J . Med. Chem. 1980,23, 1113-1122 mmol) was added. This mixture was cooled to 0 “C and pyridine (0.81 mL, 10 mmol) was added dropwise. After stirring for 10 min at the same temperature, the mixture was concentrated and diluted with AcOEt. The solution was washed successively with dilute aqueous NaHC03, 1 N HCl, and brine, dried over MgSO,, and evaporated to give an oil, which was chromatographed on a silica gel column, eluting with CH,Cl,-AcOEt (gradient elution),
1113
to give 1.69 g (87%) of 19 as an amorphous solid (see Table I).
Acknowledgment. The authors are grateful to Y. Miyazaki for technical assistance, Dr. T. Takaya who provided US some samples of a-(alkoxyimino)acetic acid, and T. Kamimura for biological testing.
Novel Analogues of Enkephalin: Identification of Functional Groups Required for Biological Activity Fredric A. Gorin, T. M. Balasubramanian, Theodore J. Cicero, John Schwietzer, and Garland R. Marshall* Departments of Physiology and Biophysics, of Psychiatry, and of Neurobiology, Washington University, St. Louis, Missouri 63110. Received April 14, 1980 Novel tri- and tetrapeptide analogues of enkephalin, in conjunction with earlier structure-activity data, confirm that chemical substituents present in the first and fourth residues of enkephalin are required for in vitro biological activity. A class of arylamino and alkylamino derivatized tripeptides also were found to have signficiant in vitro opioid-like activity indistinguishable from [~-Ala~,~-Leu~]enkephalin and morphine.
appears that naloxone binds to the morphine ( w ) binding The enkephalins are the smallest members of a family site with 30-fold greater affinity than the enkephalin of opioid-like peptides endogenous to the mammalian binding site.3 The analogues of enkephalins cited in this central nervous system. The in vivo pharmacology of the article have been evaluated in both bioassay systems, and enkephalins and opiates is complex, and there is growing excellent correlation has been found between the ranked evidence that these compounds act in vivo at several types potencies of compounds relative to [~-Ala’,~-Leu~]enkeof In in vitro systems such as the guinea pig phalin tested in the two in vitro systems. This article uses ileum,4 mouse vas defer en^,^ neuroblastoma glioma the information derived from the structure-activity data NG108-15cell line,6and rat brain binding assays,’ opiates of these analogues of enkephalin to deduce requirments and the enkephalins produce analogous pharmacologic about the morphine ( p ) receptor. effects which are reversibly blocked by the opiate antagonist naloxone. These in vitro observations have motivated Experimental Section investigators to compare chemical and conformational Materials. All Boc’O amino acids, except Boc-Tyr-OH, Bocsimilarities between the enkephalins and opiate comTyr(aMe)-OH, and Boc-Phe(aMe)-OH, were purchased from pounds. Numerous conformational studies have proposed Bachem. L-Tyr was obtained from Fluka. The preparation of receptor-bound conformations for enkephalins whereby Boc-Phe(aMe)-OH is described elsewhere.”* The Boc-Leu-resin these mammalian pentapeptides and plant opiate alkaloids was prepared by the method of Gisinllb using Lab Systems 1% pharmacologically compete for the same in vitro receptom8 cross-linked polymer (0.90 mequiv/g). Di-tert-butyl dicarbonate This article describes novel, conformationally conwas obtained from Fluka. 1-Aminoindan, 2-aminoindaq benzylamine, 1-(aminomethyl)cyclopropane,and 1-(aminomethyl)strained analogues of enkephalin which are evaluated cyclobutane were purchased from Aldrich. 0-Phenylethylamine pharmacologically in the guinea pig ileum and which was purchased from Sigma. [3H]Naloxone (26 Ci/mmol) was displace [3H]naloxone in the rat brain binding assay. obtained from New England Nuclear, and bacitracin was purThere is no assurance that the pharmacologic receptors chased from P. L. Biochemicals. [Aib2,Met-NH2]Enkephalinwas in these two in vitro assays are equivalent, and recent obtained from Peninsula Laboratories. investigation has suggested the existence of separate high Analytical Methods. Melting points are uncorrected. DMF affinity “enkephalin receptors” and “morphine ( p ) was purified according to the procedure described by Stewart and receptors” present in the rat brain binding a s ~ a y . ~It* ~ ? ~Young.12 Ascending TLC was performed on 0.25-mm silica gel G plates (Analtech) using the following solvent systems: (I) J. N. Jaffe and W. R. Martin, in “The Pharmacological Basis of Therapeutics”, L. S. Goodman and A. Gilman, Eds., MacMillan, New York, 1975, p 245. J. A. Lord, A. A. Waterfield, J. Hughes, and H. W. Kosterlitz, Nature (London),267,495 (1977),and references cited within. K.-J. Chang and P. Cuatrecasas, J . Biol. Chem., 254, 2610 (1979). H. W. Kosterlitz and A. J. Watt, Br. J . Pharmacol., 33, 266 (1968). G. Henderson, J. Hughes, and H. W. Kosterlitz, Br. J.Pharmacol., 46, 764 (1972). A. L. Lampert, M. Nirenberg, and W. A. Klee, Proc. Natl. Acad. Sci. U.S.A., 73, 3165 (1976). C. B. Pert and S. H. Snyder, Mol. Pharmacol., 10, 868 (1974). F. A. Gorin, T. M. Balasubramanian, C. D. Barry, and G. R. Marshall, J. Supramol. Struck, 9, 27 (1978), and references cited within. 0022-2623/80/ 1823-1113$01.OO/O
chloroform-acetone, 151; (11) chloroform-acetone, 7:l; (111) chloroform-acetone, 72; (IV) chloroform-acetone, 1:l; (V) chlo(9) P. E. Gilbert and W. R. Martin, J. Pharmacol. E z p . Ther., 198, 66 (1976). (IO) Abbreviations used are: Boc, tert-butyloxycarbonyl; TLC, thin-layer chromatography; LC, liquid chromatography; DCC, dicyclohexylcarbodiimide;Phe(aMe), a-methylphenylalanine (yl-);DMF, dimethylformamide; TFA, trifluoroacetic acid; Bzl, benzyl; TEA, triethylamine; DPPA, diphenylphosphoryl azide; HOBT, 1-hydroxybenzotriazole; DEPC, diethylphosphoryl cyanidate; Cha, L-cyclohexylalanine;Aib, aminoisobutyric acid. (11) (a) J. Turk, P. Needleman, and G. R. Marshall, Mol. Pharmacol., 12, 217 (1976); (b) B. F. Gisin, Helu. Chim. Acta, 56, 1476 (1973). (12) J. M. Stewart and J. D. Young, “Solid Phase Peptide Syntheses”, W. H. Freeman, San Francisco, 1969. 0 1980 American Chemical Society
1114 Journal of Medicinal Chemistry, 1980, Vol. 23, No. 10
roform-methanol, 7:l; (VI) chloroform-methanol, 3:l; (VII) chloroform-methanol, 1:l; (VIII) chloroform-methanol, 1:4;(IX) 1-butanol-acetic acid-water, 4:l:l; (X) I-butanol-ethyl acetateacetic acid-water, 2:2:1:1. The loads of compounds applied on the TLC plates were 25 pg (in 5 p L of methanol), and the distances of the solvent fronts were usually 14 cm. Plates were developed by spraying with buffered ninhydrin12 and Chlorox-starchI3 reagents. Purified peptides were analytically chromatographed using high-pressure liquid chromatography (LC) as described in the next section. Using isocratic conditions listed in Table I, the optical isomers of the 1-aminoindan analogues of enkephalin were resolved. All analogues of enkephalin demonstrated impurities of less than 0.1% by analytical high-pressure LC with UV monitoring a t 210 k 8 and 280 k 8 nM. Optical rotations were measured using a Perkin-Elmer Model 241 polarimeter. Peptide hydrolyses of protected amino acids were performed in vacuo using concentrated hydrochloric acid-propionic acid, 1:1, at 130 "C for 2 h.14 Unprotected peptides were hydrolyzed in vacuo using constant-boiling hydrochloric acid for 18 h at 110 "C.15 Phe(aMe) and Tyr(aMe) content was measured by enzymatic treatment of a portion of the hydrolysate with L-amino acid oxidase as described elsewhere.ll8 Purification by High-pressure LC. Deprotected peptides synthesized by solution phase techniques were purified by high-pressure LC on a semipreparative scale (ca. 10 mg) using two Waters 6000 A pumps, an M660 solvent programer, a Rheodyne injector with a 100-pL injection loop, and a Varichrom UV variable detector. Analytical and preparative separations were performed using two 30 cm X 4 mm reverse-phase C18 pBondapak columns (Waters) connected in series. All organic solvents were of W spectroscopic grade (Burdick and Jackson) and were filtered and degassed prior to use. The eluted peaks were monitored at 210 h 8 nm. The deprotected peptide (20 to 30 mg) was dissolved in 2 to 3 mL of UV spectroscopic grade methanol, filtered with a Millipore-Swinney apparatus, and evaporated to a residue with nitrogen. Analytical determination of purification conditions for each peptide was obtained by running a linear, 30-min gradient from 5% acetonitrile95% 0.01 M ammonium formate, pH 4.00, to 70% acetonitrile-30% 0.01 M ammonium formate as a flow rate of 2.3 mL/min. The desired peptides eluted in the range of 20 to 40% acetonitrile. A semipreparative run was then performed by injecting 100 p L of an 80 to 100 mg/mL solution of peptide in aqueous buffer with a miniial amount of acetonitrile when necessary to clarify the solution. A series of linear, stepwise gradients was programmed with a 20% increase in acetonitrile over 5 min. It was observed that, because of slight sample overloading of the stationary phase, a peak would elute on the semipreparative run at the same retention time as the analytical trial with a solvent composition containing 5 to 8% less acetonitrile. Fractions of 0.8 to 1.0 mL were collected from the desired peaks and evaporated a t 25 "C in a Buchler Vortex evaporator. Each fraction was checked on thin-layer chromatography using solvent systems M and X, and the pertinent fractions were pooled and lyophilized from an excess of glass-distilled water. Using this approach, 8 to 10 mg of peptide could be purified in a single 30to 40-min high-pressure LC run with 4&50% recovery of purified peptide. The residual ammonium formate was sublimed by continuing to evaporate the fractions under vacuum at 35 'C for 3 to 6 h. Quantitative amino acid analysis demonstrated the purified peptides to have 0 to 60% residual ammonium formate. Table I summarizes the physiochemical properties of these peptides purified by high-pressure LC. Bioassay. The compounds were assayed on electrically stimulated, intact, guinea pig ileum using the conditions of Creese and Snyder.I6 Because the native enkephalins have been demonstrated to be proteolytically labile in this ~ y s t e m , ' ~all * ' ana~ (13) D. E. Nitecki and J. W. Goodman, Biochemistry, 5,665 (1966). (14) J. Scotchler, R. Lozier, and A. B. Robinson, J . Org. Chem., 35, 3151 (1970). (15) S. Moore, in "Chemistry and Biology of Peptides", Johannes
Meienhofer, Ed., Ann Arbor Science Publishers, Ann Arbor, Mich., 1972, p 629. (16) T. Creese and S. H. Snyder, J . Pharmacol. E x p . Ther., 194, 205 (1975).
Gorin et al.
m m o m ~ m w ~ c - o o c - m m m m m m
?u!????"??????Y??Y?
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
*
2 (o
2
a J B 5cL
Novel Analogues of Enkephalin
Journal of Medicinal Chemistry, 1980, Vol. 23, No. 10 1115
Iogues of enkephalin were evaluated in the presence and absence of 100 rg/mL bacitracin. This concentration of bacitracin was demonstrated not to affect the dose-response curve of morphine or [D-Ala2,D-LeuS]enkephalin. The IDW used to calculate the potency of an analogue with respect to the reference analogue [~-Ala~,~-Leu~]enkephalin is the mean of these separate determinations. Before and after every complete set of dose-response determinations, the ICa dosage of the reference enkephalin analogue was administered to assure that tissue responsiveness waa altered by less than 15%. The naloxone binding assays were carried out essentially as described previo~sly.'~Briefly, rats were killed by decapitation, and the brains (minus the cerebellum) were obtained. A crude particulate fraction was prepared by homogenizing the brains in 100 volumes of 0.05 M Tris-HC1 buffer (pH 7.6). The homogenate was centrifuged at 4oooOg for 10 min, the supernatent fluids were discarded, and the pellet was washed twice in 40 mL of the Trii-HC1 buffer and homogenized with seven strokes in a loose-fitting glass homogenizer. This preparation was used in all subsequent incubations. Incubations consisted of the following (all concentrations are final): 100 WLof the washed particulate fraction [3H]naloxone (26 Ci/mmol, 5 X M), 1 x lo-' levorphanol, 5 X M bacitracin, and enough buffer to make a total volume of 500 wL. Assays were carried out in the presence and absence of 100 mM NaCl in duplicates or triplicates. The incubations were carried out in an ice-water bath for 2.5 h, with constant agitation, and were terminated by centrifugation (40000g for 10 min). The pellet was washed three times with 3 mL of Tris-HC1 and the final pellet was dissolved in 200 pL of NCS tissue solubilizer. The dissolved pellets were then transferred to 20-mL plastic scintillation vials, and the contents of the tubes were quantitatively transferred with 3 X 1-mL washes with Scintiverse liquid scintillation cocktail. Seven milliliters of Scintiverse was placed in each vial, and 2 drops of glacial acetate acid were added to prevent quenching. The vials were then counted in a liquid scintillation counter. Specific [3H]naloxone binding was determined by subtracting the counts per minute bound in those incubations containing [3H]naloxone and 1 X M levorphanol from those containing [3H]naloxone alone. In all of the experiments reported here, approximately 65-70% of the [3H]naloxonebound was specific. Boc-Tyr-D-Ala-Gly-Phe(aMe)-Leu-resin (1). Boc-Leu-resin (1.84 g, 0.818 mequiv/g) was deprotected with 50% trifluoroacetic acid in toluene for 30 min. This was proceeded by three swelling steps in toluene, three shrinking steps in 95% tert-butyl alcohol-5% toluene, and three swelling steps in toluene. A sixfold excess of Boc-Phe(aMe) in toluene was coupled with an equivalent amount of DCC for two successive 4-h periods, with appropriate shrinkageawelling cycles preceding and following each coupling.1° A double deprotection cycle of two 30-min periods with appropriate washes preceded the coupling step which used a sixfold excess of Boc-Gly. The remaining synthetic steps were carried out by the customary cycle of operations.20 In some cases [Boc-Gly and Boc-Tyr(Bzl)], solubility problems required the addition of a small amount of distilled, dry DMF. Fifty percent TFA in toluene was made just prior to each deprotection step. Tyr-D-Ala-Gly-Phe(aMe)-Leu (2a) a n d Tyr-D-Ala-GlyPhe(aMe) (2b). Compound 1 was suspended in 40 mL of anhydrous HBr/TFA in the presence of a 50-fold excess of anisole. The resin was washed three times with 15-mL aliquots of TFA and then concentrated to a residue under vacuum. The peptide was dissolved in 50 mL of 10% HOAc, extracted four times with .ethyl ether, and lyophilized. The yield was 350 mg (36%). The peptide was purified by 198 transfers in a countercurrent distribution using solvent system X. Tubes 120-152 ( K = 0.69) contained 77.6 mg of the peptide, shown by amino analysis to be 2a [Tyr, 0.92; D-Ala, 1.02; Gly, 1.00; Phe(aMe), 0.98; Leu, 0.971, while tubes 80-100 ( K = 0.45) contained 39.3 mg of 2b [Tyr,0.92;
D-Ala, 0.96; Gly, 1.00; PhebMe), 1.00; Leu,
