Minimum structure opioids—Dipeptide and tripeptide analogs of the enkephalins

Peptides, Vol. 2, pp. 303-308, 1981. Printed in the U.S.A,

Minimum Structure Opioids Dipeptide and Tripeptide Analogs of the Enkephalins R A Y M O N D J. V A V R E K , L I - H S U E H HSI, E U N I C E J. Y O R K M I C H A E L E. H A L L A N D J O H N M. S T E W A R T

D e p a r t m e n t o f Biochemistry, Biophysics a n d Genetics, University o f Colorado M e d i c a l School 4200 East 9th A v e m t e , Denver, C O 80262 Received 24 July 1981 VAVREK, R. J., L.-H. HSI, E. J. YORK, M. E. HALL AND J. M. STEWART.Mhlhnum structure opioids-Dipeptide and tripeptide analogs of tile enkephalh:s. PEPTIDES 2(3) 303-308, 1981.--51"hrougha systematic reduction of peptide structure, a series of 25 tripeptide and 5 dipeptide amide and alcohol analogs of enkephalin were synthesized and assayed/n vitro on the stimulated guinea pig ileum. Tyr-Pro-Phe-NH2,Tyr-D-Ala-Phe-NH2,Tyr-D-Ala-Phe-oland Tyr-D-Phe-PheNH2 had 20-25% the potency of Met-enkephalin. Four aromatic alkylamides of the dipeptide Tyr-D-Ala were made with benzylamine, phenethylamine, phenylpropylamine and phenylbutylamine. All had full naloxone reversible enkephalin-like activity in the ileum assay. Tyr-D-Ala-phenylpr0pylamidehas about 80% the potency of Met-enkephalin in vitro, and is equipotent with Tyr-D-Ala-Gly-Phe-Met-NH2in producing analgesia in mice after intraventricular administration. Tyr-DPhe-NH2 is the smallest peptide to show full intrinsic enkephalin-like activity hz vitro, although its potency is very low. Enkephalin analogs

Minimumstructure opioids

Tripeptideopioids

AFTER the initial discovery and structure elucidation of the endogenous opiate peptides Met- and Leu-enkephalin (TyrGly-Gly-Phe-Met(Leu)) [8] a great number of modifications in the structure were made along the lines of classical peptide structure-activity relationships [19] in attempts to define the opiate peptide pharmacophore [3, 5, 6]. The effects were generally centered on the substitution of isosteric and isofunctional amino acids into the basic pentapeptide sequence of the parent peptides [14,16]. The postulates of the minimal peptide structure sufficient for full opiate activity were based on studies which involved the sequential removal of amino acid residues from either C-terminal or N-terminal of the enkephalin molecule [4, 10, 15, 23]. The smallest sequential analog of enkephalin to exhibit full activity was the des-Met ~ enkephalin peptide TyrGly-Gly-Phe [23]. Since this peptide had all of the necessary elements to fit the three-point model for opiate-receptor interaction [3,5] several modifications of its sequence were examined as analogs of the minimal structure sufficient for enkephalin-like opiate activity [9, 12, 18]. An important distillation of the body of the data on enkephalin analogs [16] led to the suggestion that the minimal structure could be fulfilled by the structure Tyr-Gly-Gly-phenethylamide. Gorin et al. [6] reported a series of tripelStide aromatic amides of Tyr-Giy-Gly and showed that they indeed contained sufficient functional analogies with the enkephalins to give active opiates. In a break with the generally held idea of modification of the sequential pentapeptide structure of the enkephalins, Chipkin et al. [2] reported a series of deletion tetrapeptide analogs of Met- and Leu-enkephalin with potent hz vivo and

Dipeptideopiates

h7 vitro opioid activity. The des-Gly3 analogs of [D-Ala2]Met-enkephalin amide and [D-Ala2]-Leu-enkephalin amide (i.e., Tyr-D-Ala-Phe-Met-NH2 and Tyr-D-Ala-Phe-LeuNH2) were equipotent with the parent pentapeptides in the hz vitro stimulated guinea pig ileuin assay, but less potent in the hz vivo rat tail-flick assay. An additional series of fully active tetrapeptide deletion analogs containing modifications in position 2 has been reported [25], and in many ways the structure-activity relationships of the tetrapeptide deletion analogs parallel those of the pentapeptides. The three-point model for opiate-receptor interaction [3,5] postulates strict spatial requirements for the free amino group and aromatic ring of tyrosine in position 1 of the enkephalins, and an additional hydrophobic group within the peptide which can stabilize a receptor-active conformation. In the pentapeptide enkephalins this second hydrophobic group is provided by the phenylalanine residue in position 4, and in the deletion tetrapeptide enkephalins by the aromatic group of phenylalanine in position 3. Thus the peptide backbone spacing between the two aromatic groups which are required for potent opiate activity is by no means clearly established and has probably not yet been optimized in the peptide opiates examined so far. It was felt important to attempt to define more exactly the ' spatial and functional requirements both necessary and sufficient for small peptides to act as potent opioids, and to that end a series of 25 enkephalin-related tripeptides containing double deletions and truncated sequences was made. Additionally, 5 dipeptide amides containing the three elements postulated as necessary for opiate peptide-receptor interaction were also made and tested.

Copyright 9 1981 A N K H 0 International Inc.--0196-9781/81/030303-06501.10/0

VAVREK ET AL.

304 METHOD Peptide Synthesis All of the peptides were synthesized by the solid phase method [13] on a Beckman 990 Peptide Synthesizer (Beckman, Palo Alto. CA), on either a methylbenzhydrylamine resin [11] when simple amides were required, or on a hydroxymethyl resin [26] when C-terminals acids, alkyl amides, or alcohols were desired. Standard procedures were used throughout [21]. Boc-amino acids were commercially available (Bachem, Torrance, CA, and Beckman, Palo Alto, CA); the synthesis of Boc-Aib is reported elsewhere [24]. Peptide acids and simple amides were removed from the appropriate resins with anhydrous HF in the presence of anisole [20]. Protected peptide alkylamides were removed from the hydroxymethyl resin by transesterification in MeOH followed by aminolysis with the appropriate alkyl amine (benzylamine, phenethylamine, phenylpropylamine, phenylbutylamine; Aldrich, Milwaukee WI) for periods up to 24 hr at room temperature. For the C-terminal peptidc alcohol, the tripeptide hydroxymethyl resin was treated directly with LiBH4 (20 Eq) in tetrahydrofuran (THF) for 30 min [22]. HF treatment of the protected peptide amides and protected peptide alcohol gave the appropriate analogs. All of the peptides were purified by counter-current distribution (CCD) (100 upper phase transfers in the system nBuOH:AcOH:H20 (4:1:5)). Peptide purity was determined by TLC (Merck silica gel coated glass plates) in two different systems (nBuOH:AcOH:H20 8:3:4, and EtOAe:Pyridine:AcOH:HzO 5:5: 1:3), and by high voltage (10 v/cm) paper electrophoresis (Whatman #1 paper) at pH 2.8 (N AcOH) and pH 5.0 (pyridine acetate buffer). Peptides were visualized with the chlorine-tolidine identification spray. Peptide identity was confirmed by quantitative amino acids analysis (Beckman 120C) after acid hydrolysis in sealed glass tubes under N2. Bioassay The stimulated guinea pig ileum assays were performed essentially as described [2]. For the in vivo mouse tail-flick assay male and female HS mice (18-25 g) were maintained under controlled light conditions and given free access to food and water. The peptide to be tested and the standard peptide (Tyr-D-Ala-Gly-Phe-Met-NH2) were dissolved in 0.9% saline at 1 mg/ml concentrations and diluted with saline for serial dilutions. The mice were lightly anesthesized with ether and a total of 10 tzl of peptide solution was administered by bilateral intracerebral injections via a 27 gauge needle, 5 /.tl on either side of the midline, at the level of the external auditory meatus and a depth of 3 mm. N=5 or 6 animals per dose. Analgesia was measured using tail-flick method as described [2]. Tail-flick latencies were determined 20 min. before injection and again at 10, 30, 60, 90 and 150 min after injection. A 6-see cutoff was used to avoid tissue damage to the tail. To determine reversibility of analgesia, naloxone was given intraperitoneally (4 mg/kg) at the time of peptide injection. RESULTS The physical properties of the tripeptides and dipeptide amides are listed in Table 1. Acid hydrolysates of all the analogs showed the expected amino acid ratios within experimental error. Table 2 contains the guinea pig ileum assay results for the peptide analogs and includes results reported elsewhere for

related peptides [2]. All molar values are related to that of methionine enkephalin (Tyr-Gly-Gly-Phe-Met) as 100. The inhibition of stimulated contractions was fully naloxone reversible in all cases. Figure 1 contains the results of the ht vivo assay of TyrD-Ala-phenylpropylamide for analgesia in mice by the tailflick method using Tyr-D-Ala-Gly-Phe-Met-NHz as standard. DISCUSSION Peptides 1 and 2 are des-Gly 2.3 analogs of Met- and Leuenkephalin amide. None of these double deletion analogs show any enkephalin-like activity on the stimulated guinea pig ileum. Tyr-Gly-Phe-Met and Tyr-Giy-Phe-Met-NH~, the single deletion analogs, had 0.4% and 2% of Met-enkephalin potency [2], showing that both the deletion tetrapeptide and its amide contained sufficient information to give full intrinsic activity in our assay. Three other deletion tetrapeptide amides (Tyr-Ala-Phe-Met-NHz, Tyr-Aib-Phe-Met-NHz, Tyr-Pro-Phe-Met-NH2) showed quite significant ileum activity, with the Pro s tetrapeptide amide exhibiting 37% of the potency of Met-enkephalin [25]. This series of three analogs was an attempt to examine the steric requirements in position two of the deletion tetrapeptide amides since it was noted that a D-Ala substitution in the deletion tetrapeptide amides caused a 60-fold increase in potency over that of the parent tetrapeptide amide Tyr-Gly-Phe-Met-NH2 [2]. The same substitution in position two of the pentapeptide amides gave little change in potency. It is known that neither the C-terminal methionine nor leucine residue is necessary for full enkephalin-like activity in the pentapeptide enkephalins [10, 15, 23]. With this in mind, peptides 5, 6, and 7, which are the des-Met s analogs of Tyr-Ala-Phe-Met-NH2, Tyr-Aib-Phe-Met-NH~ and TyrPro-Phe-Met-NH2, were made and tested for enkephalin-like activity. Tyr-Ala-Phe-NHz (peptide 5) had no activity, whereas the parent Tyr-Ala-Phe-Met-NH2 had about 10% the potency of Met-enkephalin. Tyr-Aib-Phe-NHz (peptide #7) had low but definite activity (2%), about 20% of the potency of the deletion tetrapeptide with methionine. Clearly, the C-terminal Met or Leu residue is not necessary in either the pentapeptide or in the deletion tetrapeptide analogs of the enkephalins. The most interesting peptide in this series is Tyr-ProPhe-NHz (peptide 6) which has 19% activity, and which is about half as potent as the methionine containing deletion tetrapeptide amide. Pentapeptide analogs of the enkephalins which contain Pro in position 2 exhibit very low activity, with the Pro * analogs of Met-enkephalin, Leu-enkephalin and met-enkephalin amide having less than 1% of Metenkephalin potency in both the mouse vas deferens and receptor binding assays [16]. But a Pro substitution in position 2 of the deletion tetrapeptides (Tyr-Pro-Phe-Met-NH2) and in the tripeptide amides (Tyr-Pro-Phe-NHz, 6) gives analogs with good activity. The sequence Tyr-Pro-Phe is part of the N-terminal fragment of beta-casomorphin which shows enkephalin-like activity [7]. A tetrapeptide amide analog of beta-casomorphin known as morphiceptin (Tyr-Pro-Ph'ePro-NH2) is reported to be highly selective in binding to the putative/z opiate receptor [1]. When a D-amino acid is put in position 2 of the inactive tripeptide amide 5 to give Tyr-D-AIa-Phe-NHz (peptide 12), a potent analog is produced. Tyr-D-Ala-Phe-NHz has 25% the

305

MINIMUM S T R U C T U R E OPIOIDS TABLE I PHYSICAL PROPERTIESOF ENKEPHALINANALOGS Peptide

Tyr-Phe-Met Tyr-Phe-Leu Tyr-Phe-Met-NH2 Tyr-Phe-Leu-NHz Tyr-Ala-Phe-NH2 Tyr-Pro-Phe-NH2 Tyr-Aib-Phe-NH2 Tyr-D-Ala-Phe Tyr-D-Phe-Met Tyr-D-Phe-Leu Tyr-D-Phe-Phe Tyr-D-Ala-Phe-NHz Tyr-D-Ala-Phe-ol Tyr-D-Pro-Phe-NH2 Tyr-D-Phe-Met-NHz Tyr-D-Phe-Leu-NHz Tyr-D-Phe-Phe-NHz Tyr-D-Phe-D-Met Tyr-D-Phe-D-Leu Tyr-D-Ala-D-Phe Tyr-D-Phe-D-Phe Tyr-D-Phe-D-Met-NH2 Tyr-D-Phe-D-Leu-NHz Tyr-D-AIa-D-Phe-NH2 Tyr-D-Phe-D-Phe-NHz Tyr-D-Ala-BA Tyr-D-AIa-PEA Tyr-D-Ala-PPA Tyr-D-Ala-PBA Tyr-D-Phe-NHz

k(CCD)*

2.33 3.17 1.22 1.78 0.61 1.00 I. 17 1.44 2.70 3.76 4.00 0.82 1.86 1.22 1.63 1.94 2.85 2.33 3.76 1.38 3.55 1.33 2.03 0.89 2.45 1.56 1.70 3.00 4.26 1.13

HVEt

TLC:I:

pH 2.8

pH 5.0

834

5513

0.71 insw 0.41 0.45 0.49 0.49 0.47 0.37 0.71 ins 0.67 0.49 0.50 0.45 0.36 0.46 0.38 0.31 0.39 0.35 0.29 0.46 0.45 0.48 0.43 0.54 0.54 0.49 0.41 0.54

0.19 ins 0.55 0.53 0.58 0.57 0.55 0. I0 0.18 ins 0.13 0.56 0.57 0.54 0.52 0.52 0.49 0.14 0.08 0.11 0.00 0.56 0.58 0.56 0.47 0.62 0.60 0.54 0.53 0.65

0.63 ins 0.64 0.65 0.51 0.50 0.57 0.57 0.58 ins 0.60 0.57 0.64 0.55 0.62 0.64 0.62 0.60 0.64 0.63 0.65 0.66 0.67 0.57 0.64 0.67 0.62 0.65 0.67 0.56

0.72 ins 0.85 0.85 0.83 0.82 0.91 0.64 0.71 ins 0.71 0.68 0.81 0.85 0.82 0.83 0.78 0.69 0.75 0.64 0.70 0.78 0.84 0.67 0.84 0.81 0.81 0.87 0.90 0.72

*The experimentally determined partition coefficient from counter-current distribution in the system n-BuOH:AcOH:H20 (4:1:5). iMobilities relative to Lys = 1.00 during high voltage electrophoresis in N AcOH (oH 2.8) and pyridine acetate buffer (pH 5.0). :~Rt values from thin layer chromatography on Merck silica gel coated glass plates in the system 8:3:4 (n-BuOH:AcOH:H20) and 5:5:1:3 (EtOAc: Pyridine:AcOH:H20). w the peptide was insoluble in the solvents used to dissolve samples for TLC and HVE. Abbreviations: BA=benzylamide; PEA=pheaethylamide; PPA=phenylpropylamide; PBA = phenylbutylamide.

potency of Met-enkephalin. (Tyr-D-AIa-Trp-NH2 was made by Dr. K. Channabasavaiah by similar methods; it has 6% of Met-enkephalin potency). Again, as in the case of deletion tetrapeptide amides, a D-AIa in position 2 causes a g r e a t increase in activity over that of the L-AIa residue, and the smaller the peptide analog, the greater the increase produced by changing the configuration of the alanine. The C-terminal carboxyl function of methionine was reduced to the alcohol in the pentapeptide Tyr-D-Ala-GlyPhe-Met to give Tyr-D-Ala-Gly-Phe-Met-ol, an analog with 2.5 times the potency of the parent peptide acid and a prolonged activity. The alcohol was also reported to be effective when taken orally [17]. The deletion tetrapeptide Tyr-D-

Ala-Phe-Met-ol had only half the activity of Met-enkephalin and Tyr-D-Ala-Phe-Met-NHz [2], but the D-Ala tripeptide alcohol Tyr-D-AlaoPhe-ol (peptide 13) is equipotent with the tripeptide amide (peptide 12) on the guinea pig ileum. It is possible that C-terminal alcohol analogs of peptides related to Tyr-D-Ala~ would be more resistent to e n z y m a t i c attack and thus could find use as orally active opiates. The influence of protecting the C-terminal amino acid residue as an amide can be seen in the tripeptides as well as in the deletion tetrapeptides and in the pentapeptides. Peptides 8, 9 and 10 are [des-GiyZ'a]-enkephalinanalogs with free carboxylic acids at the C-terminal. They show no activity in the guinea pig ileum assay. The corresponding amides 12, 15

VAVREK E T A L .

306

TABLE 2 POTENCIES OF ENKEPHALINANALOGSON THE GUINEA PIG ILEUM* Peptide Number Peptide Structure

1 2 3 4 5 6 7 8 9 10 lI 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Tyr-GIy-Gly-Phe-Met Tyr-Gly-Gly-Phe-Met-NHz Tyr-D-Ala-Gly-Phe-Met-NH2 Tyr-D-Ala-Gly-Phe-Met-ol Tyr-D-Ala-Phe-Met-NH2 Tyr-D-Ala-Phe-Leu-NHz Tyr-D-Ala-Phe-Met-ol Tyr-Phe-Met Tyr-Phe-Leu Tyr-Phe-Met-NHz Tyr-Phe-Leu-NHz .Tyr-Ala-Phe-NHz Tyr-Pro-Phe-NH2 Tyr-Aib-Phe-NH2 Tyr-D-Ala-Phe Tyr-D-Phe-Met Tyr-D-Phe-Leu Tyr-D-Phe-Phe Tyr-D-AIa-Phe-NH2 Tyr-D-Ala-Phe-ol Tyr-D-Pro-Phe-NHz Tyr-D-Phe-Met-NH2 Tyr-D-Phe-Leu-NH2 Tyr-D-Phe-Phe-NH2 Tyr-D-Phe-D-Met Tyr-D-Phe-D-Leu Tyr-D-Ala-D-Phe Tyr-D-Phe-D-Phe Tyr-D-Phe-D-Met-NHz Tyr-D-Phe-D-Leu-NHz Tyr-D-AIa-D-Phe-NHz Tyr-D-Phe-D-Phe-NH2 Tyr-D-Phe-NH2 Tyr-D-Ala-BA (benzylamide) Tyr-D-AIa-PEA (phenethylamide) Tyr-D-Ala-PPA (phenylpropylamide) Tyr-D-Ala-PBA (phenylbutylamide)

Relative Potencies 100 160 140 360 120 120 50 0 m 0 0 0 19 2 0 0 (a) 0 (a) 0.1 (t) 25 24 0. I 9 4 23 (b,g) 0 (a) 0 (b) 0. l (t) 0 (c) 1 (e) 0.2 (b) 2 (b) 1 (b) 0.1 8 6 77 13

*Potencies are relative to Met-enkephalin (Tyr-Gly-Gly-PheMet) = 100. Data of reference peptides taken from [2]. A typographical error in that publication gave the potency of Tyr-D-Ala-PheLeu-NHz as 20. It is equipotent with Tyr-D-Ala-Phe-Met-NH2. Unless otherwise indicated, peptide stock solutions for assay were made up at 1 mg/ml concentration in distilled water and serial dilutions made with Tyrode's solution. Peptide 2 was insoluble in all solvents tried. Solvent mixtures used had no effect on tissue responses to Met-enkephalin: (a) 5% DMSO + 5% MeOH. (b) l(~/bMeOH. (c) 10% DMSO + 10% MeOH. (d) 10% DMSO. (e) 5% MeOH. (f) at high doses only very low activity was seen; the ED~o response was never reached; the slope of the dose-response curve was less steep than that of the standard. (g) delayed onset of peak effect; standard peptide effect reached peak response within 30 see to I min; analog required 2 min before peak effect was seen.

-so

~ ,o

1o

6'0

9"0

"

,'=o

TIME A F T E R I N J E C T I O N (rain)

FIG. 1. hi vivo assay of Tyr-D-Ala-phenylpropylamide for analgesia on the rat tail-flick [2] after intracerebroventricular(ICV) injection, with Tyr-D-Ala-Gly-Phe-Met-NHz as the standard. Latencies for tail-flick determined 20 min before injectioiaofpeptide, and at 10, 30, 60, 90 and 150 min after injection. A 6-sec cutoff time was used to avoid tissue damage. To test reverslbdlty, naloxone (4 mg/kg) was administered IP at the time of ICV peptide injection of 6 ,ug of Try-D-Ala-phenylpropylamide per mouse (N=6). I. Standard --10 /.tg/mouse (N=6); 2. Standard--1 #g/mouse (N=6); 3. Tyr-D-Alaphenylpropylamide--6 pg/mouse (N=5); 4. Tyr-D-Ala-phenylpropylamide--0.6 ~g/mouse (N=5); 5. Tyr-D-Ala-phenylpropylamide--0.1 ~g/mouse (N=5). (On a molar basis, 6 ~g of Tyr-D-Alaphenylpropylamide is equivalent to 10 ~g of standard peptide.)

and 16 show a good amount of activity. The D-Phe in position 2 of the tripeptide amides Tyr-D-Phe-Met-NHz (peptide 15) and Tyr-D-Phe-Leu-NH2 (peptide 16) appears to be fulfilling a dual function in both analogs, enabling them to show significant enkephalin-like activity. In both of these analogs the second hydrophobic function which is postulated to be necessary for potent opioid activity by the three point theory of peptide-receptor interaction is on the amino acid residue adjacent to the tyrosine. Additionally, the D-Phe provides the protective function of a D-amino acid in the second position (note that neither Tyr-Phe-Met-NHz (peptide 3) nor Tyr-Phe-Leu-NHz (peptide 4) has any activity). Since Met or Leu are not necessary at the C-terminal residue in the pentapeptides or in the deletion tetrapeptides it was decided to put an additional hydrophobic amino acid in the tripeptide at the C-terminus. Tyr-D-Phe-Phe-NHz (peptide 17) had the same activity as Tyr-D-AIa-Phe-NH2 (peptide 12), about one-fourth the potency of Met-enkephalin. Tyr-D-PhePhe-NH2 had a slightly different onset of effect in the ileum assay than any of the other analogs tested. The doubling of the time necessary for the analog to reach its peak response may indicate a possible metabolic processing of the tripeptide amide. One would expect that the C-terminus of the analog would be more susceptible than the N-terminus to enzymatic cleavage by membrane bound enzymes in the ileum preparation. If this were so, then Tyr-D-Phe might be seen as the active peptide moiety interacting at the receptors on the ileum. This intriguing possibility led to the synthesis of several other D-Phe containing tripeptide amides with D-amino acids in position 3 (peptides 18, 19, 21, 22, 23 and 25). None of the analogs containing two D-amino acids with

M I N I M U M S T R U C T U R E OPIOIDS

307

free carboxylic groups at the C-terminus showed any activity, but all o f the amides of these analogs did, although of very low magnitude (1% or less). As a final check on the possibility that the two hydrophobic functions postulated for strong opiate receptor interaction could be on adjacent amino acid residues, Tyr-D-Phe-NHz (peptide 26) was made. Remarkably, it showed full intrinsic activity on the guinea pig ileum, with full naloxone reversibility, at doses 1000 times that o f Met-enkephalin. Tyr-D-Phe-NH2 is the smallest peptide we have found to give a full dose-response curve parallel to that o f Met-enkephalin in the stimulated guinea pig ileum assay, and may represent the smallest peptide which is capable of exhibiting full enkephalin-like activity. Morley [16] had postulated a "minimal fragment" of the enkephalins as the tripeptide amide Tyr-Gly-Glyphenethylamide, which is the decarboxylated analog o f Tyr-Gly-Gly-Phe, and Kosterlitz [9] showed that the phenethyl amide of Tyr-D-Ala-Gly had h~ vitro activity similar to that o f the tetrapeptide Tyr-D-Ala-Gly-Phe. Based on the significant hz vitro activity found in Tyr-D-AIa-Phe-NH2 (peptide 12), we decided to make a series of amides using various aralkylamines. The benzyl-, phenethyl-, phenylpropyl- and phenylbutyl-amides of the dipeptide Tyr-D-Ala showed full intrinsic activity in the ileum assay. The potencies of Tyr-D-Ala-benzylamide (peptide 27) and Tyr-D-Alaphenethylamide (peptide 28) were 6--8% o f Met-enkephalin, and about one-third as potent as Tyr-D-Ala-Phe-NH2 (peptide 12). However, by extending the alkyl chain separating the aromatic group o f the amide from the peptide backbone by incorporating phenyipropylamine, and at the same time increasing the flexibility of the alkylamide chain, the most potent enkephalin analog in this series o f tripeptide and dipeptide amides was obtained. Tyr-D-Ala-phenylpropylamide (peptide 29) had close to 80% of the enkephalin-like activity o f Met-enkephalin, three times the potency of Tyr-D-AlaPhe-NH2 (peptide 12). Extending the chain by an additional methylene group (Tyr-D-Ala-phenylbutylamide 30) de-

creased potency, but apparently the added flexibility did allow for higher potency than that seen with the benzyl- and phenethylamides. Recently is was found that the deletion tetrapeptide amide Tyr-D-Ala-Phe-Met-NHz is less potent hz vivo than the parent pentapeptide Tyr-D-Ala-Gly-Phe-Met-NH2 even though the in vitro activities are comparable [2]. The high hz vitro activity of Tyr-D-Ala-phenylpropylamide (peptide 29) prompted a preliminary look at its hz vivo analgesic activity as measured by the mouse tail-flick assay after intraventricular injection. The standard, Tyr-D-Ala-Gly-Phe-Met-NH2, produced a profound and long-lasting analgesia at a dose of 10 /.tg/ mouse. Surprisingly, at a molar equivalent dose (6 rig/mouse) Tyr-D-Ala-phenylpropylamide exhibited a similar profound and long lasting analgesia. When both peptides are administered at doses 1/10th as large, the analgesic effects are of similar magnitude but the duration o f action o f Tyr-D-Ala-phenylpropylamide is shorter than that of the standard. At a dose o f 0.1 rig/mouse Tyr-D-Ala-phenylpropylamideproduced a slight and transient analgesia, demonstrating a normal dose-response relationship. The analgesic effect o f Tyr-D-Ala-phenylpropylamide was fully reversible when peptide and naloxone were administered together. Undoubtly, part o f the in vivo potency of the dipeptide aralkylamides is due to their resistance of enzymatic degradation, since all of the amide bonds involve the D-Ala residue. But this does not explain the almost 10-fold greater activity of Tyr-D-Ala-phenylpropylamide over the benzyl-, phenethyl- and phenylbutylamides. Further experiments are under way to delineate more precisely the requirements for highly effective opioid-receptor interaction.

ACKNOWLEDGEMENTS The authors thank Ms. Virginia Sweeney for the amino acid analyses.

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