Cyclic Retro-lnverso Dipeptides with Two Aromatic Side Chains. 1. Synthesis KEN-ICHI NUNAMI," TOSHIMASA YAMAZAKI, and MURRAY G O O D M A N ' Department of Chemistry, 0343, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0343

SYNOPSIS

A series of cyclic retro-inverso dipeptides-2- [ ( 4-hydroxy ) benzyl] -5-benzyl4,6 ( lH,2H,3H,5H) -pyrimidinedione (c [ mPhe-gTyr] ) , 2-benzyl-5- [ (4-hydroxy)benzyl] 4,6 ( 1H,2H,3H,5H) -pyrimidinedione ( c [ mTyr-gPhe] ) , and 2-benzyl-5-amino-5- [ (4-hydroxy)benzyl] -4,6( lH,2H,3H,5H) -pyrimidinedone { c [ (a-amino)mTyr-gPhe] 1 -were synthesized in order to define the minimum structural requirements for binding affinity with opiate receptors and biological activity. Although the first two compounds lack a free amine proposed to be necessary for receptor recognition, the c [ mPhe-gTyr ] and c [ mTyrgPhe] analogues serve as model molecules in conformational studies of the target analogue, c[ (a-amino)mTyr-gPhe]. The cis- and trans-c [ (a-amino)mTyr-gPhe] contain all the functional groups such as the amine and phenolic groups in the tyrosine, and the aromatic group in the phenylalanine, necessary for opiate activity. In addition, the c [ (&-amino)mTyrgPhe] analogues possess similar geometries to the Tyr-Pro part of morphiceptin (Tyr-ProPhe-Pro-NH,) whose high preceptor activity is attributed to conformations with the TyrPro amide bond in a cis conformation because the peptide bonds assume a cis conformation. However, both analogues are inactive in the guinea pig ileum and the mouse vas deferens assays. This may result from wrong orientation of the benzyl group of the gPhe residue with respect to the (a-amino)mTyr residue. Conformational studies of these molecules using 'H-nmr spectroscopy and molecular mechanics calculations will be reported in the following paper. Results of conformational analysis should provide information about backbone-side-chain interactions in the retroinverso peptide chains since all the fundamental structural elements of the retro-inverso peptides are included in these model systems even though the peptide bonds must assume a cis conformation.

INTRODUCTION There have been numerous studies of peptide opioids such as enkephalins (Tyr-Gly-Gly-Phe-Leu/MetOH), dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-ProSer-NH2), and morphiceptin (Tyr-Pro-Phe-ProNH,), all of which function as neurotransmitters and neuromodulators. The results of these efforts have led to the general agreement that the amine Biopolymen, Vol. 31, 1503-1512 (1991) 0 1991 John Wiley & Sons,Inc. CCC 0006-3525/91/131503-10$04.OO

* Present address: Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-Chome. Yodogawa-ku, Osaka 532, Japan. To whom correspondence should be addressed.

'

and phenolic groups in the tyrosine at the first position and the aromatic group in the phenylalanine at the third or fourth position are required for opiate receptor recognition. It has been suggested that there exists at least three different receptors, p, 6, and K , and perhaps others as well. Each receptor requires different conformations with specific arrays of the functional groups. In terms of molecular conformations, spacer residues, which joins the biologically important Tyr and Phe residues, plays a significant role to orient these residues in the correct array necessary for opiate activity. Each class of opioid shows distinct chiral requirements for spacer residues. Incorporation of an L-amino acid at position 2 of dermorphin results in a remarkable reduction in bioac1503

1504

NUNAMI, YAMAZAKI, AND GOODMAN

tivity. Morphiceptin requires an L-chirality for proline at the second position. In enkephalins, the glycine at the third position cannot be readily substituted with a D- or an L-amino acid, whereas the glycine a t the second position can be replaced with most D-amino acids without significantly affecting bioactivity.2 The existence of a D residue at the second position enhances the stability against the cleavage of the peptide bond between the Tyr and second residue by an aminopeptidase. In an attempt to inhibit the enzymatic cleavage and degradation of the native enkephalins, we have developed the retro-inverso modification reversing the direction of the peptide bond. The partially retro-inverso modified enkephalin analogues are found to be 2-15 times more active than the parent enkephalins and exhibit a much longer duration of acti~n.~?~ The development of structure-activity relationships for the linear opioid peptides has been hampered by the inherent flexibility of the linear molecules and lack of high specificity for the multiple opiate receptors, e.g., p , 6, and K receptors. The incorporation of constraints for the linear peptides through cyclization not only reduces the conformational flexibility, but also leads to more potent and/or highly p or 6 receptor selective anal o g u e ~ . ~Utilizing -~ both the retro-inverso modification and cyclization techniques, we have investigated the synthesis and conformational analysis of a series of cyclic retro-inverso modified enkephalin analogues.'-13 Of the constrained peptidomimetics synthesized, 14-membered cyclic enkephalin analogues showed superactivity, high receptor selectivity, and greater resistance against enzymatic degradation. In the series of our integrated studies on opioid peptides incorporating peptidomimetics, another

H

S O

N

W

o

n

part of our main subject involves defining the minimum structural requirements for binding affinity with the opiate receptors and biological activity. The linear dipeptide kyotorphin, Tyr-Arg, isolated from bovine brains, has an analgesic effect that is abolished by nalo~one.'~-'~ Unlike other opioid peptide analgesics, kyotorphin is devoid of activity on the stimulated guinea pig ileum bioassay and similarly does not bind with specific opiate re~eptors.'~.'' Takagi et al.14r1' and Jaicki et a1.l' have independently reported that [Met'] -enkephalin is released by kyotorphin from the slices of striatum and spinal cord of guinea pigs and rats. Therefore, kyotorphin may produce its enkephalin-like analgesic effect indirectly, through the release of enkephalins. Because the aromatic side chains of the tyrosine and the phenylalanine have been shown to play important roles in biological activity and receptor selectivity of opioid peptides, we focused on cyclic retro-inverso dipeptides with aromatic rings ( Figure 1) . These six-membered peptidomimetics, which have similar structures to barbiturates, are also expected to interact with barbiturate receptors and perhaps to clear the blood brain barrier in uiuo. The target compound, 2-benzyl-5-amino-5- [ (4hydroxy ) benzyl] - 4,6 ( 1H,2H,3H,5H ) -pyrimidinedione { c [ (a-amino)mTyr-gPhe] ; Figure l a } , contains all the structural requirements for opiate activity. In order to estimate preferred conformations of the above molecule, the two molecules of this series-2- [ (4-hydroxy)benzyl] 5 - benzyl - 4,6(1H,2H,3H,5H) - pyrimidine dione ( c [ mPhe-gTyr] ; Figure l b ) and 2-benzyl5 - [ ( 4 - hydroxy ) benzyl] - 4,6 (lH,2H,3H,5H)- py rimidinedione ( c [ mTyr-gPhe] ; Figure l c ) -serve as model compounds in spite of lacking a free amine, which has been proposed to be a necessary component for biological activity. In this paper, we report

\=/

H

%

HN

W

H

(b)

(c)

Figure 1. Structural formulae of cyclic retro-inverso dipeptides with two aromatic side chains: ( a ) c [ ( a-amino)mTyr-gPhe] , (b) c [ mPhe-gTyr] , and ( c ) c [ mTyr-gPhe] .

CYCLIC RETRO-INVERSO DIPEPTIDES. I

the syntheses of these cyclic retro-inverso dipeptides. Conformational analysis of these molecules using ‘H-nmr spectroscopy and energy calculations will be reported in the following paper.20 Conformational studies of these cyclic retro-inverso dipeptides with aromatic side chains should provide information regarding backbone-side-chain interactions in the retro-inverso peptide chains as all the fundamental structural elements of the retro-inverso peptides are included in these model systems even though the peptide bonds must assume the cis conformation.

BJ EtO-m 1) Zn I AcOH

2) DCHA EtO-m

RESULTS AND DISCUSSION Compound (6) (c [ mPhe-gTyr] ) was synthesized by the route shown in Figure 2. Thus, EtO-(R,S)mPhe-L-Tyr ( But) -NH2 ( 3 ) , which was derived by the reaction of monoethyl benzylmalonate ( 1)with 0-tert-butyl-L-tyrosine amide hydrochloride ( 2 ) in the presence of N-hydroxysuccinimide ( HOSu) and N,N-dicyclohexylcarbodiimide (DCC ) , was converted to EtO- ( R,S ) -mPhe- ( S ) -gTyr ( B u t ) H * TFA ( 4 ) (TFA: trifluoroacetic acid) using [ bis (trifluoroacetoxy ) iodo] benzene (IBTFA) .21*22 Cyclization was then achieved with a saturated solution of NH3 in MeOH to give a mixture of transc [ mPhe-gTyr ( But) ] [ trans- ( 5 ) ] and cis-c [ mPhegTyr(But)] [cis-(5)] in an approximate ratio of 1 : 1 in an 30.3% yield. The stereochemistry of these isomers was elucidated by comparison of the methine proton signals of the mPhe and gTyr ( But) res-

Figure 2. Scheme for the synthesis of c[ mPhe-gTyr] ( 6 ) . The “m” and “g” notations denote the malonyl and gem-diamino analogues of the named amino acids.

!

.L

/

BJ

YOH’DCHA

(10)

Figure 3. Alternate synthetic route for c [ mPhe-gTyr ] ( 6 ) . The “m” and “g” notations denote the malonyl and gem-diamino analogues of the named amino acids.

idues in the ‘H-nmr spectra. The methine proton signals of the trans isomer appear a t a much higher field than those of the corresponding cis isomer because of the magnetic anisotropy of the adjacent aromatic rings. Recrystallization of the mixture from aqueous MeOH gave pure trans- ( 5 ) in 20.5% yield. This result shows that each isomer is interconvertible in solution. The trans compound came out of an equilibrated solution in aqueous MeOH. Compound trans- ( 5 ) was deprotected with TFA and thioanisole, giving a mixture of trans- ( 6 ) and cis( 6 ) . Pure cis- ( 6 ) was obtained by recrystallization of the mixture from MeOH. These facts suggest that protonation on amide carbonyl groups in a protic solvent such as TFA or MeOH leads to facile enolization of the malonyl residue. Based on this hypothesis, we attempted to synthesize trans- ( 6 ) by hydrogenolysis of transc [ mPhe-gTyr (Bzl)] ( 12) in an aprotic solvent. Since oxidative cleavage of benzyl phenyl ethers by IBTFA has been reported, an alternate synthetic method shown in Figure 3 was adopted. The O-ben-

1506

NUNAMI, YAMAZAKI, AND GOODMAN

zyl-L-tyrosine phenacyl ester trifluoroacetate was coupled with monoethyl benzylmalonate ( 1) using ethyl-3- ( 3-dimethylamino) propylcarbodiimide hydrochloride (EDAC * HC1). The Curtius rearrangement of this analogue with diphenylphosphorylazide (DPPA) generated the isocyanate, which was trapped with p-methoxybenzyl alcohol (pMZ) to yield the linear retro-inverso dipeptide ( 11) . Acidolytic cleavage of the pMZ group followed by cyclization using a saturated solution of NH3 in MeOH gave a mixture of cis- and trans-c [ mPhe-gTyr (Bzl) ] [cis- and trans- (12)]. Recrystallization from MeOH afforded trans- ( 12). Catalytic hydrogenation of trans- (12) in the presence of Pd-black in dimethylformamide (DMF) unexpectedly produced a mixture of cis- ( 6) and trans- ( 6 ) . Racemization of an optically pure 2-substituted malonyl residue has been observed for linear retroinverso peptides,’l yet for cyclic compounds such as the 14-membered enkephalin analogues, racemization has not been detected.a11Since the 6-membered pure compounds will racemize, the amide bonds play an important role for racemization of the malonyl residue. Compound ( 19) (c [ mTyr-gPhe] ) ,which is a reversed analogue of (6 ), was synthesized according to the scheme given in Figure 4. Ethyl 4-tert-butoxybenzylmalonate ( 14), which was converted from

EtO-m

diethyl 4-benzyloxybenzylmalonate ( 13), was coupled to the L-phenylalanine amide ( 15). Rearrangement with IBTFA followed by cyclization with a saturated solution of NH3 in MeOH was carried out yielding c [ mTyr ( But) -gPhe] ( 18). Recrystallization of the deprotected c [ mTyr-gPhe] (19) from MeOH gave trans- ( 19). Figure 5 shows the synthetic route of ~ [ ( a amino)mTyr-gPhe] (25), which contains all the functional groups required for opiate activity. Ethyl tert-butoxycarbonylaminomalonate( 20) was condensed with L-phenylalanine amide (15) using EDAC-HC1 to give the dipeptide analogue (21). Compound (21) was converted to N- [ (R,S)-N-Boca-EtOCO-Gly ] - ( S )-gPhe-H * TFA using IBTFA, and then cyclized with a saturated solution of NH3 in MeOH to give a mixture of cis- and trans- (22). Alkylation of compound ( 22) with 4-benzyloxybenzyl chloride was investigated under several conditions using NaH, ButOK, EtONa, DBU, or inorganic reagents as a base. The alkylation using 1.2 equivalents of NaOEt in EtOH at 45°C proceeded in 54% yield. Under this condition (22) and (23) were hardly soluble, while an intermediate enolate form of (22) was soluble in EtOH. This difference in solubility avoided further alkylation of the product. Recrystallization of crude crystals gave trans- ( 23), which was subsequently deprotected stepwise, re-

Phe

kBzl OEt

(13)

2) CH,=C(CH,), / H+

3) KOH

But

EtO-

m But

1 1

TFA*H

m

m

I

NHz

(16)

H*TFA

(17)

IBTFA g

1

(15)

EDAC*HCI

EtO-m But

NH,

NH, / MeOH g

(18)

g

(19)

TFA Thioanisole

Figure 4. Synthetic scheme for c [ mTyr-gPhe] ( 19). The “m” and “g” notations denote the malonyl and gem-diamino analogues of the named amino acids.

CYCLIC RETRO-INVERSO DIPEPTIDES. I

1507

0 Recrystallization _______)

from O

I

H

(4-0Bzl)QCH;

cis- and ?rum-(23)

1) H2l Pd

2) TFA

O

H

rrum-(23)

1

H2 I Pd

3) Reverx Phase HPLC 4) HCI / dioxane

cis-(25)

?ram-(25)

Figure 5. Synthetic scheme for cis and trans-c [ (cy-amino)mTyr-gPhe] (25). The “m” and “g” notations denote the malonyl and gem-diamino analogues of the named amino acids.

sulting in the expected trans-c [ (a-amino)mTyrgPhe] hydrochloride [trans-(25) 1. The mother liquor of the above recrystallization underwent hydrogenation followed by acidolysis and was then subjected to reverse-phase preparative high performance liquid chromatography (HPLC) using CIScoated silica gel to give cis- (25) as crystals. None of the cyclic retro-inverso dipeptides examined in this investigation, including cis- and trans-c [ (a-amino)mTyr-gPhe] , which contain all the functional groups such as the amine and phenolic groups in the tyrosine and the aromatic group in the phenylalanine necessary for opiate activity, display biological activity for either the guinea pig ileum or mouse vas deferens assays. From conformational studies of morphiceptinrelated analogues, we have recently reported that a cis conformation about the peptide bond between residues 1 (Tyr) and 2 (Pro) is required for biolog-

ical a~tivity.’~ Since the peptide bonds in c[ (aamino) mTyr-gPhe] assume a cis conformation, this analog possesses similar geometries to the Tyr-Pro part of the morphiceptin analogues. Therefore, the lack of biological activity for both the cis- and transc [ (a-amino) mTyr-gPhe ] may result from the wrong orientation of the benzyl group of the gPhe residue relative to the amine and phenolic groups in the ( aamino ) mTyr residue. It has been reported that dipeptides with structures of Tyr-D-Ala-NH ( CH2),Ph ( n = 1-4) display opiate activity and that Tyr-D-Ala-NH( CH2)3Phis the most active among this series of corn pound^.^^ These observations prompted us to carry out conformational energy calculations of the series of cyclic retro-inverso dipeptides containing ( CHp),Phe ( n = 2-4) in place of the benzyl group of the trunsc [ (a-amino)mTyr-gPhe] analogue. The n = 2 and 4 analogues assume similar topologies to the pre-

1508

NUNAMI, YAMAZAKI, AND GOODMAN

conformations of morphiceptin and thus are expected to show opiate activity. The results will be reported elsewhere together with experimental data.

EXPERIMENTAL Measurements

Melting points were determined in open glass capillaries using a Thomas-Hoover melting point apparatus and were uncorrected. Specific rotations were measured on a Perkin-Elmer 141 polarimeter using a 10-cm path-length water-jacketed cell. Infrared spectra were obtained on a Nicolet 7199 Fourier transform ir spectrophotometer with an MTC detector. Proton nmr (lH-nmr) spectra were recorded on a General Electric GN-500 spectrometer using tetramethylsilane as an internal standard. Elemental analyses were performed by Desert Analytics (Tucson, Arizona). Fast atom bombardment mass spectra (FAB MS) were carried out at the University of California in Riverside. Materials EtO-(R,S)-mPhe-r-Tyr(Bu ')-NH, (3). To a solution of ethyl benzylmalonate (1) (1.11 g, 5 mmol) and HOSu (0.69 g, 6 mmol) in DMF ( 10 mL) was added

DCC (1.24 g, 6 mmol) under ice cooling. After stirring for 30 min O-tert-butyl-L-tyrosine amide hydrochloride ( 2 ) ( 1.52 g, 5 mmol) and triethylamine (0.84 ml, 6 mmol) in DMF (10 mL) were added to the solution and stirred overnight at room temperature. Insoluble materials were filtered off and the filtrate was dried under reduced pressure. The residue was dissolved in ethyl acetate ( AcOEt ) ,washed with 0.5M citric acid, saturated aqueous NaHC03, and brine; dried over MgSO,; and taken to dryness under reduced pressure. The residue was subjected to column chromatography of silica gel eluting with CHC13-AcOEt( 1: 1) and evaporated under reduced pressure. The residue was triturated with isopropyl ether ( i P r 2 0 ) ,giving ( 3 ) as a crystalline solid; 1.9 g (86.4%). 'H-nmr in DMSO-&, 6: 1.08, 1.11 (t, t, J = 7 Hz, 3H, CHzCH3),1.23, 1.28 (s, s, 9H, B u t ) , 2.61-3.15 [ dd, 4H, CH24, CH2( 4-OBut)6],3.50-3.85 (dd, lH, COCHCO ) ,3.90-4.15 (q, q, J = 7 Hz, 2H, CH2CH3),4.36-4.55 (m, l H , NCHCO), 6.78-7.45 [m, 11H, (4-OBut)4,&,NH2],8.32,8.44 (d,d, NH). Anal. Calculated for C25H32N205: C, 68.16; H, 7.32; N, 6.36%. Found C, 67.89; H, 7.51; N, 6.31%. c[rnPhe-gTyr(Bu')] ( 5 ) . A mixture of ( 3 ) (0.88 g, 2 mmol) and IBTFA (1.03 g, 2.4 mmol) in aceto-

nitrile (CH3CN) (10 mL) and H20 (4 mL) was stirred for 1 h at room temperature. After removal of the solvent under reduced pressure, AcOEt was added to the residue and dried over MgSO,. The solvent was evaporated under reduced pressure and the residue was dissolved in MeOH (30 mL) and then saturated with NH3 gas at -50°C. The solution stood for 2 days a t room temperature and was taken to dryness under reduced pressure. AcOEt was added to the residue giving crystalline solids as a mixture of cis- and trans- (5); 220 mg (30.3% ) . 'H-nmr in DMSO-& (the chemical shift for the trans isomer is given first in each case), 6: 1.26, 1.27 (s, s, 9H, But), 2.21, 3.47 (t, J = 4.0 Hz, t, J = 5.4 Hz, l H , COCHCO),2.79, 2.62 ( d , J = 4.0 H z , d , J = 5.4 Hz, 2H, CH24), 2.98, 2.95 [d, J = 4.9 Hz, d, J = 5.5 Hz, 2H, CH2(4-OBut)4],4.55,4.99 (m, m, lH, NCHN), 6.88 and 7.07,6.87 and 7.05 [ AzB2,J = 8.5 Hz, A2Bz, J = 8.5 Hz, 4H, (4-OBut)4],7.10-7.23 (m, 5H, 4 ) , 8.39, 8.36 ( d , J = 3 Hz, bs, 2H, 2NH). Recrystallization of these crystals from aqueous MeOH gave trans- (5); 150 mg (20.5% ). mp 235237°C. Infrared (KBr) vmaX:3189,3058,1681, 1650 cm-' , ir ( MeOH) v,: 1708 cm-' . Anal. Calculated for C22H26N203: C, 72.11; H, 7.15; N, 7.65%. Found C, 72.36; H, 7.27; N, 7.71%. c[mPhe-gTyr] (6). A mixture of trans-(5) (0.10 g,

0.27 mmol) and thioanisole (0.5 mL) in TFA (10 mL) was stirred for 10 min under ice cooling and stirred for 50 min at room temperature. The solvent was removed under reduced pressure and then the residue was triturated with EtzO to give a mixture of cis- and trans-(6) as a crystalline solid; 70 mg (82.6%). 'H-nmr in DMSO-d, (the chemical shift for the trans isomer is given first in each case), 6: 2.24, 3.46 (t, J = 4.1 Hz, t, J = 5.3 Hz, lH, COCHCO), 2.70, 2.58 ( d , J = 4.1 H z , d , J = 5.3 Hz, d, J = 6.6 Hz, 2H, CH&), 2.98, 2.97 [d, J = 5.2 Hz, 2H, CHz(4-OH)4],4.48,4.93 (m, m, lH, NCHN), 6.66 and 6.93,6.67 and 7.05 [ AzBz,J = 8.6 Hz, A2B2, J = 8.6 Hz, 4H, (4-OH)4], 7.10-7.24 (m, 5H, 4), 8.33, 8.27 (d, J = 2.1 Hz, bs, 2H, 2NH), 9.26, 9.22 (bs, bs, l H , OH). Recrystallization of the crystals from MeOH gave cis- ( 6 ) ; 50 mg (59.0% ); mp 279-281°C (dec.). Infrared (KBr) vmax: 3320,1657,1616,1594,1513 cm-'. FAB MS (m/e): 310 (M'). Anal. Calculated for Cl8HI8N2O3:C, 69.66; H, 5.85; N, 9.03%. Found C, 69.59; H, 5.91; N, 8.95%. The same result was obtained using a mixture of cis- and trans- (5) as a starting material. N-Boc-r-Tyr(B+I)-OPac (8). A mixture of O-benzyl-

N-tert-butoxycarbonyl-L-tyrosine (3.71 g, 10 mmol) ,

CYCLIC RETRO-INVERSO DIPEPTIDES. I

l-bromoacetophenone (2.91 g, 11 mmol), and triethylamine (1.54 ml, ll mmol) in DMF (20 mL) was stirred overnight at room temperature. The solvent was removed under reduced pressure and AcOEt was added to the residue. The insoluble materials were filtered off and the filtrate was washed with saturated aqueous NaHC03 and brine, dried over MgS04, and then evaporated to dryness under reduced pressure. Trituration of the residue with hexane gave (8) as crystals that had been recrystallized from AcOEt-hexane; 4.2 g (85.7% ); mp 102103°C. [a]? -20.6" ( c l , DMF). 'H-nmr in DMSO4, 6: 1.32 (s, 9H, B u t ) , 2.87, 3.15 [dd, J -14.2 and 11.0 Hz, dd, J = -14.2, 3.6 Hz, 2H, CH2(4OBzl)$], 5.07 (s, 2H, OCH&), 5.50, 5.64 (d, J = -16.9 Hz, d, J = -16.9 Hz, 2H, OCH&O4), 6.94 and 7.23 [ AzB2,J = 8.6 Hz, 4H, (4-OBzl)41, 7.307.99 [m, 11H, NH, (4-OCHz4)4 and OCHzC04]. Anal. Calculated for C29H31NOS: C, 71.14; H, 6.38; N, 2.86%. Found: C, 70.91; H, 6.52; N, 2.69%. 7

EtO- (R, S) -mPhe-r- Tyr (Bzl)-0Pac (9). A solution of (8) (2.45 g, 5 mmol) in TFA (20 mL) was stirred for 10 min under ice cooling and stirred for 50 min

at room temperature. The solution was evaporated to dryness under reduced pressure and then triturated with EtzO, giving crystals that were used for the next reaction without purification. To a solution of the above crystals, EtO-mPheOH (1) (1.11 g, 5 mmol), 1-hydroxybenzotriazole (HOBt; 0.81 g, 6 mmol), and EDAC HC1 ( 1.18 g, 6 mmol) in DMF ( 15 mL) and triethylamine (0.84 mL, 6 mmol) was added at -20°C. Stirring was continued overnight a t room temperature; then the solvent was removed under reduced pressure. The residue was dissolved in AcOEt, washed with 0.5Mcitric acid, saturated aqueous NaHC03, and brine, and dried over MgSO,. After removal of MgS04 by filtration, the solution was evaporated to dryness under reduced pressure. The residue was triturated with hexane giving ( 9 ) as Crystals; 2.4 g (80.8%). 'H-nmr in DMSO-4, 6: 1.02, 1.12 (t, t, J = 7 Hz, 3H, CH2CH3),2.72-3.30 [ m, 4H, CH2$ and CH2(4OBzl)4], 3.65, 3.80 (m, m, lH, COCHCO), 3.95, 4.04 (4, q, J = 7 Hz, 2H, CH2CH3),4.58, 4.80 (m, m, l H , NCHCO), 5.10 (s, 2H, OCH&), 5.60 (m, 2H, OCH,CO$), 6.90-8.10 [m, 19H, 4, (4OCH24)4, and OCH2C04],8.69-8.83 (m, lH, NH ) Anal. Calculated for C36H35N07:C, 72.77; H, 5.94; N, 2.36%. Found C, 72.81; H, 5.81; N, 2.25%.

-

.

€ t o - (R, S) -mPhe-r- Tyr (Bzl)-OH N, N-Dicyclohexylamine Salt (10). To a solution of ( 9 ) (2.20 g, 3.7

mmol) in AcOH (20 mL) was added portionwise zinc powder ( 1.21 g, 18.5 mmol) a t 50°C. After stir-

1509

ring was continued overnight at room temperature, the insoluble materials were filtered off. The filtrate was evaporated to dryness under reduced pressure and dissolved in EtzO. To the solution, N,Ndicyclohexylamine (0.67 g, 3.7 mmol) was added, giving (10) as crystals; 1.64 g (67.5%).Anal. Calculated for C40H52N206: C, 73.14; H, 7.98 N, 4.27%. Found C, 73.01; H, 8.11, N, 4.15%. EtO-(R,S)-mPhe-(S)-gTyr(Bz1)-pMZ ( 1 I ) . A suspension of (10) (1.50 g, 2.28 mmol) in AcOEt was treated with 2 N NaHS04 ( 10 mL) , giving the free carboxylic acid as an oil. To a solution of this oil and DPPA (0.75 g, 2.74 mmol) in benzene, triethylamine (0.4 mL, 2.86 mmol) was added at room temperature. After stirring was continued for 4 h, 4methoxybenzyl alcohol (0.63 g, 4.56 mmol) was added. The solution was refluxed for 2 h, washed with saturated aqueous NaHC03 and brine, and dried over MgSO,. After removal of MgS04,the filtrate was evaporated to dryness under reduced pressure. The residue was subjected to column chromatography of silica gel eluting with CHCl3-AcOEt ( 19 : 1) and triturated with iPrzO to give ( 11 as crystals, which were recrystallized from AcOEtiPr20;0.83 g (59.7% ). 'H-nmr in DMSO-4,6: 1.05, 1.07 (t, t, J = 7 Hz, 3H, OCHzCH3),2.61-3.04 Em, 4H, CH24 and CH,( 4-OBzl) $1, 3.58-3.67 (m, IH, COCHCO), 3.72 (s, 3H, OCH3), 3.91-4.05 (9, q, 2H, OCH&H3), 4.89 [ S, 2H, OCH2 (4-OBzl)$],5.05 (s, 2H, OCH24), 5.18-5.30 (m, m, lH, NCHN), 6.86-7.54 [ m, 18H, ( 4-OCHz4)4, 4, and OCHz (4OMe)&],8.42-8.64 (m, H, CONH). Anal. Calculated for C36H38N207: C, 70.80; H, 6.27; N, 4.59%. Found C, 70.59; H, 6.37; N, 4.39%. Trans-c[mPhe-gTyr(Bz/)] [trans-(12)]. A solution of (11) (0.72 g, 1.18 mmol) in TFA (20 mL) was

stirred for 10 min under ice cooling and for 20 min at room temperature. The solution was evaporated to dryness under reduced pressure, dissolved in MeOH (50 mL) , and saturated with NH3 gas at -40°C. The mixture stood for 2 days at room temperature and then the solvent was removed under reduced pressure. The residue was subjected to column chromatography of silica gel eluting with CHC13-MeOH ( 9 : l ) , giving crystals that were recrystallized from MeOH to afford trans- (12); 120 mg (25.5%); mp 219-220°C. Infrared (KBr) urnan: 3195,1678, 1646, 1512 cm-'. 'H-nmr in DMSO-4, 6: 2.28 (t, J = 3.7 Hz, l H , COCHCO), 2.75 (d, J = 3.7 Hz, 2H, CH&), 2.99 [d, J = 4.9 Hz, 2H, CHz(4-OBzl)d], 4.48 (m, lH, NCHN), 5.07 (s, 2H, OCH&), 6.93-7.49 [ m, 14H, ( 4-OCHz4)4 and 41, 8.45 (d, J = 2.6 Hz, 2H, 2NH). FAB MS (m/e):

1510

NUNAMI, YAMAZAKI, AND GOODMAN

401 (MH'). Anal. Calculated for Cz5HZ4N2O3: C, 74.98; H, 6.04; N, 7.00%. Found: C, 74.90; H, 5.89; N, 6.94%. Hydrogenation of (12). A mixture of (12) (0.10 g,

0.25 mmol) and Pd-black (10 mg) in DMF ( 5 mL) was stirred for 4 h under atmospheric pressure of hydrogen. The catalyst was filtered off and the filtrate was dried under reduced pressure. The residue was triturated with EtzO to yield a mixture of cisand trans- ( 6 ) as crystals; 60 mg ( 77.4% ) .

DiethyI4-benzyloxybenzylmalonate( 1 3). To a solution of diethyl malonate (1.60 g, 10 mmol) in tetrahydrofuran (20 mL), sodium hydride (0.29 g, 12 mmol) was added at room temperature. Stirring was continued for 15 min at the same temperature. 4Benzyloxybenzyl chloride (2.32 g, 10 mmol) was added to the reaction mixture, and allowed to stand overnight. The solvent was removed under reduced pressure. The residue was subjected to column chromatography of silica gel eluting with benzene-hexane ( 19 : 1) to afford ( 13) as a colorless oil; 3.0 g (84.3%) . 'H-nmr in DMSO-4, 6: 1.17 (t, J = 7 Hz, 6H, 2CH,CH,), 3.19 (s, 2H, CH2+), 4.12 (9, J = 7 Hz, 4H, 2CH2CH3),5.06 ( s, 2H, OCH2+),6.88 and 7.05 [A2B2,J = 10 Hz, 4H, (4-OBzl)4], 7.31-7.48 (m, 5H, 4). FAB MS (m/e): 356 ( M + ) . €to-(R,S)-mTyr(8u ')-r-Phe-NH, (16). A mixture of (13) (2.53 g, 7.1 mmol) and Pd-black (0.1 g) in EtOH (20 mL) was stirred for 1h under atmospheric pressure of hydrogen. The catalyst was filtered off and the filtrate was evaporated to dryness under reduced pressure. The residue was dissolved in a mixture of CH2C12(50 mL), concentrated H2S04(0.2 mL) , and liquid isobutylene (20 mL) , stirred overnight at room temperature, washed with saturated aqueous NaHC03 brine and, dried over MgSO,. A mixture of the residue, obtained by removal of the solvent under reduced pressure, and KOH (0.5 g, 7.9 mmol) in EtOH ( 7 mL) was stirred for 6 h at room temperature. The solvent was removed in vacuo and 5% of NaHS04 was added to the residue and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO, , and concentrated in uucuo, giving (14) as a colorless oil, which was used for the next reaction without purification. To a mixture of the above oil, L-phenylalanine amide trifluoroacetate (15) (1.97 g, 7.1 mmol), HOBt (1.15 g, 8.5 mmol) , and EDAC HCI ( 1.62 g, 8.5 mmol) in DMF and triethylamine (1.19 ml, 8.5 mmol) was addeddropwise at -20°C. After the stirring was continued overnight a t room temperature, the solvent was removed under reduced pressure. AcOEt was added to the residue, washed with 0.5M

-

citric acid, saturated aqueous NaHCO,, and brine, dried over MgSO,, and evaporated in uucuo. The residue was subjected to column chromatography of silica gel eluting with CHCl,-AcOEt (1 : 1 ) . The eluate was concentrated in uucw and triturated with iPr20giving ( 16) as a white powder; 2.5 g (79.9%). 'H-nmr in DMSO-d,, 6: 0.99, 1.07 (t, t, J = 7 Hz, 3H, OCH,CH,), 1.24,1.25 (6, S, 9H, B u t ) , 2.61-3.04 [ m, 4H, CH24and CH2( 4-OBut)d], 3.68,3.72 (t, t, J = 7.5 Hz, IH, COCHCO), 3.87-4.05 (m, 2H, OCH,CH,), 4.36-4.51 (m, l H , NCHN), 6.81, 6.82, 7.00 [A2B2, J = 8.4 Hz, 4H, (4-OBut)4],7.00-7.40 (m,7H,4andCONH2),8.27,8.37 ( d , d , J = 8.4 Hz, C, 68.16; lH, NH) .Anal. Calculated for C25H32N205: H, 7.32; N, 6.36%. Found C, 68.01; H, 7.34; N, 6.25%. c[mTyr(Bu')-gPhe] (18). A mixture of (16) (0.72 g, 1.64 mmol) and IBTFA (0.85 g, 2 mmol) in CH&N (10 mL) and H20 ( 4 mL) was treated as described for the synthesis of ( 5 ) , giving a mixture of cis- and trans- ( 18) as crystals; 170 mg (28.3% ) . 'H-nmr in DMSO-d, (the chemical shift for the trans isomer is given first in each case), 6: 1.25,1.21 (s, s, 9H, But), 2.29, 3.30 ( t , J = 4.9 Hz, t, J = 5.4 Hz, lH, COCHCO), 2.81, 2.59 (d, J = 4.0 Hz, d, J = 5.4 Hz, 2H, CH2$), 2.94, 2.94 [d, J = 4.9 Hz, d, J = 4.9 Hz, 2H, CH2(4-OBut)+],4.45, 4.97 (m, m, lH, NCHN), 6.78 and 6.96, 6.80 and 7.04 [A2B2,J = 8.4, A2B2, 10.4 Hz, 4H, (4-OBut)4], 7.17-7.31 (m, 5H, 4 ) , 8.43, 8.38 (d, J = 2.5 Hz, d, J = 1.8 Hz, 2H, 2NH). Recrystallization of the crystals from aqueous MeOH gave trans- ( 18); 120 mg ( 20.0% ) ; mp 231232°C. Infrared (KBr) v,,: 3280,1687,1655,1625, 1506 cm-'. c[mTyr-gPhe] ( 1 9). A mixture of ( 18) (30 mg, 0.08

mmol) and thioanisole (0.25 mL) in TFA ( 5 mL) was treated as described for the synthesis of ( 6 ) , giving a mixture of cis- and trans-(19); 20 mg (78.7%). 'H-nmr in DMSO-d, (the chemical shift for the trans isomer is given first in each case), 6: 2.24, 3.29 (t, J = 4.7 Hz, t, J = 5.1 Hz, l H , COCHCO),2.80, 2.58(d,J = 3.9 H z , d , J = 5.4 Hz, 2H, CH2$), 2.88, 2.87 [d, J = 4.7 Hz, d, J = 5.1 Hz, 2H, CH2(4-OH)4],4.44,4.95 ( m , m , lH, NCHN), 6.56 and 6.86,6.60 and 6.93 [ A2B2,J = 8.4 Hz, AzBz, J = 8.4 Hz, 4H, (4-OH)4], 7.15-7.31 (m, 5H, +), 8.35, 8.30 (d, J = 1.8 Hz, bs, 2H, 2NH), 9.09, 9.06 (bs, bs, l H , OH). The crystals were recrystallized from aqueous MeOH giving truns- ( 19); 10 mg (34.9% ) ; mp 249250°C. FAB MS (m/e) : 310 ( M + ) .Anal. Calculated for C18H18N203:C, 69.66; H, 5.85; N, 9.03%. Found C, 69.53; H, 5.91; N, 8.89%.

CYCLIC RETRO-INVERSO DIPEPTIDES. I

N- [ (R,S) -N-tert-Butoxycarbonyl-a-ethoxycarbon-

ylglycyl]-1-Phenylalanine Amide (21). To a solution of ethyl N-tert-butoxycarbonyl-aminomalonate (20) (4.50 g, 18.2 mmol), L-phenylalanine amide trifluoroacetate ( 15) (5.00 g, 18.2 mmol), HOBt (2.96 g, 21.9 mmol), and EDAC HC1 (4.18 g, 21.9 mmol) in DMF (30 mL) and triethylamine (2.8 mL, 20 mmol) was added dropwise at -20°C. After the stirring was continued overnight at room temperature, the solvent was removed under reduced pressure. The residue was dissolved in AcOEt, washed with 0.5M citric acid, saturated aqueous NaHCO, and brine, dried over MgSO, ,and taken to dryness under reduced pressure. The residue was triturated with iPr20 giving (21) as a white powder; 5.81 g (81.3%). 'H-nmr in DMSO-$, 6: 1.07, 1.08 (t, t, J = 7 Hz, 3H, CH2CH,), 1.38, 1.39 (s, s, 9H, But), 2.80-3.20 (m, 2H, CH.&), 3.97-4.20 (m, 2H, CH2CH,), 4.40-4.58 (m, l H , NCHCO), 4.80-4.90 (m, lH, COCHCO), 7.07-7.60 (m, 5H, 4), 8.48,8.58 (d, d, l H , NH ) . FAB MS (m/e) : 394 (MH' ) .Anal. Calculated for C19H2,N306:C, 58.00; H, 6.91; N, 10.68%. Found C, 57.69; H, 7.11; N, 10.85%.

-

cia - (tert - Bufoxycarbony/amino)mCly - gPhe] (22). Compound (21) (3.80 g, 9.7 mmol) was treated as described for the synthesis of ( 5 ) . The residual oil was subjected to column chromatography of silica gel eluting with CHCI,-MeOH ( 15 : 11. The eluate was evaporated under reduced pressure and triturated with Et20, giving a mixture of cis- and trans-(22) as crystals; 1.3 g (42.1%). 'H-nmr in DMSO-$, 6: 1.16, 1.22, 1.32, 1.40 (s, S, S, S, 9H, But), 2.84-3.03 (m, 2H, CH24),4.76,5.94 (d, J = 9.0 Hz, d, J = 8.5 Hz, l H , COCHCO), 5.16-5.34 (m, lH, NCHN), 6.88, 7.01 (d, J = 8.5 Hz, d, J = 9.0 Hz, lH, BocNH), 7.15-7.50 (m, 5H, 4 ) , 8.45-8.60 (m, 2H, 2NH). FAB MS (m/e): 320 (MH'). Anal. Calculated for C16H21N304:C, 60.17; H, 6.63; N, 13.16%.Found C, 59.98; H, 6.77; N, 13.31%. c [ a-(tert- Bufoxycarbonylamino)m Tyr (Bzl)-gPhe]

(23). A solution of (22) (0.32 g, 1 mmol), NaOEt (82 mg, 1.2 mmol), and 4-benzyloxybenzylchloride (0.26 g, 1.1mmol) in EtOH ( 10 mL) was stirred for 1.5 h at 45°C. After the solvent was removed under reduced pressure, 0.5M citric acid and AcOEt was added to the residue. The crystals were collected by suction and recrystallized from MeOH giving trans(23); 120 mg (23.3%); mp 239-240°C. Infrared (KBr) v,: 3250,3220,1711,1687,1659,1513cm-'. 'H-nmr in DMSO-&, 6: 1.34 (s, 9H, But), 2.65 (d, 2H, CH24),2.83 [s, 2H, CH2(4-OBzl)4],3.69 (m, lH, NCHN), 5.06 (s, 2H, OCHz4), 6.88 and 7.05

1511

[A2B2,J = 8.4 Hz, 4H, (4-OBzl)r$], 7.10-7.46 (m, 10H, CHCH24 and OCH24), 7.93 (bd, 2H, 2NH 1, 8.12 (bs, lH, BocNH). FAB MS (m/e): 516 (MH+). Anal. Calculated for C30H33N305: C, 69.88; H, 6.45; N, 8.15%. Found C, 69.73; H, 6.50; N, 8.11%. The organic layer was washed with brine and dried over MgSO, .The residue obtained by removal of the solvent under reduced pressure was subjected to column chromatography of silica gel eluting with CHC13-MeOH (15 : 1).The eluate was evaporated under reduced pressure and triturated with Et2O to give cis-rich product (23) ; 160 mg (31.0% ) . 'H-nmr in DMSO-& (the chemical shift for the cis isomer), 6: 1.25 ( s , 9H, But), 2.01 (d, 2H, CH24), 2.72 [s, 2H,CH2(4-OBzl)4],4.76(m, lH,NCHN),5.05 (s, 2H, OCH&), 6.82-7.20 [ AzB2, 4H, (4-OBzl)41, 6.82-7.46 (m, 10H, CHCH24and OCH24),8.04 (bd, 2H, 2NH), 8.16 (bs, l H , BocNH). Trans-c[ a-(tert-Butoxycarbonylamino)mTyr-gPhe] [trans-(24)]. A mixture of trans-(23) (0.10 g, 0.2

mmol) and Pd-black ( 5 mg) in AcOH (10 mL) was stirred overnight under atmospheric pressure of hydrogen. After the catalyst was filtered off, the filtrate was evaporated under reduced pressure and triturated with Et20 to give trans- (24) as crystals. The product was recrystallized from MeOH; 80 mg (94.1% ) ; mp 223-224°C (dec.) . 'H-nmr in DMSO4, 6: 1.04, 1.33 (s,S, 9H, Bu'), 2.50 (s, 2H, CH24), 2.87 [ s, 2H, CH2(4-OH)41, 3.61 (m, l H , NCHN), 6.60-7.28 [m, 9H, (4-OH)cP and $1, 7.87 (s, 2H, 2NH), 8.11 (bs, lH, BocNH), 9.35 (bs, l H , OH). Anal. Calculated for C23H2,N305:C, 64.93; H, 6.40; N, 9.88%. Found C, 64.79; H, 6.51; N, 9.73%.

Trans-c[(a-Amino)mTyr-gPhe] Hydrochloride [trans-(25)]. A solution of (24) (80 mg, 0.19 mmol) in TFA (15 mL) was stirred for 10 min under ice

cooling and for 50 min at room temperature. After the solvent was removed under reduced pressure, 4 N HCl in dioxane (0.1 mL) was added to the residue. The residue obtained by removal of the solvent under reduced pressure was triturated with Et20giving trans- ( 2 5 ) as crystals, which were recrystallized from MeOH-Et20; 65 mg (94.6%); mp 230-231°C (dec.). 'H-nmr in DMSO-$,6: 2.83 (d, J = 3.8 Hz, 2H, CH&), 3.09 [s, 2H, CH2(4-OH)4],4.36 (m, l H , NCHN), 6.71 and 7.00 (A2BZ,J = 8.4 Hz, 4H, (4-OH)4], 7.20-7.36 (m, 5H, 4 ) , 8.29 (bs, 2H, NH2), 9.18 (5, 2H, 2NH), 9.49 (s, l H , OH). FAB MS (m/e): 326 (MH+-HCI).Anal. Calculated for C1aH19N303*HCl:C, 59.75; H, 5.57; N, 11.61; C1, 9.80%. Found C, 59.57; H, 5.65; N, 11.37; C1,10.11%.

1512

NUNAMI, YAMAZAKI, AND GOODMAN

Cis-c[ (a-amino)mTyr-gPhe] Hydrochloride [cis(25)]. A mixture of cis- and trans-(23) (0.1 g 0.2 mmol) and Pd-black ( 10 mg) in AcOH ( 10 mL) was stirred for 2 h under atmospheric pressure of hydrogen. After filtration of the catalyst, the filtrate was evaporated under reduced pressure. A solution of the residue in TFA (15 mL) was stirred for 10 min under ice cooling and for 50 min at room temperature. The residue obtained by removal of TFA under reduced pressure was triturated with E t 2 0 giving a mixture of cis- and trans- (25) as crystals; 70 mg (96.7% ) . The two diastereomers (50 mg) were separated by reverse-phase preparative HPLC using Cls coated silica gel with a gradient (10% aqueous CH3CN 0.1% TFA + 35% aqueous CH3CN 0.1% TFA). The fractions were taken to dryness under reduced pressure and 4 N HC1 in dioxane (0.1 mL) was added to the residue. Trituration with E t 2 0 gave cis- and trans- (25) as crystals. The products were recrystallized from MeOH-EtzO; cis- (25): 50 mg (69.4%) ; mp 234-235°C (dec.). FAB MS (m/e): 326 ( M H + HC1). 'H-nmr in DMSO-4, 6: 2.17 (d, J = 5.4 Hz, 2H, CH,$), 2.70 [s, 2H, CHz(4-OH)q5], 4.79 (m, lH, NCHN), 6.73 and 6.87 [A2B2,J = 8.4 Hz, 4H, (4-OH)$], 7.09 (d, J = 7.4 Hz, 2H, 42.6 of CH2$), 7.22 (t,J = 7.4 Hz, l H , $4 of CH&), 7.31 (t,J = 7.4, 2H, 43,s of CH2$), 8.53 (bs, 2H, NHz), 9.15 (d, J = 2.1 Hz, 2H, 2NH), 9.45 (bs, l H , OH). Anal. Cal* HC1: C, 59.75; H, 5.57; N, culated for CISHl9N3O3 11.61; C1, 9.80%. Found C, 59.61; H, 5.71; N, 11.39; C1,9.99%. Trans-(25): 10 mg (13.9%).

+

+

The authors gratefully acknowledge the support of the NIH DK 15410 and NIH GM 18694. One of us ( K N ) would like to thank Tanabe Seiyaku Co., Ltd.

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F., Vane, J., Wilkinson, S., Chang, K., Cuatrecasas, P. & Miller, R. (1977) Proc. Roy. SOC.Lond. 198, 249-253. 2. Morley, J. (1980) Ann. Rev. Phurmacol. Toxicol. 20, 81-110. 3. Goodman, M. & Chorev, M. (1981) in Perspectiues in Peptide Chemistry, Eberle, A., Geiger, R., & Wieland, T., Eds., Karger, Basel, pp. 283-294. 4. Chorev, M., Shavitz, R., Goodman, M., Minick, S. & Guillemin, R. (1979) Science 204,1210-1212. 5. DiMaio, J., Nguyen, T. M.-D., Lemieux, C. & Schiller, P. W. (1982) J . Med. Chem. 25,1432-1438.

6. Mosberg, H. L., Hurst, R., Hruby, V. J., Galligan, J. J., Burks, T. F., Gee, K. & Yamamura, H. L. ( 1983) Life Sci. 32, 2565-2569. 7. Spatola, A. F., Mapelli, C., Humphrey, J., Edwards, J. V., Burks, T. & Shook, J. E. (1987) in Peptides 1986, Proceedings of the 19th European Peptide Symposium, Theodoropoulos, D., Ed., Walter de Gruyter, Berlin, pp. 381-384. 8. Berman, J. M., Jenkins, N., Hassan, M., Goodman, M., Nguyen, T. M.-D. & Schiller, P. W. (1983) in Peptides: Structure and Function, Pierce Chemical Co., Rockford, IL, pp. 283-286. 9. Berman, J. M., Goodman, M., Nguyen, T. M.-D. & Schiller, P. W. (1983) Biochern. Biophys. Res. Commun. 115,864-870. 10. Berman, J. M. & Goodman, M. (1984) Znt. J. Peptide Protein Res. 23,610-620. 11. Richman, S. J., Goodman, M., Nguyen, T. M.-D. & Schiller, P. W. (1985) Znt. J . Peptide Protein Res. 2 5 , 648-662. 12. Hassan, M. & Goodman, M. (1986) Biochemistry 2 5 , 7596-7606. 13. Mammi, N. J. & Goodman, M. (1986) Biochemistry 2 5 , 7607-7614. 14. Takagi, H., Shiomi, H., Ueda, H. & Amano, H. ( 1979) Nature 282,410-412. 15. Takagi, H., Shiomi, H., Ueda, H. & Amano, H. (1979) Eur. J . Pharmacol. 55,109-111. 16. Vaught, J. L. & Chipkin, R. E. (1982) Eur. J. Pharmacol. 79,167-173. 17. Rackham, A., Wood, P. L. & Hudgin, R. L. (1982) Life Sci. 30,1337-1342. 18. Shiomi, H., Kuraishi, Y., Ueda, H., Harada, Y., Amano, H. & Takagi, H. (1981) Brain Res. 221,161169. 19. Janicki, P. K. & Lipkowski, A. W. (1983) Neuroscience 43,73-77. 20. Yamazaki, T., Nunami, K. & Goodman, M., the fol-

lowing paper. 21. Pallai, P., Richman, S. J., Struthers, R. S. & Goodman, M. (1983) Znt. J. Peptide Protein Res. 21,84-92. 22. Pallai, P. & Goodman, M. (1982) Chem. Commun. 280-281. 23. Yamazaki, T., Probstl, A., Schiller, P. W. & Goodman, M. (1991 ) Znt. J . Peptide Protein Res. 37,364-381. 24. Casiano, F. M., Cumiskey, W. R., Gordon, T. D.,

Hansen, P. E., Mckay, F. C., Morgan, B. A., Pierson, A. K., Rosi, D., Singh, J., Terminiello, L., Ward, S. J. & Wescoe, D. M. (1983) in Peptides: Structure and Function; Proceedings of the 8th American Peptide Symposium, Hruby, V. J. & Rich, D. H., Eds., Pierce Chemical Co., Rockford, IL, pp. 311-314.

Received October 6, 1990 Accepted July 9, 1991