the dimeric enkephalin analogues of the present invention comprise symmetrical dimers of precursor carboxyl - terminal enkephalin polypeptide monomers linked at the c - termini thereof with a diamino bridging group especially hydrazine , 2 , 7 - diaminofluorene or diaminoalkane . the enkephalin polypeptide monomer precursors are leucine enkephalin ( h - tyr - d - ala - gly - phe - leu - oh ), or di -, tri -, or tetrapeptide acid fragments thereof characterized by the following formula : wherein r is selected from one of the following α - amino acid residue sequences : h - tyr - d - ala - gly - phe - leu ; h - tyr - d - ala - gly - phe ; h - tyr - d - ala - gly ; and phe - leu ; wherein tyr is tyrosine , ala is alanine , gly is glycine , phe is phenylanine , and leu is leucine . the monomeric precursors r - oh are dimerized or linked with a diamino bridging compound of the formula h 2 n -- x -- nh 2 , to form compounds of the formula : ## str1 ## wherein the r groups are the same , and are as defined above . the bridging moiety -- x -- is a direct bond , ## str2 ## or alkylene having from 1 to 22 carbon atoms . the alkylene group may be branched or unbranched ; substituted or unsubstituted ; or uninterrupted or interrupted by heteroatoms , especially -- o --, -- s --, or -- nh --, or groups such as carbonyl , carboxy , or carboxyamide , with the caveat that the dimers retain the capability of binding to neural opiate receptors . particularly preferred groups are unbranched , unsubstituted , uninterrupted alkyl groups comprising polymethylene chains --( ch 2 ) n --, wherein n is from 1 to 22 . ( a ) dimeric pentapeptide enkephalins ( dpe ) of the formula : ## str3 ## wherein r is h - tyr - d - ala - gly - phe - leu and n is 1 to 22 , preferably 1 to 12 ; ( b ) dimeric tetrapeptide enkepahlins ( dte ) of the formula : ## str4 ## wherein r is h - tyr - d - ala - gly - phe and n is 1 to 22 ; ( c ) dimeric tripeptide enkephalins ( dtre ) of the formula : ## str5 ## wherein r is h - tyr - d - ala - gly and n is 1 to 22 ; ( d ) dimeric dipeptide enkephalins ( dde ) of the formula : ## str6 ## wherein r is phe - leu and n is 1 to 22 , preferably 1 to 12 ; ( e ) pentapeptide enkephalin dimers of the formula : ## str7 ## wherein r is h - tyr - d - ala - gly - phe - leu ; and ( f ) pentapeptide enkephalin dimers of the formula : ## str8 ## wherein r is h - tyr - d - ala - gly - phe - leu . compounds exhibiting a particularly good affinity and selectivity for the delta opiate receptor are ## str9 ## wherein r is the pentapeptide h - tyr - d - ala - gly - phe - leu ; and ## str10 ## wherein r is the tetrapeptide h - tyr - d - ala - gly - phe . owing to the selectivity and relative activities of these compounds they are especially useful as probes of the opiate receptor for membranes when radio - labelled by well known techniques such as tritium exchange or ( 125 i ) iodination . the compounds of the invention are broadly synthesized by linking two n - protected polypeptide acid precursors via the desired diamino bridging group to form the corresponding diamide . preferably , the dimeric penta - and tetrapeptides are prepared in two stages : by condensing an appropriate n - protected dipeptide acid with the requisite bridging group , and then elongating the α - amino acid sequences of the resultant intermediate after deprotection . the α - amino acid sequences of the precursor polypeptides are obtained in a known fashion by conventional liquid phase techniques , as exemplified by bodanszky , et al ., in peptide synthesis , wiley interscience , n . y . ( 1966 ). various methods are known for protecting the terminal α - amino groups from undesired reaction during synthesis , most especially the use of blocking groups such as tert - butoxycarbonyl ( boc ) or carbobenzoxy . the enkephalin polypeptide acid precursors synthesized according to the prior art are condensed with the bridging groups according to the following scheme : ## str11 ## preferably , at least when r is h - tyr - d - ala - gly - phe - leu or h - tyr - d - ala - gly - phe , the dimers are prepared by two - step coupling . in this method of preparation , an n - protected dipeptide acid corresponding to the two ( 2 ) c - terminal amino acids of the α - amino acid sequence r is first reacted with the diamino bridging compounds to form an enkephalin amide intermediate . the α - amino acid sequences of the intermediate are then elongated to form the desired r group by deprotecting the α - amino groups of the intermediate followed by coupling with the appropriate n - protected di - or tripeptide acid . the following is an exemplary scheme for dimeric pentapeptide enkephalins according to the invention : ## str12 ## suitable coupling agents are well known in the art and include , for example , water - soluble carbodiimides which activate the terminal carboxyl groups of the reactive peptide acids . the most widely used carbodiimides for this purpose are edc [ 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide ] and dcci ( dicyclohexylcarbodiimide ). the coupling reaction is generally carried out in an organic solvent such as methylene chloride , dimethylformamide , or dioxane . a particularly useful system for the dimerization of the present invention is edc in dimethylformamide . to minimize racemization during coupling , additives such as 1 - hydroxybenzotriazole ( hobt ) are included in the reaction mixture . this is particularly important in the first step of the two - stage procedure when leucine is present . the n - protecting groups are removed by any customary deprotecting agent such as trifluoroacetic acid or hbr in acetic acid . the product dimers are purified by conventional methods , usual gel filtration over sephadex polydextran gels , suitably a sephadex g - 25 gel . the gels are prepared as usual and eluted with a suitable eluant such as 30 % acetic acid . the dimeric enkephalin analogues bridged by methylene chains are particularly useful in the elucidation of the physiological role of ` δ ` ( peptide ) and ` μ ` ( alkaloid ) opiate receptors of the brain and other neural tissues , owing to the extremely high δ selectivity of many of the compounds . these analogues also exhibit a strong affinity for the δ receptors , and a loss of binding activity for the μ receptors . since the corresponding monomers generally exhibit a relatively low affinity and little or no selectivity for δ receptors over μ receptors , it is believed that the affinity and selectivity of these dimeric ligands is at least in part attributable to the property of selectively crosslinking δ but not μ receptor , sites . the result is particularly surprising owing to the known phenomenon that structural modifications or abolition of the c - terminal free carboxyl group in monomeric enkephalins invariably leads to loss of δselectivity and gain of μ activity . while the diaminofluorene and hydrazine bridged compounds exhibit little or no delta selectivity they are useful as biochemical probes of the properties and structure - activity relationship of the enkaphalin receptors . the enkephalin analogues are thus useful as radioligands in probes of the opiate receptor membranes . further , the dimers function as high - potency endogenous opiate peptide agonists or antagonists , with pharmaceutical analgesic activity organtagonistic activity toward known opiate drugs . the bioactivity of the present dimeric enkephalins was established using the classical bioassays for opiate activity , the guinea pig ileum ( gpi ) assay and the mouse vas deferens ( mvd ) assay . there was an excellent general correlation between activity in delta receptor assays ( infra ) and the mvd assay . dimeric pentapeptide enkephalin dpe 2 [ x =--( ch 2 ) 2 --] was the most potent in the mvd assay , with activity surpassing that of the corresponding pentapeptide monomer . dpe 2 also exhibited analgesic activity when administered to vivo and when administered intracerebroventricularly . in standard hot plate and tail flick assays , dpe 2 was more potent than the corresponding monomer , producing a dramatic stupor and persistent somnolence in test animals . dimeric tetrapeptide enkephalin dte 12 [ x = 13 ( ch 2 ) 12 - ] exhibited good analgesic activity but was less potent than dpe 2 . in other bioassays , dpe 2 produced a rise in rat serum prolactin , after intracerebroventricular injection , which corresponds to that observed for other opiates and enkephalins . in an in vitro bioassay employing ng108 - 15 cells ( a neuroblastoma - glioma hybrid cell line ) stimulated by prostaglandin e 1 ( pge . sub . 1 ), dpe 2 showed an ic 50 of about 0 . 15 nm , consistent with the receptor affinity in binding assays . for use as analgesics , the enkephalin dimers of the invention can be administered by either oral or parenteral routes , optionally in the form of their pharmaceutically acceptable salts , to relieve pain in mammals . the dosages are formulated for the chosen route of administration , and vary , inter - alia , according to the potency of the drug , the sensitivity of the patient , and the incidence of pain . typically , dosages of active ingredient from about 0 . 1 mg to about 3 mg per kilogram of body weight are sufficient to achieve analgesia in ordinary therapeutic applications . the compounds are generally administered in conjunction with a conventional pharmaceutically acceptable carrier , either solid or liquid , with or without customary adjuvants . the abbreviations used are : dpe n , dimeric pentapeptide enkephalins [ xd =( ch 2 ) n --], where n denotes the length of the methylene chain ; tlc , thin - layer chromatography ; boc , tert - butoxycarbonyl ; dd , dimeric dipeptides ; dmf , n , n - dimethylformamide ; hobt , 1 - hydroxybenzotriazole ; edc - hcl , 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide hydrochloride ; dp , dimeric pentapeptides ; tfa , trifluoroacetic acid ; nal , naloxone ; damea , [ d - ala 2 , met 5 ] enkephalin amide ; dadle , [ d - ala 2 , d - leu 5 ] enkephalin ; dalea , [ d - ala 2 , leu 5 ] enkephalin amide . synthesis of dimeric pentapeptide enkephalins [ x =--( ch 2 ) n - , n is 2 , 4 , 6 , 8 , 10 , or 12 ]. melting points were measured utilizing a 6427 - h10 thomas - hoover melting point apparatus and were uncorrected . tlc was carried out utilizing silica gel g ( 250 μm , analtech ). optical rotations were measured with a perkin - elmer model 241 mc polarimeter . mass spectra were obtained using californium - 252 plasma desorption mass spectrometry , amino acid analyses were performed on a beckman model 121mb amino acid analyzer . step 1 . ( boc - phe - leu - nh -) 2 . (-- ch 2 --) n ( n = 2 , 4 , 6 , 8 , 10 and 12 ). the dimers of the boc - phe - leu dipeptide are designated dd n , where n denotes the length of the methylene chain . to a solution of boc - phe - leu - oh ( 833 mg , 2 . 20 mmoles ) and α , ω - diaminoalkanes , h 2 n --( ch 2 ) n -- nh 2 ( 1 . 00 mmole ) in dmf ( 20 ml ) were added hobt ( 404 mg , 2 . 64 mmole ) and edc . hcl ( 464 mg , 2 . 42 mmole ) at - 10 °. the reaction mixture was stirred for 2 hr at 0 °, followed by incubation for 2 days at room temperature . the solvent was evaporated in vacuo , and ice - water was added to precipitate a solid . the solid collected was washed successively with 4 % nahco 3 , 10 % citric acid , water , and petroleum ether . it was taken dried , and recrystallized twice from dmf - etoac - ether . step 2 . ( boc - tyr - d - ala - gly - phe - leu - nh -) 2 . (-- ch 2 --) n ( n = 2 - 12 ). the dimers of the boc - protected derivatives of the pentapeptide analogues are designated dp n , wherein n again indicates the length of the methylene chain . compound dd n ( 0 . 5 mmole ) was dissolved in tfa ( 5 ml ) at 0 °. after 30 min . at 0 °, the solution was evaporated to leave an oil , which was solidified by the addition of anhydrous ether , yielding ( tfa . h - phe - leu - nh -) 2 . (-- ch 2 --) n . this tfa salt ( 0 . 5 mmole ), et 3 n ( 0 . 14 ml , 1 . 0 mmole ) and boc - tyr - d - ala - gly - oh ( 409 mg , 1 . 0 mmole ) were dissolved in dmf ( 15 ml ). to the solution were added hobt ( 184 mg , 1 . 2 mmoles ) and edc . hcl ( 211 mg , 1 . 1 . mmoles ) at - 10 °, and the reaction mixture was treated as described for the dd n series in step 1 . purifications were carried out by gel filtration on a sephadex lh - 20 column ( 2 . 2 × 96 cm ) eluted with dmf , and then by recrystallization from dmf - etoac - ether . step 3 . acoh . h - tyr - d - ala - gly - phe - leu - nh -) 2 . (-- ch 2 --) n ( n = 2 - 12 ). the liberated dimetric pentapeptide analogues are designated as dpe n , where n again indicates the methylene chain length . the compound dp n ( 0 . 25 mmole ) was treated with tfa ( 5 ml ) at 0 ° for 30 min . after evaporation of tfa the residual oil was dissolved in 30 % acoh and subjected to gel filtration on a column ( 2 . 2 × 145 cm ) of sephadex g - 25 in 30 % acoh . the fractions containing a pure product by tlc were pooled and lyophilized repeatedly with aqueous acoh . homogeneity of the peptides was verified by ascending tlc in three different solvent systems ( table 1 ). for amino acid analysis , peptides were hydrolyzed in 6 m hci for 24 hr at 110 ° in de - aerated tubes ( data not given ). table 1______________________________________physical properties of ( acoh . h -- tyr -- d - ala -- gly -- phe -- leu -- nh --). sub . 2 :(-- ch . sub . 2 --). sub . n , ( dpe . sub . n ) yield [ α ] . sub . d . sup . 20 tlc . sup . adpe . sub . n % melting point ( co . 5 , 95 % acoh ) r . sub . f______________________________________dpe . sub . 2 98 126 ° + 22 . 6 ° 0 . 28dpe . sub . 4 98 149 ° + 21 . 0 ° 0 . 29dpe . sub . 6 96 118 ° + 22 . 6 ° 0 . 32dpe . sub . 8 87 111 ° + 23 . 4 ° 0 . 35dpe . sub . 10 96 109 ° + 22 . 8 ° 0 . 36dpe . sub . 12 98 108 ° + 22 . 2 ° 0 . 38______________________________________ . sup . a solvent : nbuoh -- acoh -- h . sub . 2 o ( 4 : 1 : 5 , v / v , organic phase .) the synthesis of dimeric analogues by the conventional solution methods as described here offers several advantages . ( a ) cross linkages are built up by amide bond formation which makes the splitting of dimers by metabolic processes unlikely . ( b ) synthesis of dimeric analogues can be carried out exactly like that of a monomeric analogue except that 2 equivalents of peptide acids are used . this means that a dimeric analogue can be obtained with essentially the same effort as monomeric pentapeptide enkephalins . no differences of reactivity were observed among the series of dimers . ( c ) coupling reactions can be conducted by the water - soluble edc - hobt method and using the fragment intermediate boc - tyr - d - ala - gly - oh , which avoids the problem of racemization at the cooh terminus . this resulted in high yields of well characterized final products . three binding assays using tracers of different selectivity were used to assess the activity of dpen :[ 3 h ] naloxone ( 3 h - nal ), which labels μ receptors ; [ 125 ]-[ d - ala 2 , d - leu 5 ]- enkephalin ( 125 i - dadle ), which labels δ ; and [ 3 h ]-[ d - ala 2 , met 5 ]- enkephalin amide ( 3 h - damea ), which labels both μ and δ . binding was studied in rat brain membranes at 26 ° c . in the presence of bacitracin ( 100 mg / ml ) in absence of sodium ion ( table 2 ). table 2______________________________________ed . sub . 50 values for dpen in three radioligandassays : ` μ `, using h -- naloxone , ` δ `, using . sup . 125 i -- dadle ; or ` mixed δ and μ `, using . sup . 3 h -- damea . ed . sub . 50 ( nm ) enkephalins . sup . 3 h -- nal . sup . 3 h damea . sup . 125 i -- dadle______________________________________ ( monomer ) dalea 1 . 74 1 . 99 2 . 17 ( dimers ) dpe . sub . 2 1 . 78 1 . 01 0 . 27dpe . sub . 4 2 . 28 1 . 40 0 . 33dpe . sub . 6 2 . 61 1 . 53 0 . 43dpe . sub . 8 3 . 37 2 . 06 0 . 83dpe . sub . 10 5 . 82 3 . 99 1 . 95dpe . sub . 12 8 . 51 6 . 50 9 . 12relative ± 22 % 28 % ± 37 %% error______________________________________ the ed 50 values of dimeric enkephalin are clearly lower in the 125 t - dadle binding assay than in the 3 h - nal assay , suggesting that their affinity for δ receptor is very high . if the potency of dpen is normalized to that of the parent monomer ( standard dalea = 1 ), then the activities of dimeric enkephalins in the three binding assays can be compared as a function of the chain length ( fig1 ). the most potent analog , dpe2 , is seven times more potent than the monomer standard in binding to δ receptors ( 125 i - dadle ), but equivalent to monomer when binding to μ sites ( 3 h - nal ). elongation of the methylene chain reduces activity in all the assays . for 4 & lt ; n & lt ; 10 , dimeric enkephalins are more active than dalea in a δ assay but less potent than dalea in the μ assay . a chain length of 12 produces a severe drop of potency in both cases . in the 3 h - damea radioligand assay , the activity - length relationship is intermediate . the ratio of the ed 50 values for any peptide when using 3 h - nal as a tracer relative to its ed 50 when using 125 i - dadle furnishes a measure of the selectivity of peptides for δ and μ receptors . a completely nonselective compound with the same potency in both the assays would have a ratio of 1 . the shorter the chains , the higher the selectivity of dimeric enkephalins for δ receptors ( fig2 ) compounds dpe2 , dpe4 , dpe6 show greater selectivity for δ than dadle itself . dpe12 shows no selectivity for δ relative to μ receptors .
the compounds of the invention are symmetrical dimers of enkephalin polypeptides comprising enkephalin polypeptide monomers linked at the c - termini thereof with a difunctional amino bridging group . the compounds are useful as investigative tools for probing the opiate receptor membranes , particularly as radiolabelled ligands for exploring the delta opiate receptor , and also have particular use as narcotic and / or analgesic agents .