. in Hanks Balanced Salt Solution (HBSS) supplemented to contain 138 mM NaCl, 5 mM KCl, 1.26 mM MgSO4, 2.5 mM CaCl₂-2H₂O, 1 mM phosphates, 4 mM NaHCO₃, 10 mM glucose, 10 mM Hepes with 0.1% DMSO or 0.1% DMSO plus test compounds at their final concentrations (0.1-100 μM). Briefly, plates of cells were washed 3× with 100 ul with HBSS with 0.1% DMSO with a Multiwash Plus (Molecular Device) plate washer. Washed plates were placed into a plate warmer with an air:carbon dioxide mixture of 95:5.

A silanized 96 well plate was prepared as a mother plate for treating the cells. For each test compound a dilution sheet was generated to encompass a range of concentrations around a predicted IC₅₀ of 500 nM.

To the motherplate, 150 ul of a 2× dilution of test compound was added to two rows columns 1-12 or the 96 well mother plate. Add 150 ul of HBSS with 0.1% DMSO to each well of one of ROW A (label this row as pretreatment). To row B add 150 ul per well of [³H]anandamide 100 or 1000 nM and label this row as treatment. This results in a 1× concentration of test compounds and a 50 or 500 nM concentration final concentration of anandamide.

Take the mother plate and set the electrapipette to fill 225 ul and dispense 50 ul of the pretreatment to the appropriate wells. Next decant the 96 well plate to remove the 100 ul of wash buffer. Add 50 ul per well for an n=4 columnwise down for rows a-d for test compound number 1. Then add 50 ul of compound 2 per well for 4 columnwise transfers to rows e-h. Place the plates back into the plate warmer/incubator.

After the 10 minute preincubation period, decant the plates. With the mother plate, set the electrapipette to fill 225 ul and dispense 50 ul of the treatment to the appropriate wells. Place the plate back into the plate warmer for 4 minutes. Then decant the plate into the hot sink and immediately aspirate the incubation media using the Filtermate 196 Cell Harvester (Packard Instruments, Meriden, Conn.), followed by rinsing the cells 6× with ice-cold HBSS containing 0.1% fatty acid free bovine serum albumin (Sigma).

Reactions were stopped by removing the incubation media and rinsing the cells three times with 0.1 ml of ice-cold HBSS containing 0.1% fatty acid-free BSA (Sigma). A final wash of the plate in HBSS was performed to remove any traces of albumin for the following protein analysis.

Cells were then solubilized by the addition of 50 ul/well of 1.2N NaOH/0.1% Triton X-100 and shaken on a plate shaker for 10 minutes. Aliquots of 15 ul were removed for protein analysis using the Biorad DC protein kit. To the remaining cell extracts in the viewplates, 215 ul of Microscint-20 were added and radioactive material was measured by liquid scintillation counting. Preliminary analyses carried out by TLC demonstrated that >95% of this radioactive material was nonmetabolized [³H]anandamide, suggesting that our astrocytoma cell preparation contains no significant anandamide amidohydrolase activity.

Some of the inhibitors have been identified as competitive since they are recognized as substrates by the transporter and will undergo membrane translocation.

The IC₅₀ data in Table I and Table II provide the affinity data for ligand recognition by the anandamide transporter, but do not provide information on whether the ligands also may serve as substrates for the transporter. To investigate substrate translocation we used a representative set of radioactively labeled compounds. We tested four key analogs that compete with anandamide for uptake: [³H]N-(4-hydroxyphenyl)arachidonamide designated as AM404, and the materials designated AM1172, AM 1177 and AM1191 arachidonylglycerol. As shown in FIGS. 1 and 2, all of the analogs are transported as rapidly and effectively as [³H]anandamide at levels of 50 mM and 500 mM. These findings suggest that the anandamide transporter also may participate in the inactivation of 2-arachidonylglycerol, which was thought to be primarily mediated by enzymatic hydrolysis.

TABLE I
Structure IC50
AM1191    0.8
AM1177    5.0
AM1172    2.0

TABLE II
		IC50
		(AT inhib.)
Compound	Structure	μM)
1		7.1
2		6.4
3		1.5
4		0.4
5		21.9
6		15.5
7		15.5
8		33.0
9		6.6
10		8.6
11		16.1
12		1.78
13		5.92
14		7.21
15		4.50
16		3.29
17		1.7
18		2.8
19		1.5
20		44.2
21		92.3
22		14.1
23		11.2
24		26.0
25		24.1

Modifications of the hydrophobic fatty acid tail reveal unexpectedly distinct requirements for recognition and translocation of substrates by the anandamide transporter. Substrate recognition requires the presence of at least one cis double bond situated at the middle of the fatty acid chain, pointing to a preference for ligands in which the hydrophobic tail can fold in the middle and adopt a bent U-shaped conformation. Indeed, analogs with fully saturated chains or those incorporating trans double bonds do not interact significantly with the transporter. By contrast, substrate translocation requires a minimum of four cis nonconjugated double bonds, as ligands containing one, two, or three olefins are transported either very slowly or not at all. This finding suggests that for transmembrane transport to occur substrates must be capable of adopting a tightly folded conformation, one that is not energetically favorable for ligands containing an insufficient number of cis double bonds.

Molecular modeling studies of fatty acid ethanolamides differing in the degree of unsaturation of their hydrophobic carbon chains provides insight into these distinctive conformational requirements. Possible low-energy conformers of these molecules are significantly different. The presence of one or more nonconjugated cis double bonds in the middle of the chain or the use of a biphenyl group leads to the formation of a turn that brings in closer proximity the head and tail of the molecule. The shape of this turn is determined by the number and position of the cis double bonds. Conversely, the introduction of a central trans double bond yields a more extended chain conformation and hinders the ability of the molecule to undergo folding. Thus one of the low-energy conformers of anandamide displays a folded hairpin shape with the two halves of the molecule facing each other. The cis-triene analog may adopt an analogous conformation, though one that is wider than that of anandamide. The width of the turn increases considerably in the cis-dienes and the two monoalkenes due to the marked increase in distance between the head group and tail of the molecule. In the corresponding trans alkene analog, the distance between the head and tail is much greater. It is important to point out that, whereas anandamide like arachidonic acid may adopt either a closed-hairpin or a U-shaped conformation depending on the properties of the surrounding milieu, the hairpin conformation may be thermodynamically unfavorable to fatty acid ethanolamides containing only one or two double bonds.

A plausible interpretation of our results is that recognition and translocation of substrates by the anandamide transporter are governed by distinct conformational preferences. Although the initial recognition step may require that substrates assume a bent U-shaped conformation of variable width, the subsequent step of translocation across the cell membrane may impose a more tightly folded hairpin conformation.

A “therapeutically effective amount” of a compound, as used herein, is the quantity of a compound which, when administered to an individual or animal, results in a sufficiently high level of anandamide in the individual or animal to cause a discernable increase or decrease in a cellular activity affected or controlled by cannabinoid receptors. For example, anandamide can stimulate receptor-mediated signal transduction that leads to the inhibition of forskolin-stimulated adenylate cyclase (Vogel et al., J. Neurochem. 60:352 (1993)). Anandamide also causes partial inhibition of N-type calcium currents via a pertussis toxin-sensitive G protein pathway, independently of cAMP metabolism (Mackie et al., Mol. Pharmacol. 47:711 (1993)).

A “therapeutically effective amount” of an anandamide inhibitor can also be an amount which results in a sufficiently high level of anandamide in an individual or animal to cause a physiological effect resulting from stimulation of cannabinoid receptors. Physiological effects which result from cannabinoid receptor stimulation include analgesia, decreased nausea resulting from chemotherapy, sedation and increased appetite. Other physiological functions include relieving intraocular pressure in glaucoma patients and suppression of the immune system. Typically, a “therapeutically effective amount” of the compound ranges from about 10 mg/day to about 1,000 mg/day.

As used herein, an “individual” refers to a human. An “animal” refers to veterinary animals, such as dogs, cats, horses, and the like, and farm animals, such as cows, pigs, guinea pigs and the like.

The compounds of the present invention can be administered by a variety of known methods, including orally, rectally, or by parenteral routes (e.g., intramuscular, intravenous, subcutaneous, nasal or topical). The form in which the compounds are administered will be determined by the route of administration. Such forms include, but are not limited to, capsular and tablet formulations (for oral and rectal administration), liquid formulations (for oral, intravenous, intramuscular, subcutaneous, ocular, intranasal, inhalation based or transdermal administration and slow releasing microcarriers (for rectal, intramuscular or intravenous administration). The formulations can also contain a physiologically acceptable vehicle and optional adjuvants, flavorings, colorants and preservatives. Suitable physiologically acceptable vehicles may include saline, sterile water, Ringer's solution, and isotonic sodium chloride solutions. The specific dosage level of active ingredient will depend upon a number of factors, including, for example, biological activity of the particular preparation, age, body weight, sex and general health of the individual being treated.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the invention.

Claims

1. A compound having the structure

2. The compound of claim 1 having the structure of any of compounds 17 to 25:

3. A compound selected from the following structures and physiologically acceptable salts thereof:

4. A compound from claim 3 selected from the following structures and physiologically acceptable salts thereof:

5. A pharmaceutical composition prepared from a compound as disclosed in claim 1.

6. A pharmaceutical composition prepared from a compound as disclosed in claim 3 and physiological acceptable salts thereof.

7. The pharmaceutical composition of claim 6 selected from the following structures and physiologically acceptable salts thereof: