Patent Application: US-41353903-A

Abstract:
compounds of the formula : wherein r , r1 and r4 are defined in the specification , and pharmaceutically acceptable salts , esters and tautomers thereof , having activity at peripheral cannabinoid receptors , commonly designated the cb2 receptor class . the compounds are useful for therapy , especially in the treatment of pain , inflammation and autoimmune disease .

Description:
male icr mice ( 25 – 32 g ), obtained from harlan ( dublin , va . ), were housed in groups of five . all animals were kept in a temperature - controlled ( 20 – 22 ° c .) environment with a 12 - hour light - dark cycle ( lights on at 7 a . m .). separate mice were used for testing each drug dose in the in vivo behavioral procedures . brain tissue for binding studies was obtained from male sprague - dawley rats ( 150 – 200 g ) purchased from harlan laboratories ( dublin , va .). measurement of spontaneous activity in mice occurred in standard activity chambers interfaced with a digiscan animal activity monitor ( omnitech electronics , inc ., columbus , ohio ). a standard tail - flick apparatus and a digital thermometer ( fisher scientific , pittsburgh , pa .) were used to measure antinociception and rectal temperature , respectively resorcinols were synthesized in our labs ( organix , inc ., woburn , mass .) according to the procedure specified below and were suspended in a vehicle of absolute ethanol , emulphor - 620 ( rhone - poulenc , inc ., princeton , n . j . ), and saline in a ratio of 1 : 1 : 18 . drugs were administered to the mice intravenously ( i . v .) in the tail vein at a volume of 0 . 1 ml / 10 g . analogs o - 1376 and o - 1532 listed in table 1 were synthesized as previously described ( mahadevan et al ., 2000 incorporated herein by reference — see bibliography herein ). analog o - 1601 was synthesized from 1 - deoxy - 9 - carbomethoxy cannabinol dmh analog ( mahadevan et al ., 2000 ) by lithium / liquid ammonia reduction as described 1 for the preparation of o - 1376 . the compounds listed in tables 2 and 3 were prepared using a three step sequence ( fig1 ). the 2 - lithio derivative of 1 , 3 - dimethoxy - 5 -( 1 ′, 1 ′- dimethylheptyl ) resorcinol ( 1 ) was prepared using n - buli / hexane in thf . it was condensed with the appropriate ketone to give the tertiary alcohol 2 , which on treatment with trifluoroacetic acid / et 3 sih gave the dimethoxy precursors 3 . demethylation 2 with bbr 3 / ch 2 cl 2 gave the target compounds ( crocker et al ., 1999 incorporated herein by reference ). the general procedure is illustrated in fig2 and described below . to a solution of the resorcinol 1 , ( 5 mmol ) in 25 ml of dry thf was added a 2 . 5 m solution of n - buli in hexane ( 5 . 5 mmol ) at 0 ° c . with stirring / n 2 . after additional stirring for 1 h at 0 ° c ., added a solution of the ketone ( 7 . 5 mmol ) in 3 ml of dry thf all at once . the solution was stirred for 0 . 5 h at 0 ° c . and then at 23 ° c . for 18 h . the reaction was worked up by the addition of sat nh 4 cl solution and extracted with ether . after washing ( h 2 o ) and drying ( na 2 so 4 ) the solvent was evaporated to give the crude tertiary alcohol 2 , which was used as such in the subsequent reaction . a solution of the tertiary alcohol 2 ( 5 mmol ) in 10 ml of dry ch 2 cl 2 was treated with cf 3 cooh ( 27 . 5 mmol ) followed by et 3 sih ( 12 . 5 mmol ). the solution was stirred / n 2 for 1 h or more ( followed by tlc ) and then quenched by the addition of sat nahco 3 solution . the organic layer was separated and after washing ( h 2 o ) and drying gave the crude dimethoxy precursor 3 of the target compound . this material was used as such for the demethylation step . treatment of 3 , as a solution in dry ch 2 cl 2 at 0 ° c ., with 3 equivalents of 1 n bbr 3 solution in ch 2 cl 2 , using the standard procedure and work up 2 . gave the crude target compound , which was purified by chromatography , generally using hexane / ethyl acetate mixtures . in the case of o - 1662 ( table 2 ), the corresponding tertiary alcohol 2 on treatment with cf 3 cooh / et 3 sih gave the unsaturated compound ( dehydrated but not reduced ) which on catalytic reduction ( pto 2 / c / h 2 ) in acetic acid gave the desired dimethoxy precursor 3 . the final compound was purified by chromatography using 5 % et 3 nh 2 / etoac mixture . the unsaturated analog o - 1423 ( table 2 ) was prepared by treatment of the corresponding tertiary alcohol 2 with cf 3 cooh alone in ch 2 cl 2 followed by demethylation . in table 3 , compounds o - 1797 - a and o - 1798 - b were diastereomeric mixtures and showed as two distinct spots in tlc which were separated by column chromatography , whereas o - 1657 was a sample of the mixture of diastereomers o - 1797 - a and o - 1798 - b . the dimethoxy compounds listed in tables 4 and 5 were prepared ( fig2 ) from 1 and the appropriate ketones using buli , as in the preparation of 2 , and isolating and purifying the compounds by chromatography ( ethyl acetate / hexane mixtures ). deprotection of o - 2092 was carried out by treatment with 10 % hcl in a ether / thf ( 5 : 4 ) mixture for 0 . 5 h at 23 ° c . to give a mixture of o - 2115 ( major ) and the dehydrated compound o - 2114 ( minor ). sodium borohydride reduction of o - 2115 furnished a mixture of diastereomeric compounds which were separated by chromatography to give the target compounds o - 2116 - a and o - 2117 - b . similarly o - 1966 - a and o - 1967 - b were separated from a diastereomeric mixture by chromatography . epoxidation of o - 2114 followed by nabh 4 reduction gave the target compound o - 2122 . in the preparation of o - 2090 the corresponding diethoxy resorcinol derivative of 1 was used in place of 1 . all compounds showed appropriate 1 hnmrs ( jeol eclipse 300 mhz ) and were characterized on the basis of their 1 hnmrs , tlc , and elemental analyses . prior to testing in the behavioral procedures , mice were acclimated to the experimental setting ( ambient temperature 22 – 24 ° c .) overnight . pre - injection control values were determined for rectal temperature and tail - flick latency ( in sec ). five min after i . v . injection with drug or vehicle , mice were placed in individual activity chambers and spontaneous activity was measured for 10 min activity was measured as total number of interruptions of 16 photocell beams per chamber during the 10 - min test and expressed as % inhibition of activity of the vehicle group . tail - flick latency was measured at 20 min post - injection . maximum latency of 10 sec was used . antinociception was calculated as percent of maximum possible effect {% mpe =[( test − control latency )/( 10 - control )]× 100 }. control latencies typically ranged from 1 . 5 to 4 . 0 sec . at 30 min post - injection , rectal temperature was measured this value was expressed as the difference between control temperature ( before injection ) and temperatures following drug administration (° c .). different mice ( n = 5 – 6 per dose ) were tested for each dose of each compound . each mouse was tested in each of the 3 procedures . the methods used for tissue preparation and binding have been described previously ( compton et al ., 1993 ) and are similar to those described by devane et al . ( 1988 ). all assays , as described briefly below , were performed in triplicate , and the results represent the combined data from three to six individual experiments . following decapitation and rapid removal of the brain , whole brain was homogenized and centrifuged . the resulting pellet was termed p 1 . the supernatant was saved and combined with the two subsequent supernatants obtained from washing of the p 1 pellet . the combined supernatant fractions were centrifuged , resulting in the p 2 pellet . after further incubation and centrifuging , this pellet was resuspended in assay buffer to a protein concentration of approximately 2 mg / ml . the membrane preparation was quickly frozen in a bath solution of dry ice and 2 - methylbutane ( sigma chemical co ., st . louis , mo . ), then stored at − 80 ° c . for no more than 2 weeks . prior to performing a binding assay an aliquot of frozen membrane was rapidly thawed and protein values determined by the method of bradford ( 1976 ). binding was initiated by the addition of 150 μg of p 2 membrane to test tubes containing 1 nm of [ 3 h ] cp 55 , 940 ( 79 ci / mmol ) and a sufficient quantity of buffer to bring the total incubation volume to 1 ml . nonspecific binding was determined by the addition of 1 μm unlabeled cp 55 , 940 . following incubation at 30 ° c . for 1 hr , binding was terminated by addition of ice cold buffer and vacuum filtration through pretreated filters in a 12 - well sampling manifold ( millipore , bedford , mass .). after washing , filters were placed into plastic scintillation vials ( packard , downer grove , ill .) and shaken . the quantity of radioactivity present was determined by liquid scintillation spectrometry . membranes for cb 2 binding were obtained from cho cells . the transfected cell line was maintained in dublecco &# 39 ; s modified eagle medium ( gibco brl , grand island , n . y .) with 10 % fetal clone ii ( hyclone laboratories , inc ., logan , utah ) plus 0 . 3 to 0 . 5 mg / ml g418 ( to maintain selective pressure ) under 5 % co 2 at 37 % c . cells were harvested with 1 mm edta in phosphate - buffered saline and were centrifuged at 1000 × g for 5 min at 4 % c . the supernatant was saved and the p1 pellet was resuspended in centrifugation buffer . homogenization and centrifugation were repeated twice and the combined supernatant fractions were centrifuged at 40 , 000 × g for 30 min at 4 % c . the p2 pellet was resuspended in centrifugation buffer 2 ( tris hcl , 50 mm ; edta , 1 mm ; and mgcl 2 , 3 mm , ph 7 . 4 ) to a protein concentration of approximately 2 mg / ml . protein concentrations were determined by the method of bradford ( 1976 ) using bio - rad protein assay ( bio - rad , richmond , calif .) and bsa standards ( fatty acid free , sigma chemical co ., st . louis , mo .). the membrane preparation was divided into amounts convenient for binding assays and frozen rapidly in dry ice and stored at − 80 % c . binding was initiated by the addition of 50 μg of quickly thawed p2 membranes to test tubes containing [ 3 h ] cp - 55 , 940 ( final reaction concentration 0 . 5 nm ), an appropriate concentration of unlabeled cp - 55 , 940 or test drug , and sufficient quantity of assay buffer ( 50 mm tris - hcl , 1 mm edta , 3 mm mgcl 2 , 5 mg / ml bovine serum albumin , ph 7 . 4 ) to bring the total incubation volume to 0 . 5 ml . concentration of [ 3 h ] cp - 55 , 940 in saturation studies ranged from 50 to 10 , 000 pm . nonspecific binding was determined by the addition of 1 μm unlabeled cp - 55 , 940 . cp - 55 , 940 and all cannabinoid analogs were prepared by suspension in assay buffer from a 1 mg / ml ethanolic stock without evaporation of the ethanol ( final concentration of no more than 0 . 4 %). in competition studies , analog concentrations ranged from 0 . 1 nm to 10 μm . after incubation at 30 % c for 1 hr , binding was terminated by the addition of 2 ml of ice - cold wash buffer ( 50 mm tris - hcl and 1 mg / ml bsa ) and vacuum filtration through pre - treated filters in a 12 - well sampling manifold ( millipore , bedford , mass .). reaction vessels were washed once with 2 ml of ice - cold wash buffer . filters were placed into 7 - ml plastic scintillation vials ( rpi corp ., mount prospect , ill .) with 4 ml of budget - solve ( rpi corp .). after shaking for 30 min , the radioactivity present was determined by liquid scintillation spectrometry . three reaction vessels were used for each drug concentration in each assay . the results represent the combined data of three independent experiments . all assays were performed in siliconized test tubes , which were prepared by air drying ( 12 hr ) inverted borosilicate tubes after two rinses with a 0 . 1 % solution of aquasil ( pierce , rockford , ill .). the gf / c glass - fiber filters ( 2 . 4 cm , baxter , mcgaw park , ill .) were pre - treated in a 0 . 1 % solution of ph 7 . 4 polyethylenimine ( sigma chemical co .) for at least 6 hr . based on data obtained from numerous previous studies with cannabinoids , maximal cannabinoid effects in each procedure were estimated as follows : 90 % inhibition of spontaneous activity , 100 % mpe in the tail flick procedure , and − 6 ° c . change in rectal temperature . ed 50 &# 39 ; s were defined as the dose at which half maximal effect occurred . for drugs that produced one or more cannabinoid effect , ed 50 &# 39 ; s were calculated separately using least - squares linear regression on the linear part of the dose - effect curve for each measure in the mouse tetrad , plotted against log 10 transformation of the dose . for the purposes of potency comparison , potencies were expressed as μmol / kg . pearson product - moment correlation coefficients ( with associated significance tests ) were calculated between cb 1 binding affinity ( expressed as log k i ) and in vivo potency for each measure ( expressed as log ed 50 in μmol / kg ) for all active cannabinoid compounds that bound to the cb 1 receptor . in addition , multiple linear regression was used to calculate the overall degree of relationship between cb 1 binding affinity and potency in the mouse measures for all active cannabinoids . a correlation between cb 1 and cb 2 binding affinities was calculated for all compounds that had measurable k i &# 39 ; s for cb 1 and cb 2 binding ( k i & lt ; 10 , 000 nm ). ki values for cb 1 and cb 2 binding were obtained from scatchard displacement analysis as determined via ebda program of the kell software package ( biosoft , milltown , n . j .). the cb 1 and cb 2 binding affinities for substituted biphenyl analogs are shown in table 1 . these compounds contain a phenolic hydroxyl and a lipophilic side chain in the same orientation as in cannabinol . in addition , the pyran oxygen is absent and the analogs have substituents in the phenyl ring ( ring c ) of cannabinol . two of the analogs ( o - 1376 and o - 1601 ) have a dimethylheptyl side chain ; each possessed good cb 1 and cb 2 binding affinities and in vivo activity . o - 1601 , the more potent of the two active compounds , had a hydroxymethyl group in the phenyl ring . this substitution increased cb 1 affinity and in vivo potencies compared to o - 1376 , but did not affect affinity for cb 2 receptors . a similar effect is observed in the cannabinol series where the substitution of a hydroxymethyl group for a methyl at c - 9 in cannabinol increased binding affinity and potency ( mahadevan et al ., 2000 ) shortening the side chain of o - 1376 to dimethylbutyl ( o - 1532 ) severely decreased affinity for both receptors and resulted in loss of in vivo activity . table 2 presents binding and in vivo data for a series of 2 - cyclic ring substituted - 5 - dimethylheptyl resorcinols . manipulation of the size of the cyclic structure attached at position 2 of the resorcinol ring resulted in changes in binding affinities and potencies . substitution of a cyclopentane ring ( o - 1424 ) resulted in moderate affinity for the cb 1 receptor with excellent affinity for the cb 2 receptor . although this compound was active in all three in vivo assays , potency was relatively poor . in addition , potencies across the measures were not equal ; i . e ., potency for reducing spontaneous activity was approximately half that for producing antinociceptive and hypothermic effects . increasing ring size to a cyclohexane ( o - 1422 ), cycloheptane ( o - 1656 ), or adamantyl ( o - 1660 ) improved affinity 5 - to 14 - fold for both cannabinoid receptors and greatly increased potencies in vivo . substitution of a sulfur for a carbon in a cyclohexane ring ( o - 1425 ) decreased cb 1 affinity by 14 - fold and cb 2 affinity by 8 - fold ( compared to o - 1422 ) as well as reducing in vivo potencies . similarly , sulfur substitution in a cyclopentane ring ( o - 1661 ) also attenuated binding to both cannabinoid receptors . when a methylated nitrogen ( o - 1662 ) was inserted into the cyclohexane ring in the same position as the sulfur of o - 1425 , binding to cb 1 receptors did not occur . in addition , cb 2 binding was drastically decreased and the compound was not fully active in vivo . in contrast , placing a double bond in the cyclohexane ring ( o - 1423 ) decreased affinities and potencies , but the compound remained active . however , moving the lipophilic side chain of o - 1422 from c - 5 to c4 and replacing the dmh with a n - hexyl chain ( o - 2010 ) produced a 865 - fold decrease in cb 1 affinity and a loss of activity in vivo . table 3 shows results of tests with cyclohexane substituted resorcinols in which the position of the substituent at the cyclohexane ring attached to the core resorcinol was varied . all compounds were diastereomeric mixtures . all of these analogs had good ( k i = 2 nm ) to moderate ( k i = 144 nm ) affinity for cb 1 receptors and were cb 2 - selective ( k i range = 0 . 3 – 13 nm ). methylation at the 2 position of the cyclohexane ring ( o - 1658 ) did not dramatically alter affinity for either cannabinoid receptor or in vivo potencies compared to the corresponding cannabinoid with a non - methylated cyclohexane ( o - 1422 in table 2 ). moving the methyl to position 4 of the cyclohexane ring ( o - 1659 ) decreased affinity for both cannabinoid receptors by about 5 - fold and produced an even greater decrease ( 11 - to 24 - fold ) in potencies in vivo . substituting a phenyl group for the methyl at this same position ( o - 1663 ) resulted in 2 - to 3 - fold decreases in cb 2 and cb 1 affinities , respectively , and a loss of activity in vivo . in the next five analogs shown in table 3 , the methyl was attached at position 3 of the cyclohexane ring . o - 1657 exhibited cb 1 and cb 2 affinities that were similar to those of o - 1658 ; however , the profiles of in vivo potencies differed . whereas the two analogs showed approximately equal potencies in suppressing spontaneous activity , o - 1658 was twice as potent in producing antinociception and three times as potent in reducing body temperature . by careful chromatography , compound o - 1657 was separated into two distinct entities which were designated o - 1797 - a and o - 1798 - b . these analogs were still mixtures affinities of o - 1797 - a and o - 1798 - b were 2 - 3 times greater than those of o - 1657 . while potencies of these isomers for suppression of locomotor activity and hypothermia were not notably different from those of o - 1657 , antinociceptive potencies were reduced by about half . the 3r isomer of this series ( o - 1826 ) showed decreased affinity for cb 1 receptors compared to o - 1657 ; however , affinity for cb 2 receptors was identical for both compounds . not surprisingly , given its decreased cb 1 affinity , o - 1826 was less potent than o - 1657 in vivo . substitution of a dimethylbutyl for the dmh side chain at c5 of the resorcinol component ( o - 1890 ) decreased affinities for both cannabinoid receptors . this compound was active in vivo , although potency was notably low for all measures in contrast , addition of a gem - dimethyl group at the 3 position of the cyclohexane ring with retention of the dmh side chain of the resorcinol component ( o - 1871 ) resulted in the best cb 1 and cb 2 affinities of this series . in vivo potencies , however , were lower than expected for this compound , given its higher cb 1 binding affinity . in order to develop cb 2 selective ligands , we examined cyclic ring substituted dimethoxy resorcinols . the cb 1 and cb 2 binding affinities of these analogs are shown in tables 4 and 5 . although most of the compounds shown in tables 4 and 5 possessed a dimethylheptyl side chain , all had poor cb 1 affinity ; hence , they were not tested in vivo . the bicyclic structure of o - 1999 ( table 4 ) was almost identical to that of o - 1657 ( table 3 ), an analog with good cb 1 and cb 2 affinities and potent in vivo effects . both compounds had a dimethylheptyl side chain attached to the 5 position of a resorcinol core that was attached at position 2 to a cyclohexane ring . each compound had a methyl group at the 3 position of the cyclohexane ring . the major structural difference between the two compounds was that o - 1999 was a dimethoxy derivative of the resorcinol o - 1657 . this seemingly minor structural change from a phenol to a methoxy derivative resulted in complete loss of affinity for cb 1 receptors and an almost 600 - fold reduction in affinity for cb 2 receptors . similarly , the other analogs that were dimethoxy derivatives of the corresponding resorcinols had poor affinity for cb 1 receptors ( k i ranged from 1716 to & gt ; 10 , 000 ), regardless of the cyclic ring substitution at position 2 . in contrast , cb 2 binding affinities for some of these analogs remained high , as described in more detail below . table 4 presents binding data for 2 - cyclic ring substituted dimethoxy - resorcinol - dmh analogs that contain at least one oxygen inserted into or attached to the non - resorcinol cyclohexane ring . compared to o - 1999 which did not contain an oxygen in the cyclohexane ring , conversion of the cyclohexane ring to a pyran ring ( o - 1964 ) decreased cb 2 affinity almost 2 - fold without effect on cb 1 binding . further addition of a double bond at position 3 of the pyran ring resulted in o - 1965 which did not bind to either cannabinoid receptor . in contrast , the introduction of a tertiary hydroxyl group at c4 of the pyran ring ( o - 1962 ) increased cb 2 affinity by 3 - fold adding additional oxygens such as a ketol group attached at c - 4 to the point of attachment of the dimethoxy resorcinol substituent ( o - 2092 ) also increased cb 2 affinity whereas adding an oxygen as an epoxide ( o - 2122 ) decreased it . the presence of a ketone group at c - 4 of the cyclohexane ring and having unsaturation in the ring ( o - 2114 ) resulted in a compound with poor affinity for either cannabinoid receptor ; however , if a tertiary hydroxyl group was added at the site of dimethoxy resorcinol attachment ( o - 2115 ), cb 2 affinity improved . retention of the tertiary hydroxyl , methylation at position 5 and the presence of a ketone at position 3 of the cyclohexane ring increased affinity for both receptors and resulted in a compound ( o - 2123 ) with the best affinity ( k i = 125 nm ) in this series . table 5 shows cb 1 and cb 2 affinities for 2 - cyclic ring substituted dimethoxy - resorcinol - dmh analogs in which the ring size and the position of the methyl or hydroxyl substituent on the cyclohexane ring are varied . the first analog ( o - 2072 ) contains one hydroxyl attached to the cyclohexane at the same position at which the resorcinol core is attached . this compound is cb 2 - selective . while it had poor affinity for cb 1 receptors , it bound with moderate affinity to cb 2 receptors . introduction of a methyl substituent in the 3 position of the cyclohexane ring gave a diastereomeric mixture from which two distinct entities were separated by careful chromotography , these analogs ( o - 1966 - a and o - 1967 - b ) were still mixtures . this substitution resulted in a 5 - fold increase in affinity for cb 2 receptors with continued poor affinity for cb 1 receptors . however , one of these isomers ( o - 1966 - a ) showed the best cb 2 selectivity ( 225 - fold ) in the series and had high binding affinity for the cb 2 receptor ( k i = 22 . 5 nm ). addition of an extra hydroxyl group to the cyclohexane ring ( o - 2121 ) reduced both selectivity and binding affinity for the cb 2 receptor comparable to those obtained with o - 1967 - b . removal of the methyl at position 3 and addition of an hydroxyl at position 4 resulted in two diastereomeric mixtures which could be separated that were designated as o - 2116 - a and o - 2117 - b . both of these isomers had poor affinity for cb 1 receptors , but while the b isomer also had poor affinity for cb 2 receptors , the a isomer bound to cb 2 receptors with moderate affinity attachment of a gem - dimethyl group to position 3 of o - 2072 ( i . e ., o - 2068 ) did not significantly alter affinities for cb 1 or cb 2 receptors ; however , replacement of the dmh group of o - 2068 with a methyl group ( o - 2139 ) produced loss of affinity at both receptors . changing the dimethyoxy groups of the resorcinol by adding diethoxy groups ( o - 2090 ) drastically decreased affinities for cb 1 and cb 2 receptors ( compare o - 2090 to o - 1966 - a or o - 1967 - b ). enlarging the cyclohexane ring in o - 2072 to a cycloheptane ring ( o - 2091 ) resulted in little change in affinity for cb 1 receptors and an almost 2 - fold increase in cb 2 affinity . as stated in the introduction , the lack of cb 1 binding affinity of cannabidiol compared to other pyran - ring open analogs such as cp 55 , 940 prompted us to examine the structure - activity relationships of resorcinol derivatives for in vitro and in vivo cannabinoid activity . our results show that many of the structural changes that affect cb 1 receptor recognition and activation in traditional bicyclic and tricyclic cannabinoids similarly alter binding and activity in this resorcinol series . previous research has shown that the length and branching of a lipophilic substituent is important for cb 1 receptor recognition in all of the major cannabinoid agonist classes , including tetrahydrocannabinols ( compton et al ., 1993 , incorporated herein by reference ), bicyclic cannabinoids ( compton et al ., 1993 ), indole - derived cannabinoids ( wiley et al ., 1998 incorporated herein by reference ), and anandamides ( ryan et al ., 1997 ; seltzman et al ., 1997 both incorporated herein by reference ). in the tricyclic and bicyclic series , a 1 ′, 1 ′- dimethylheptyl side chain is optimal ( compton et al ., 1993 ) and is contained in most of the resorcinols presented here . reducing the length of this substituent to 1 ′, 1 ′- dimethylbutyl ( o - 1532 and o - 1890 ) or methyl ( o - 2139 ) or a hydrogen ( o - 2010 ) resulted in a concomitant elimination or decrease in cb 1 receptor recognition , as occurs in other cannabinoid series with similar structural manipulations ( see references above ). other structural features affecting cb 1 receptor recognition and activation in this resorcinol series are related to the size , saturation , substitution , and methylation of the second , non - resorcinol ring of these bicyclic cannabinoids . in most tricyclic and bicyclic cannabinoids , the ring corresponding to the non - resorcinol ring in the current series is a cyclohexane . reducing this size to a cyclopentane decreases cb 1 affinity and potency whereas increasing it to a cycloheptane has little effect . substitution of an adamantyl results in better cb 1 affinity ; however , potency is decreased . similar modifications of tricyclic and bicyclic cannabinoids have not been reported . the degree of saturation of the cyclohexane ring , however , has been manipulated in several cannabinoid classes . in the resorcinol series , the presence of a cyclohexane ring appeared optimal , although a thorough investigation of this issue was not undertaken . introduction of a single double bond ( o - 1423 ) within the ring decreased cb 1 affinity and potency to the same extent as did a reduction in the size of the ring to a cyclopentane . hence , most structural manipulations were performed upon a bicyclic resorcinol - cyclohexane template . degree of saturation of , as well as the position of the double bond in the cyclohexane ring of tricyclic and bicyclic cannabinoids and in the polyolefin loop of the anandamides , has also been shown to affect cb 1 receptor recognition and activity in these cannabinoid classes . greatest affinity and potency within the anandamides is achieved with four double bonds , with greater or lesser saturation resulting in a reduction in cb 1 binding and / or in vivo activity ( adams et al ., 1995 ; sheskin et al ., 1997 ; thomas et al ., 1996 ). similarly , number and position of double bonds within the cyclohexane ring of tetrahydrocannabinols and bicyclic cannabinoids affect activity . for example , moving the double bond of δ 9 - thc to position 8 ( as in δ 8 - thc ) decreases cb 1 affinity three - fold and somewhat reduces potency ( compton et al ., 1993 ). unsaturation of the cyclohexane ring results in cannabinol with its greatly reduced cb 1 affinity ( showalter et al ., 1996 ). in contrast , cp 55 , 940 , with a completely saturated cyclohexane ring , is several fold more potent than δ 8 - thc - dmh which has a single double bond in the cyclohexane ring , but δ 8 - thc with its single double bond binds with better cb 1 affinity than does δ 9 ( 11 ) - thc which has a completely saturated cyclohexane ring ( compton et al ., 1993 ). the most remarkable structural features of the resorcinol series affecting cb 1 affinity , however , are the length of the lipophilic side chain at position 5 and the size of the cyclic ring substituent at position 2 of the resorcinol core . thc and cp 55 , 940 contain two oxygens : one as a phenol ( one hydroxyl in the aromatic ring ) with a second oxygen incorporated into a separate ring ( pyran oxygen in thc ) or a hydroxyl group attached as a substituent in the cyclohexane ring as in cp 55 , 940 . previous research has shown that eliminating the phenolic hydroxyl of δ 8 - thc - like cannabinoids results in deoxy - thc analogs that are cb 2 - selective ( huffman et al ., 1999 ). although some of these deoxy - thc analogs also retain reasonable affinity for cb 1 receptors , orientation of their binding to cb 1 receptors may be inverted such that the pyran oxygen substitutes for the absent phenolic hydroxyl in hydrogen bonding ( huffman et al ., 1996 ). in the absence of a pyran oxygen , as in the resorcinols , the nature of the substituent at position 2 of the resorcinol core is important to maintain adequate cb 1 affinity for in vivo activity an acyclic ring was found to be better than a heterocyclic ring with a cyclohexane ring being optimal for in vivo activity . in addition , the size and the position of the substituent on the cyclic ring is important to maintenance of cb 1 affinity . the presence of a methyl substituent at position 3 enhanced activity in some cases . further , the 3r analog ( o - 1826 ; table 2 ) has a poorer cb 1 binding affinity ( k i = 40 nm ) compared to the diastereomeric mixture o - 1657 ( k i = 14 nm , table 2 ), suggesting that cb 1 binding affinity is enhanced when the orientation of the methyl substituent at position 3 in the cyclohexane ring is 3s compared to 3r . methylation of the phenols of the resorcinols drastically decreased or eliminated cb 1 affinity , perhaps because hydrogen donation is less likely from a methoxy group than from thc &# 39 ; s free hydroxyl group ( b . r . martin , unpublished observations ). similarly , methoxy substitution for the phenolic hydroxyl in the methyl esters of δ 8 - and δ 9 ( 11 ) - thc - dmh resulted in analogs that were cb 2 - selective and had little affinity for cb 1 receptors ( gareau et al ., 1996 ; huffman et al ., 1999 ; ross et al ., 1999 ). notably , with the exception of a few compounds , the dimethoxyresorcinols tested here were cb 2 - selective . most of the structural features that affected recognition at cb 1 receptors also affected cb 2 receptor recognition , although not always to the same degree or in the same manner . these factors included length and branching of the side chain and size and degree of saturation of the non - resorcinol cyclohexane ring . in a sar study on a series of cb 2 - selective deoxy - δ 8 - thc analogs , huffman et al . ( 1999 ) reported that length and branching of the c3 side chain affected cb 2 binding in a manner similar to its effect on cb 1 affinity , as it did in the present study ; however , the range of chain lengths for which moderate to good cb 2 affinity was retained for the deoxy - δ 8 - thc analogs was greater than the range for cb 1 affinity . similar results were obtained with a series of cb 2 - selective indole - derived cannabinoids in which length of the nitrogen substituent was varied ( aung et al ., 2000 ). to date , anandamide analogs appear to be cb 1 - selective with relatively little affinity for cb 2 receptors across several types of manipulations ( showalter et al ., 1996 ). insufficient research is available to determine the effect of substitution on a cyclohexane ring on cb 2 affinity across cannabinoid classes . other structural manipulations eliminated or drastically reduced cb 1 receptor recognition , but did not necessarily alter cb 2 receptor binding in an identical manner . as mentioned , cb 2 selectivity was most evident in the dimethoxy analogs , primarily as a consequence of severe reductions in cb 1 affinity . hu - 308 , the most selective cb 2 agonist to date , has a dimethoxy resorcinol core structure and does not bind to cb 1 receptors at all ( hanu et al ., 1999 ). in addition , greater tolerance in cb 2 ( vs . cb 1 ) receptor recognition was observed with other c2 substitutions in the resorcinols . huffman et al . ( 2001 ) recently reported that bicyclic pyridone analogs with carbonyl substitution at c1 and a nitrogen substituent substitution at c2 of thc had little affinity for cb 1 receptors . in contrast , moderate cb 2 affinity ( ki — 53 nm ) was retained . differences in allosteric regulation of cb 1 and cb 2 receptors by ions and guanine nucleotides has been noted previously ( showalter et al ., 1996 ). together , results presented here and elsewhere ( see above ) suggest incomplete overlap of the pharmacophores for cb 1 and cb 2 receptors . in summary , structure - activity relationships of the resorcinol series presented here are consistent with the cb 1 and cb 2 pharmacophores of other cannabinoid classes , including tetrahydrocannabinols , bicyclic cannabinoids , aminoalkylindoles , and anandamides . in this series of resorcinols , several structural features were essential for maintenance of cb 1 receptor recognition and in vivo activity , including the presence of a branched lipophilic side chain ( dmh ) at c5 , the presence of free phenols , and substitution of a cyclohexane ring at c2 . an important structural feature for receptor recognition at cb 2 receptors was side chain length , as reduction of the chain length to a methyl eliminated cb 2 binding affinity . the cb 2 selectivity observed with some resorcinols was maximized in the dimethoxyresorcinol analogs and this selectivity was greatly enhanced when a tertiary hydroxyl group was present in the cyclohexane ring in the same position at which the resorcinol core is attached . in contrast , the presence of unsaturation or a ketone group or an additional hydroxyl substitution in the cyclohexane ring adversely affected the cb 2 selectivity . methyl ethers were optimal for cb 2 selectivity since ethyl ethers reduced selectivity . in conclusion , although resorcinol derivatives with cyclic ring substituents at c2 are closely related to the nonactive cannabinoid cannabidiol , many of these analogs have high cb 1 and / or cb 2 binding affinity as well as potent in vivo activity . in addition , because dimethoxyresorcinols are cb 2 selective , they have potential to offer insight into similarities and differences between requirements for receptor recognition at cb 1 versus cb 2 receptors . one such difference noted here was the greater tolerance found for substitution at position 2 , in the resorcinol series , for cb 2 receptor recognition compared to that for cb 1 receptors . the results presented here suggest that the resorcinol series represent a novel template for the development of cb 1 and cb 2 selective cannabinoid agonists . while the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment , it is to be understood that the invention is not to be limited to the disclosed embodiment , but on the contrary , is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims . adams i b , ryan w , singer m , thomas b f , compton d r , razdan r k , martin b r ( 1995 ) evaluation of cannabinoid receptor binding and in vivo activities for anandamide analogs . j pharmacol exp ther 273 : 1172 – 1181 . aung m m , griffin g , huffman j w , wu m j , keel c , yang b , showalter v m , abood m e , martin b r ( 2000 ) influence of the n - 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308 : a specific agonist for cb2 , a peripheral cannabinoid receptor . proc nat acad sci usa 96 : 14228 – 14233 . huffman j w , lu j , hynd g , wiley j l , martin b r ( 2001 ) a pyridone analogue of traditional cannabinoids . a new class of selective ligands for the cb 2 receptor . bioorg med chem , in press . huffman j w , liddle j , yu s , aung m m , abood m e , wiley j l , martin b r ( 1999 ) 3 -( 1 ′, 1 ′- dimethylbutyl )- 1 - deoxy - 8 - thc and related compounds , synthesis of selective ligands for the cb 2 receptor . bioorg med chem 7 : 2905 – 2914 . huffman j w , yu s , showalter v , abood m e , wiley j l , compton d r , martin b r , bramblett r d , reggio p h ( 1996 ) synthesis and pharmacology of a very potent cannabinoid lacking a phenolic hydroxyl with high affinity for the cb2 receptor j med chem 39 : 3875 – 3877 . little p j , compton d r , johnson m r , melvin l s , martin b r ( 1988 ) pharmacology and stereoselectivity of structurally novel cannabinoids in mice . j pharmacol exp ther 247 : 1046 – 1051 . mahadevan a , siegal c , martin b r , abood m e , beletskaya i , razdan r k ( 2000 ) novel cannabinol probes for cb1 and cb2 cannabinoid receptors . j med chem 43 : 3778 – 3785 . munro s , thomas k l , abu - shaar m ( 1993 ) molecular characterization of a peripheral receptor for cannabinoids . nature 365 : 61 – 65 . reggio p h , wang t , brown a e , fleming d n , seltzman h h , griffin g , pertwee r g , compton d r , abood m e , martin b r ( 1997 ) importance of the c - 1 substituent in classical cannabinoids to cb 2 receptor selectivity , synthesis and characterization of a series of o , 2 - propano - 8 - tetrahydrocannabinol analogs . j med chem 40 : 3312 – 3318 . rinaldi - carmona m , barth f , heaulme m , shire d , calandra b , congy c , martinez s , maruani j , neliat g , caput d , ferrara p , soubrie p , breliere j c , le fur g ( 1994 ) s r 141716a , a potent and selective antagonist of the brain cannabinoid receptor . febs lett 350 : 240 – 244 . ross r a , brockie h c , stevenson l a , murphy v l , templeton f , makriyannis a , pertwee r g ( 1999 ) agonist - inverse agonist characterization at cb 1 and cb 2 cannabinoid receptors of l759633 , l759656 and am630 . br j pharmacol 126 : 665 – 672 . ryan w j , banner w k , wiley j l , martin b r , razdan r k ( 1997 ) potent anandamide analogs : the effect of changing the length and branching of the end pentyl chain . j med chem 40 : 3617 – 3625 . seltzman h h , fleming d n , thomas b f , gilliam a f , mccallion d s , pertwee r g , compton d r , martin b r ( 1997 ) synthesis and pharmacological comparison of dimethylheptyl and pentyl anandamide analogs . j med chem 40 : 3626 – 3634 . sheskin t , hanu l , slager j , vogel z , mechoulam r ( 1997 ) structural requirements for binding of anandamide - type compounds to the brain cannabinoid receptor . j med chem 40 : 659 – 667 . sheskin t , hanu l , slager j , vogel z , mechoulam r ( 1997 ) structural requirements for binding of anandamide - type compounds to the brain cannabinoid receptor . j med chem 40 : 659 – 667 . showalter v m , compton d r , martin b r , abood m e ( 1996 ) evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor ( cb2 ): identification of cannabinoid receptor subtype selective ligands . j pharmacol exp ther 278 : 989 – 999 . song z h , bonner t i ( 1996 ) a lysine residue of the cannabinoid receptor is critical for receptor recognition by several agonists but not win - 55 , 212 . mol pharmacol 49 , 891 – 896 . thomas b f , adams i b , mascarella w , martin b r , razdan r k ( 1996 ) structure - activity analysis of anandamide analogs : relationship to a cannabinoid pharmacophore . j med chem 39 : 471 – 479 . wiley j l , compton d r , dai d , lainton j a h , phillips m , huffman j w , martin b r ( 1998 ) structure - activity relationships of indole - and pyrrole - derived cannabinoids . j pharmacol exp ther 285 : 995 – 1004 . the disclosures of all of the above - cited references are incorporated herein by reference . the k i &# 39 ; s are presented as means ± sem . all ed 50 &# 39 ; s are expressed as μmol / kg ( with 95 % confidence limits in parentheses ). for compounds that failed to produce either manual or dose - related effects , the percent effect at the highest dose ( mg / kg : in parenthesis ) is provided . sa = suppression of spontaneous activity ; mpe =% maximum possible antinociceptive effect in tail flick assay ; rt = rectal temperature . dmh = dimethylheptyl . the k i &# 39 ; s are presented as means ± sem . all ed 50 &# 39 ; s are expressed as μmol / kg ( with 95 % confidence limits in parentheses ). for compounds that filed to produce either maximal or dose - related effects , the percent effect at the highest dose ( mg / kg : in parenthesis ) is provided . sa = suppression of spontaneous activity ; mpe =% maximum possible antinociceptive effect in tail flick assay ; rt = rectal temperature . a this dose ( μmol / kg ) produced & gt ; 50 % inhibition and was the lowest dose tested . the k &# 39 ; s are presented as means ± sem . all ed 50 &# 39 ; s are expressed as μmol / kg ( with 95 % confidence limits in parentheses ). for compounds that failed to produce either maximal or dose - related effects the percent effect as the highest dose ( mg / kg ; in parenthesis ) is provided . sa = suppression of spontaneous activity : mpe = % maximum possible antinociceptive effect in tall flick assay ; rt = rectal temperature .