Int. J . Peptide Protein Res. 35. 1990. 452459

Preparation and opioid activities of N-methylated analogs of [D-Ala2 ,Leu5]enkephalin

MASAO KAWAI, NORIHITO FUKUTA, NAOKI ITO. TAKATOYO KAGA I, YASUO BUTSUGAN, MUTSUMI MARUYAMA.? and YOSHIHISA K U D O Y

Department of Applied Chemistry, Nagoya Institute of Technology, Nagoya, and Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan

Received 6 July. accepted for publication 3 November 1989

Analogs of opioid pentapeptide [D-Ala2 ,Leu']enkephalin were prepared using two kinds of N-methylation reactions, namely quaternization and amide-methylation. Quaternization reaction with CH, I-KHCO, in methanol was applied to the deprotected N-terminal group of the pentapeptide derivatives affording trimethylammonium group-containing analogs. [Me: Tyr' ,~-Ala',Leu~]enkephalin and its amide were found to show opioid activity on guinea pig ileium assay only slightly lower than the parent unmethylated peptides. Application of amide-methylation reaction using CH, I-Ag, 0 in D M F to the protected pentapep- tide yielded a pentamethyl derivative in which all of the five N atoms were methylated. Deprotection of the derivative gave pentamethyl analogs of [D-AIa' ,Leu5]enkephalin, which showed no significant activity on the guinea pig ileum assay and opiate-receptor binding assay.

Key words: N-methylated [D-AIa',Leu']enkephalin; quaternization of primary amino group; N-methylation of peptide linkage; trimethylammonium group-containing analogs of enkephalin

N-Methylated analogs of biologically active peptides have been reported frequently since they often exhibit enhanced potencies or prolonged activities (1). Such analogs are usually prepared by peptide synthesis using N-methylated amino acid derivatives in addition to normal protected amino acids as building blocks. An alternative method for synthesizing analogs con- taining N-methyl group(s), however, is modification of the parent peptides, i.e., selective methylation on N-atom(s) of the parent peptide molecules. N- Methylation of solvent-exposed amide linkages (2) and quaternization reaction of primary amino group (3) were successfully applied to the preparation of analogs of gramicidin S , an antibiotic cyclic decapep- tide (4,5). In this paper we will describe application of the methylation reactions to the preparation of the analogs of linear pentapeptide [~-Ala',Leu~]enke- phalin (Tyr-D- Ala-Gly-Phe-Leu) 1 (6) and the opioid activities of the resulting N-methylated analogs.

SYNTHESIS OF PROTECTED PENTAPEPTIDE

Fully protected pentapeptide Boc-Tyr(Bz1)-D-Ala- Gly-Phe-Leu-OMe 2 which was used for the prepara- tion of N-methyl analogs was synthesized using dicy- clohexylcarbodiimide as a coupling agent and 1- hydroxybenzotriazole as an additive. For the protec- tion of N- and C-termini and phenolic OH of Tyr residue, Boc group, Me or Bzl ester, and Bzl ether groups, respectively, were employed. The synthetic scheme of 2 is given in Fig. 1 . The corresponding Et ester Boc-Tyr(Bz1)-D-Ala-Gly-Phe-Leu-OEt 2' was

DCC-HOBI

TfoOH OM OM

DCC-HOB1

Abbreviations: Me: Tyr, N,N,N-trimethylated Tyr residue carrying a positive charge on N atom; DMF, N,N-dimethylformamide; DCC, dicyctohexylcarbodiimide; HOBt, I-hydroxybenzotriazole; Tfa, CF,CO; DCHA, dicyclohexylamine.

452

FIGURE 1 Synthetic scheme of Boc-Tyr(Bz1)-D-Ala-Gly-Phe-Leu-OMe 2.

N-methylated [D-Ala', Leu'lenkephalin

also synthesized similarly using Leu-OEt instead of Leu-OMe.

ester 4 was prepared by the quaternization of Tyr-D- Ala-Gly-Phe-Leu-OEt 3', which was obtained from 2' by catalytic hydrogenolysis followed by trifluoroacetic acid treatment. During the purification of 4' it was found that silica gel column chromatography using CHC1,-MeOH-AcOH as eluent gave a mixture of the

OF TRIMETHYLAMMoNIUM- TYPE ANALOGS

Quaternization reaction reported by Chen & Benoiton (3) consists in treatment of a compound containing free amino group with CH,I-KHCO, in methanol at room temperature to convert the H,N group into Me,N+ group.

As an attempt to prepare a trimethylammonium group-containing analog, Boc group of the protected pentapeptide 2' was removed and the resulting amino compound was subjected to the quaternization reac- tion using CH,I-KHCO, to afford I- .Me: Tyr(Bz1)- D-Ala-Gly-Phe-Leu-OEt. Removal of Bzl group of the trimethyltyrosyl residue by catalytic hydrogenolysis, however, was unsuccessful resulting in the recovery of the starting material. Model reactions using several Tyr(Bz1) derivatives were undertaken to study the observed stability of Bzl group against hydrogenoly- sis. While phenolic Bzl ether group of Boc-Tyr(Bz1)- OH was deprotected in 4 h by hydrogenolysis in MeOH in the presence of Pd black, overnight treat- ment of the trimethylammonium iodide I- .Me: Tyr(Bz1)-OMe under the same conditions did not give any deprotected product. Inhibition of catalytic re- duction by iodide ion was already described by Baltzly (7). In fact addition of 0.05 equiv. NaI to the reaction mixture containing Boc-Tyr(Bz1)-OH completely in- hibited the hydrogenolytic cleavage of the Bzl ether group. The betain Me: Tyr(Bz1)-0- , on the other hand, could be deprotected by hydrogenolysis if iodide ion was carefully removed. The pentapeptide iodide was converted into a chloride C1- *

Me: Tyr( Bz1)-D-Ala-Gly-Phe-Leu-OEt, which was still stable under the hydrogenolytic conditions. The resistance to hydrogenation of the Bzl group in the pentapeptide chloride might be attributed either to the presence of trace amounts of iodide ions or inhibitory action of trimethylammonium chloride group itself, but was not investigated further. Treatment of the trimethylated pentapeptide with HBr/AcOH-anisole, an alternative method for Bzl ether cleavage, was also found unsatisfactory, affording a mixture presumably due to electrophilic attack of Bzl cation to the phe- nolic ring. The results indicated that Bzl group must be removed prior to the quaternization.

The protected pentapeptide 2 was treated with HC1/ dioxane to remove the Boc group and the product was hydrogenated over Pd to give HCl - Tyr-D-Ala-Gly- Phe-Leu-OMe 3. The pentapeptide Me ester 3 was subjected to the quaternization reaction and column chromatographic separation of the product afforded the trimethylammonium analog Me: Tyr-D-Ala-Gly- Phe-Leu-OMe 4 in 50% yield. The corresponding Et

esters 4 and 4 due to ester exchange reaction. Hence EtOH instead of MeOH was employed for the purifi- cation of the Et ester 4'.

The zwitterionic analog Me: Tyr-D-Ala-Gly-Phe- Leu-0- 5 was prepared by alkaline hydrolysis of the ester 4 or 4 (70-80% yield). The trimethylammonium amide Me: Tyr-D-Ala-Gly-Phe-Leu-NH, 6 was also synthesized in 8 1 YO yield by quaternization of Tyr-D- Ala-Gly-Phe-Leu-NH, 7, which was obtained by methanolic NH, treatment of Boc-Tyr-D-Ala-Gly- Phe-Leu-OEt followed by the removal of the Boc group. The preparations of trirnethylammonium-type analogs of 1 are summarized in Fig. 2.

N-METHYLATION OF PROTECTED PENTAPEPTIDE

Methylation reaction using CH31 and Ag,O in DMF solution converts a peptide linkage -CO-NH- into a -CO-N(Me)- group. When the N-methylation was applied to the protected cyclic decapeptide di- phthaloylgramicidin S , only solvent-exposed N atoms were methylated while intramolecularly H-bonded NH remained intact (2,4), i.e., the N-methylation was shown to serve as a method of conformational analy- sis and also for the preparation of methylated analogs.

For the purpose of preparing partially N-methylat- ed analogs of the opioid peptide 1, methylation of the protected pentapeptide 2 was studied. From the reac- tion mixture consisting of 2, CH,I, and Ag,O in DMF, aliquots were taken out and subjected to HPLC

i l TtoOH-misole or Ha/dioxxane-onisole iil Hz-W/MeOH

iiil CHd-KHCOJMeOH ivl NoOHIW-MeOH YI NH1/MeOH

FIGURE 2 Synthetic scheme of trimethylammonium group-containing analogs of Tyr-D-Ala-Gly-Phe-Leu 1. Only protected and unprotected ter- minal amino and carboxyl groups and protected phenolic OH group of Tyr residue are shown.

45 3

M. Kawai et al.

time/h

FIGURE 3 Time course of N-methylation of each amino acid residue of Boc- Tyr(Bz1)-D-Ala-Gly-Phe-Leu-OMe 2 as analyzed by amino acid analysis.

and amino acid analysis to check proceeding of the methylation. Fig. 3 shows the time course of N- methylation of each amino acid residue in 2. Among the five methylation sites, N-atoms of D-Ala, Gly, and Phe residues were methylated faster than the urethane N of Tyr(Bz1) residue and the amide N of Leu residue. HPLC analysis, however, indicated that the methyla- tion products were complex mixtures composed of differently methylated compounds. Since it seemed difficult to isolate the partially methylated products, the attempt was confined to the isolation of only the totally methylated product, namely pentamethyl de- rivative Boc-MeTyr(Bz1)-D-MeAla-MeGly-MePhe- MeLeu-OMe 8. Thus 2 was treated with a large excess of CH,I and Ag,O in DMF solution for 42 h and the product was chromatographed over SiO, to give the pentamethyl derivative 8 in 80% yield. Amino acid

analysis of the acid hydrolyzate of 8 did not show any significant amount of unmethylated amino acids and presence of five N-methyl groups in 8 was confirmed by the 'H NMR spectrum.

The Me ester, Boc, and Bzl groups of the pen- tamethylated pentapeptide 8 were removed succes- sively by saponification, HCI treatment, and catalytic hydrogenolysis, respectively, affording the pen- tamethyl analog MeTyr-D-MeAla-MeGly-MePhe- MeLeu 9 in 58% yield. The corresponding Me ester MeTyr-D-MeAla-MeGly-MePhe-MeLeu-OMe 10 was also prepared in the same manner as above but without saponification procedure.

OPIOID ACTIVITIES OF THE N-METHYL ANALOGS

Biological activities of N-methyl group-containing analogs of [~-Ala~,Leu']enkephalin 1 were deter- mined and the results are summarized in Table 1 . The pentamethyl analog 9 and its methyl ester 10 did not show any inhibitory activity to the contraction of guinea pig ileum (GPI). The trimethylammonium analogs 5 and 6, however, were found to possess opioid activity only slightly lower than the corre- sponding unmethylated compounds 1 and 7 on the GPI assay. The activity of quaternary ammonium- containing esters 4 and 4' were very low. The effect of these analogs on the binding of 'H-labeled opioid ligands to rat brain membranes was also examined using [' HI [D-Ala' ,Me t5 Ienkephal inamide (DALAMID) (8), [,HI [~-Ala~,~-Leu']enkephalin (DADL) (6, 9, lo), and ['H][~-Ala',MePhe~,

TABLE 1 Inhibitory potencies of the N-rnethylaied analogs of [ ~ A l d , Ldlenkephalin I 01 ihe electrically induced contraction of GPI and on the

binding of 'H-labeled ligands (DALAMID. DADL, and DAGO) to ihe rat brain membranes

Analog Number GPI DALAMID DADL DAGO IC,,(DADL)

Ic , /M" I C 5 0 b / ~ I C , , b / ~ IC, ,b /~ IC,,(DAGO)

Trimethylammonium-type analogs Me, + Tyr-D-Ala-Gly-Phe-Leu 5 1.3 x ( 76) 1.0 x lo-* 3.3 x lo-* Me, + Tyr-D-Ala-Gly-Phe-Leu-NH, 6 5.2 x lo-' ( 65) 1.7 x lo-' 4.5 x lo-' Me, + Tyr-D-Ala-Gly-Phe-Leu-OMe 4 > l o - * ( 22) 1.0 4.5 x 1 0 - 8

Me, + Tyr-D-Ala-Gly-Phe-Leu-OEt 4' > 10-4 ( 7) 8.0 x 10-8 7.5 x 10-8

MeTyr-D-MeAla-MeGly-MePhe- MeLeu 9 ( 0) > I O - ~ > 10-4 MeTyr-o-MeAla-MeGly-MePhe-MeLeu-OMe 10 > ( 0) > > 10-4

N ' , N2 , N', p, P-Pentamethyl analogs

Reference peptides having no N-methyl group Tyr-D- Ala-Gly-Phe-Leu 1 8.8 x lo-' ( 78) 3.1 x lo-" 5.2 x lo-" Tyr-D- Ala-Gly-Phe-NH, 7 1.9 x ( 75) 1.7 x lo-" 7.6 x 102"

Tyr-D-Ala-Gl y-Phe-Leu-OEt 3' 1.8 x ( 63) 5.7 x lo-" 8.2 x Tyr-D- Ala-Gly-Phe-Leu-OMe 3 2.1 (100) -c -

"Inhibition (YO) induced by the analog at the concentration of 1 0 - 5 ~ is given in parentheses. bConcentration of the analog to produce 50% inhibition of the 'H-labeled ligand at 2 x 1 0 - 9 ~ . 'Not determined.

454

6.2 x I O - ~ 4.0 10-9 1.0 x 10-7

z 10-4 > 10-4

1.5 x

9.3 x 10-1° 2.0 x lo-'

8.0 x

e -

5.3 11.3 0.45 5.0

-

-

0.56 0.04

1.03

N-methylated [D-Ala2, Leu'lenkephalin

GlyolS]enkephalin (DAGO) (1 1). The trimethyl analogs 4 , 4 , 5 , and 6 showed lower affinity to opioid receptor than the unmethylated pentapeptides 1, 3', and 7, and the activity of pentamethyl analogs 9 and 10 was negligible as given in Table 1.

DISCUSSION

N-Methyl group-containing analogs of a biologically active peptide possess steric characteristics different from the parent molecule. Replacement of an H3N+ group with a trimethylammonium group introduces steric bulkiness and inhibit H-bonding interaction, if any, while keeping a positive charge on the N atom which is lost by usual acylation-type modification. In the case of N-methylated peptide linkage both s-trans and s-cis conformations are possible, while contribu- tion of the s-cis conformation is usually negligible for an unmodified peptide bond in linear peptides unless the amide N atom belongs to a Pro residue.

Nature of interaction of a peptide molecule with the receptor site is modified by N-methylation as a result of loss of intermolecular H-bonding and/or change in conformational characteristics. Since an N-methylat- ed amino acid is no more a proteineous amino acid, the analogs containing such an amino acid residue are often resistant to degradation by the proteolytic enzymes. Consequently, study of the N-methyl analogs is expected to afford new insight upon the mechanism of activity and also to lead to the prepara- tion of the analogs with higher potencies or prolonged activities.

Trimethylammonium group-containing analogs 4, 4', 5, and 6 of [~-Ala~,Leu']enkephalin 1 have been synthesized by the use of the quaternization reaction, and the zwitterion 5 and its amide 6 showed activity on the GPI assay only slightly lower than the corre- sponding unmethylated analogs. The moderate potency of the quaternary ammonium analogs 5 and 6 discovered in this study is interesting considering the loss of activity of the 3-hydroxymorphinane opiates on quaternization (12). The result seems to indicate that the presence of a H atom on the terminal N atom, which enables H-bonding interaction, is not essential for the interaction of enkephalin molecules with opioid receptors. The opioid activity of the trimethyl- ammonium analogs 4 and 4 having C-terminal ester functions, however, is very low, which is consistent with the result reported by DiMaio et al. (13) for Me: Tyr-Gly-Gly-Phe-Leu-OMe. The much lower ac- tivity of the trimethylammonium analogs of the peptide esters than those of the peptide acid or amide is in contrast to what is observed with the correspond- ing peptides with an unmodified N-terminal amino group. Although it is difficult to give a reasonable explanation for this, the least active analogs 4 and 4 of this series are largest in size and also most hydro- phobic, possibly exceeding the critical limit in size or

hydrophobicity for manifesting activity. All the tri- methylammonium-containing analogs showed much lower affinity to rat brain membranes than the un- methylated peptides. The results given in Table 1 in- dicate the absence of any significant receptor subtype- selectivity in the binding of these new analogs to opiate receptors.

Amide-methylation reaction with CH, I-Ag,O in DMF was known to methylate preferentially solvent- exposed amide nitrogens when applied to cyclic de- capeptide molecules (4,5). In the case of the protected linear pentapeptide 2, the N atoms of the terminal residues, Tyr and Leu, were methylated more slowly than those of other amino acid residues as shown in Fig. 3. The result might indicate the presence of those conformers in which some intramolecular intertermi- nal interactions exist, but no further study has been undertaken on the conformation of these molecules. At the beginning of the reaction ( < 20 min) methyla- tion yield was very low, which can be expected for this type of heterogeneous reactions (14). Isolation of par- tially methylated derivatives could not be attained, and only the fully methylated product 8 was prepared. It is known that N-monomethylation at the N-termi- nal, the 4th, or the 5th amino acid residue of enke- phalins sometimes affords a derivative with higher opioid potency (6, 15-1 7), while the derivatives having an N-methyl group at the 2nd or the 3rd residue of enkephalins exhibit very weak activity (6). The pen- tamethyl analogs 9 and 10, obtained by the deprotec- tion of 8, did not show any significant opioid activity, which might be attributed to the presence of the N- methyl groups at the D-Ala and Gly residues.

EXPERIMENTAL PROCEDURE

General Melting points were determined with a hot-stage ap- paratus and uncorrected. Purity of each product was ascertained by TLC, HPLC, and/or 'H NMR spectral analysis. Si02 column chromatography was per- formed using Silica Gel (Merck, # 7734). TLC analy- sis was performed using precoated SiO, plates (Merck, Silica Gel 60 F2%) with the solvent systems CHC1,- MeOH, CHCl, -MeOH-AcOH, and/or butanol- AcOH-H,O. HPLC was performed using FineSIL 20 (JASCO) with CHC1,-MeOH as eluent or Unisil Cl8 (Gasukuro Kogyo) with MeOH-NaC10, aqueous. 'H NMR spectra were recorded on a Varian XL-200 (200 MHz) or a JEOL JNM-GSX 400 (400 MHz) spectrometer. FAB-mass spectra were measured on JEOL-JMS-SX 102 mass spectrometer. Elemental an- alyses were performed at Elemental Analysis Center in Kyoto University. For amino acid analysis a weighed amount of a synthetic peptide was hydrolyzed in conc. HCI-AcOH (1 : 1) at 1 10" for 24 h. Equimolar aspartic

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M. Kawai et al.

acid was added as an internal standard to the hydroly- zate, which was subjected to amino acid analysis.

Synthesis of protected peptides

Boc-Phe-Leu-OMe. DCC (3.63 g, 18 mmol) and HOBt (1.08 g, 8 mmol) were added to the ice-cooled mixture of Boc-Phe (4.25 g, 16mmol), Leu-OMe-HCl (2.91 g, 16mmol), and Et,N (2.24mL, 16mmol) in CH2C1, (64mL) and the mixture was stirred for 1 h at 0" and overnight at room temperature. The solvent was evaporated in vucuo, the residue was taken in AcOEt, and precipitated DCU was filtered off. The filtrate was washed with 10% citric acid aqueous, 4% NaHCO, aqueous, and saturated NaCl aqueous and was dried over Na, SO,. Evaporation of the solvent afforded an oily mass, which was crystallized from AcOEt-hexane affording a white solid (5.35 g, yield 88%), m.p. 105- 106".

(0.29 g, 2.2 mmol) in a similar manner to the synthesis of Boc-D-Ala-Gly-Phe-Leu-OMe. Silica gel column chromatography and recrystallization from AcOEt- hexane afforded Boc-Tyr(Bz1)-D-Ala-Gly-Phe-Leu- OMe 2 as colorless needles (1.92 g, yield 54%), m.p.

Me,C-Leu), 1.22 (d, J = 7Hz, 3H, Me-Ala), 1.39 (s, 9H, Boc), 1.58 (m, 2H, CH,-Leu), 2.9-3.1 (m, 4H, CH,-Tyr and CH2-Phe), 3.72 (s, 3H, OMe), 3.88 (d, J = 6Hz, 2H, CH2-Gly), 4.22 (m, lH, CaH), 4.47 (m, lH, C H ) , 4.55 (m, IH, CH),4.79 (m, lH, C H ) , 5.07 (s, 2H, CH,-Bzl), 5.56 (m, lH, NH-Tyr), 6.52 (d, J = ~ H z , lH, NH), 6.78 (d, J = ~ H z , NH), 6.94 (d, J = 8Hz, 2H, arom-Tyr), 7.04 (m, IH, NH), 7.15 (d, J = 8 Hz, 2H, arom-Tyr), 7.3 (m, 5H, arom), 7.45 (m, 5H, arom). Anal. calc. for C,,H,,N,O,: C 65.18, H 7.16, N 9.05%. Found: C 65.08, H 7.06, N 8.92%.

147-148". 'H NMR (CDCI,) 6 0.90 (d, J = ~ H z , 6H,

Anal. calc. for CZl H3,N205 .:H20: C 62.82, H 8.28, N 6.98%. Found: C 63.15, H 8.04, N 7.07%.

Boc-Tyr(Bzl)-o-Ala-Gly-Phe-Leu-OEt 2' (m.p. 163- 165") was synthesized similarly using Leu-OEt .HC1 instead of Leu-OMe *HCl via Boc-Phe-Leu-OEt (m.p. Boc-DAla-Gb-OBzl. This compound was synthesized 1 1 80) and Boc-D-Ala-Gly-Phe-Leu-OEt (m.p. 205").

from Boc-D-Ala (3.38 g, 18 mmol), Gly-OBzl-TosOH (6.03 g, 18 mmol), EGN (2.5 mL, 18 mmol), DCC (4.30g, 21 mmol), and HOBt (1.21 g, 9mmol) in a similar manner to the synthesis of Boc-Phe-Leu-OMe. White solid (4.81 g, yield 8O%), m.p. 88-89". Anal. calc. for C,,H,,N,O,: C 60.70, H 7.19, N 8.33%. Found: C 60.80, H 7.20, N 8.31%.

Boc-D-Ala-Gly-OBzl (1.68 g, 5 mmol) in MeOH (25 mL) was subjected to catalytic hydrogenolysis over 5% Pd on activated charcoal catalyst for 18 h and the product was treated with DCHA (1 mL, 5mmol) to afford Boc-D-Ala-Gly-OH - DCHA (1.6 1 g, yield 75%) as a white solid, m.p. 137-138".

Boc-D- Alu-Gly- Phe- Leu- OMe. Boc-Phe- Leu-0 Me (1.96 g, 5 mmol) was stirred for 1 h in TfaOH at room temperature. TfaOH was evaporated and the residue dissolved in CH2C1, was neutralized by the addition of Et3N. The mixture was cooled to 0", to which BOC-D- Ala-Gly-OH - DCHA (2.14 g, 5 mmol), DCC (1.14 g, 5.5mmol), HOBt (0.34g, 2.5mmol) were added and the mixture was treated similarly to the synthesis of Boc-Phe-Leu-OMe. The crude product was purified by silica gel column chromatography using CHC1,- MeOH as eluent and the resulting oil was triturated with hexane to afford Boc-D-Ala-Gly-Phe-Leu-OMe as a white solid (2.46g, yield 82%), m.p. 187-188". Anal. calc. for C,,H,N,O,: C 59.98, H 7.74, N 10.76%. Found: C 59.71, H 7.77, N 10.81%.

Boc-Tyr(Bz1)-o-Ah-Gly-Phe-Leu-OMe 2 . BOC-D- Ala-Gly-Phe-Leu-OMe (2.22 g, 4.3 mmol) was treated with TfaOH and was subjected to the coupling reac- tion with Boc-Tyr(Bz1) (1.59 g, 4.3 mmol) using Et,N (0.60 mL, 4.3 mmol), DCC (1.06 g, 5.1 mmol), HOBt

Synthesis of trimethylammonium-type analogs

Boc- Tyr-~Ala-Gly-Phe-Leu-OMe. The protected pentapeptide 2 (1 10 mg, 0.142 mmol) in MeOH (3.5 mL) was subjected to catalytic hydrogenation in the presence of Pd-black (20 mg) under atmospheric pressure of H, at room temperature for 7 h. The cat- alyst was filtered off, the solvent was evaporated, and the residue was treated with Et,O to give Boc-Tyr-D- Ala-Gly-Phe-Leu-OMe (88 mg, yield 91 %) as color- less plates, m.p. 126-133".

Tyr-o- Ah-Gly- Phe- Leu- OMe ( [ p A l a 2 ,LeuS Ienke- phalin Me ester) 3. i) A mixture of Boc-Tyr-D-Ala- Gly-Phe-Leu-OMe (80mg, 0.1 16 mmol), anisole (0.05 mL), and TfaOH (0.2 mL) was stirred for 1 h at room temperature. After the evaporation of TfaOH, E t 2 0 was added to the residue, and Tyr-D-Ala-Gly- Phe-Leu-OMe. TfaOH was collected by filtration as white solid (66mg, yield 82%), m.p. 122-126".

ii) The protected peptide 2 (91 mg, 0.12mmol), anisole (0.05 mL), and 4 M HCl/dioxane (2 mL) were stirred for 1 h at room temperature. Usual workup afforded Tyr(Bz1)-D-Ala-Gly-Phe-Leu-OMe-HC1 (84 mg), which was hydrogenated over Pd-black (21 mg) in MeOH (5mL) for 16 h. Sephadex LH-20 column chromatography of the product using MeOH as eluent yielded Tyr-D-Ala-Gly-Phe-Leu-OMe. HC1 (72 mg, yield 98%) as a colorless semi-solid.

Tyr-o- Ah-Gly- Phe- Leu-0 Et ([o- Ala' ,Leu' Ienke- phalin Et ester) 3'. The protected pentapeptide Et ester 2' was subjected to catalytic hydrogenolysis as described above to give Boc-Tyr-D-Ala-Gly-Phe-Leu-

456

N-methylated [D-Ma2, Leu'lenkephalin

0.15 mmol) in MeOH (2 mL) was saturated with NH, . After keeping at room temperature for 76h the mixture was evaporated and the residue was crystal- lized from AcOEt-hexane to give Boc-Tyr-D-Ala-Gly- Phe-Leu-NH, as a white solid (83mg, yield 82%), m.p. 139-141'.

Tyr- D- Ah-Gly- Phe- Leu-NH, ( [D- A h Z , L e d Ienke- phalinamide) 7 . A mixture of Boc-Tyr-D-Ala-Gly-Phe- Leu-NH, (72 mg, 0.1 1 mmol), TfaOH (0.15 mL), and anisole (0.04 mL) was stirred at room temperature for 1.5h. After the evaporation of TfaOH, Et,O was added to the mixture to induce the precipitation of Tyr-D-Ala-Gly-Phe-Leu-NH, - TfaOH as a white solid (67mg, yield %?YO), m.p. 130-133".

OEt, m.p. 110-1 12', which was treated with TfaOH- anisole affording Tyr-D- Ala-Gly-Phe- Leu-OEt - TfaOH as a white solid, m.p. 119-123'.

Tyr-o- Ala-Gly- Phe-Leu ([o- Alu' ,Leu']enkephulin) I was obtained by the alkaline hydrolysis of 3 or 3'.

Me: Tyr-o-Ala-Gly-Phe-Leu-OMe ([Me: Tyr' ,D- Alu2,Led]enkephalin Me ester) 4. To a solution of Tyr-D-Ala-Gly-Phe-Leu-OMe. HCI (56 mg, 0.08 mmol) in MeOH (1.5mL) was added CH,I (0.2mL, 3.2mmol) and KHCO, (84mg, 0.84mmol), and the mixture was stirred in the dark for 46 h at room tem- perature. The insoluble material was filtered off, the filtrate was evaporated, and the residue was chro- matographed over silica gel using CHCI, -MeOH- AcOH as eluent and then over Sephadex LH-20 using MeOH. The desired product was finally obtained by lyophilization of the aqueous solution. Me: Tyr-D- Ala-Gly-Phe-Leu-OMe-HCO; (29 mg, 50%), m.p.

Anal. calc. for C,,H,,N,O, .HCO, -+H,O: C 56.42, H 7.38, N 9.68%. Found: C 56.65, H 7.39, N 9.90%.

Me: Tyr-o- Ah-Gly- Phe- Leu- OEt ( [Me: Tyr' ,D Alu2,Leu5]enkephalin Et ester) 4 was similarly syn- thesized as a chloride, m.p. 139-142'.

Me: Tyr-o-Ala-Gly-Phe-Leu (Me: Tyr' ,D-AluZ, Leu5]enkephalin) 5 . Me: Tyr-D-Ala-Gly-Phe-Leu- OEt-Cl- (17mg, 0.023mmol) in 0 . 5 ~ NaOH aqueous (2mL) was stirred for 1 h at room tem- perature. CO, was introduced to the solution, the solvent was evaporated, and the residue was chro- matographed over Sephadex LH-20 using MeOH as eluent. Crystallization from MeOH-AcOEt afforded Me: Tyr-D-Ala-Gly-Phe-Leu as white solid (1 1 mg, 78%), m.p. 190-193', 'H NMR (CD,OD) 6 0.93 (d, J = 6 Hz, 3H, Me-Leu), 0.94 (d, J = 6 Hz, 3H, Me'- Leu), 1.03 (d, J = 7Hz, 3H, Me-Ala), 1.6 (m, 2H, CBH,-Leu), 1.75 (m, lH, CYH-Leu), 2.99 (dd, J = 14 and lOHz, lH, CBH-Phe), 3.17 (t, J = 12Hz, lH, CBH-Tyr), 3.27 (dd, J = 14 and 4Hz, lH, CBH'-Phe), 3.33 (dd, J = 12 and 4Hz, IH, CBH'-Tyr), 3.55 (d,

CaH'-Gly), 3.96 (dd, J = 12 and 4Hz, lH, C"H-Tyr), 4.01 (9, J = 7Hz, lH, CH-Ala), 4.26 (dd, J = 10 and 5Hz, IH, CH-Leu), 4.64 (dd, J = 10 and 4Hz, lH, CH-Phe), 6.76 (d, J = 9 Hz, 2H, arom-Tyr), 7.08 (d, J = 9Hz, 2H, arom-Tyr), 7.19 (br t, J = 8Hz, lH, p-Phe), 7.27 (br t, J = 8Hz, 2H, m-Phe), 7.32 (br d, J = 8Hz, o-Phe). Anal. calc. for Cj2H4,N,O7 -?H,O: C 58.52, H 7.68, N 10.66%. Found: C 58.25, H 7.59, N 10.52%.

Boc- Tyr--Ah-Gly-Phe-Leu-NH,. An ice-cooled solu- tion of Boc-Tyr-D-Ala-Gly-Phe-Leu-OEt (1 05 mg,

143-149'.

J = 17Hz, lH, CH-Gly), 3.95 (d, J = 17Hz, lH,

Me: Tyr-o-Ala-Gly-Phe-Leu-NH, ([Me: Tyr' ,o- A l d ,Led ]enkephalinamide) 6. Tyr-D-Ala-Gly- Phe- Leu-NH, *TfaOH (58 mg, 0.084 mmol) was subjected to the quaternization reaction in the same manner as described for the synthesis of 4 to afford Me: T y r o Ala-Gly-Phe-Leu-NH, .HCO, as a white solid (46mg, Sly0), m.p. 159-162'. 'H NMR (CD,OD) 6 0.89 (d, J = 6Hz, 3H, Me-Leu), 0.94 (d, J = 7Hz, 3H, Me'-Leu), 1.09 (d, J = 7Hz, 3H, Me-Ala), 1.58 (m, IH, CBH-Leu), 1.59 (m, lH, CPH'-Leu), 1.65 (m, lH, C?H-Leu), 3.03 (dd, J = 14 and 9Hz, IH, CBH- Phe), 3.13 (t, J = 12Hz, lH, CPH-Tyr), 3.21 (dd, J = 14 and 5Hz, lH, CBH'-Phe), 3.29 (dd, J = 12 and 4Hz, lH, CBH'-Tyr), 3.63 (d, J = 17Hz, lH,

(dd, J = 12 and 4Hz, lH, CH-Tyr), 3.97 (9, J = 7Hz, lH, CaH-Ala), 4.29 (dd, J = 8 and 6Hz, IH, CH-Leu), 4.63 (dd, J = 9 and 5Hz, lH, C H - Phe), 6.66 (d, J = 9Hz, 2H, arom-Tyr), 6.96 (d, J = 9Hz, 2H, arom-Tyr), 7.2 (m, lH, arom-Phe), 7.3 (m, 4H, arom-Phe). Anal. calc. for C32H47N606 -HC03 .SH,O: C 53.87, H 7.53, N 11.42%. Found: C 53.90, H 7.34, N 11.12%.

Attempted hydrogenolytic debenzylation of trimethyl- ammonium-type compounds. Quaternization of Tyr(Bz1)-D-Ala-GI y-Phe-Leu-OE t * TfaOH afforded Me: Tyr(Bz1)-D-Ala-Gly-Phe-Leu-OEt -1- (m.p. 101- 105') in 60% yield. The trimethylammonium iodide (90mg, O.lOmmo1) and Pd-black (20mg) in MeOH ( 5 mL) were stirred vigorously under H, atmosphere for 30h. TLC of the reaction mixture showed no indication of the proceeding of hydrogenolysis.

Me: Tyr(Bz1)-OMe- I- (m.p. 182-1 86"), prepared by the quaternization of Tyr(Bz1)-OMe, was subjected to catalytic hydrogenolysis in MeOH in the presence of Pd black as described above to result in the com- plete recovery of the starting material.

Me: Tyr(Bz1)-0- was prepared by the quaterniza- tion of Tyr(Bz1) and was treated with AgNO, aqueous to remove I- ion. The iodode-free zwitterionic

CH-Gly), 3.92 (d, J = 17H2, lH, CH'-Gly), 3.95

457

M. Kawai et al.

trimethylammonium compound was subjected to cat- alytic hydrogenolysis and the main product was iden- tified as Me:Tyr by TLC comparison with the au- thentic sample prepared by the quaternization of Tyr.

Boc-Tyr(Bz1) (74 mg, 0.2 mmol) in MeOH (2 mL) was hydrogenolyzed in the presence of Pd black (7 mg) for 4 h to give quantitative yield of Boc-Tyr, while addition of NaI (1.5 mg, 0.01 mmol) to the reaction mixture completely in hi bited the h yd rogenolytic cleavage of benzyi group as analyzed by TLC.

Synthesis of pentamethyl analogs using amide- methylation

Boc-Me Tyr(Bz1) -D-MeAla-MeGly-MePhe-MeLeu- OMe 8. To a stirred solution of the pentapeptide 2 (310mg,0.40mmol)in DMF(4mL) wereaddedCH,I (13mL, 200mmol) and AgzO (4.6g, 20mmol) at room temperature. After stirring in the dark for 42 h, MeOH (1OmL) was added to the mixture and in- soluble material was filtered off. The filtrate was evaporated and the residue was chromatographed over silica gel using CHC1,-MeOH as eluent to afford 8 as a colorless solid (271 mg, SO%), m.p. 58-61'. Anal. calc. for C,,H,,N,O, *:H,O: C 66.18, H 7.80, N 8.21%. Found: C 66.22, H 7.70, N 8.27%.

FAB-mass (m/z): 844 (M + H)' ,866 (M + Na)+ . Amino acid analysis with equimolar aspartic acid: Tyr 0.00; Ala < 0.04; Gly < 0.02; Phe 0.00; Leu < 0.04 (Asp 1.00).

MeTyr-D-MeAla-MeGly-MePhe-MeLeu 9. Alkaline hydrolysis of the ester function of 8 (304mg, 0.36mmol) in the same manner as described for the synthesis of 5 afforded Boc-MeTyr(Bz1)-D-MeAla- MeGly-MePhe-MeLeu, m.p. 79-85" (288 mg, yield 88%), which was dissolved in 4 M HCl/dioxane (1 mL) containing anisole (0.1 mL) and was kept at room temperature for 1 h. Usual workup and Sephadex LH-20 column chromatography using MeOH as eluent gave MeTyr(Bz1)-D-MeAla-MeGly-MePhe- MeLeu-HCl as a white solid (204mg, yield 78%), m.p. 101-104'. This compound (92 mg, 0.12mmol) was hydrogenolyzed and the product was chromato- graphed over Sephadex LH-20. Lyophilization of the aqueous solution yielded the pentamethyl derivative MeTyr-D-MeAla-MeG1 y-MePhe-MeLeu * HCl as a white solid (68mg, yield 84%), m.p. 117-123". Anal. calc. for C,,H,N,O, .HCl-~H,O: C 55.24, H 7.77, N 9.47%. Found: C 55.29, H 7.53, N 9.71%.

eluent to give MeTyr(Bz1)-D-MeAla-MeGly-MePhe- MeLeu-OMe-HC1 (14mg, yield 20%), which was subjected to catalytic hydrogenolysis. Lyophilization afforded MeTyr-D-MeAla-MeGly-MePhe-MeLeu- OMe-HCl as a white solid (1 1 mg, yield 87%), m.p.

Anal. calc. for C3,H5, N,O, *:H,O: C 55.85, H 7.89, N 9.30%. Found: C 55.55, H 7.80, N 9.36%.

82-85".

Determination of rate of N-methylation of each amino acid residue. A mixture of 2 (0.05mmol), CH,I (50mmol), and Ag,O (25mmol) in DMF (2mL) was stirred in the dark at room temperature. Aliquots were removed from the reaction mixture after 0.25, 0.5, 1, 2, 3, and 24 h and poured into MeOH. Each sample was evaporated after filtration and was subjected to acid-hydrolysis followed by amino acid analysis. Methylation yield of each amino acid residue was obtained from the remaining amino acid and is given in Fig. 3 as a function of time.

Determination of biological and pharmacological activities

GPI assay. Pharmacological actions of the synthetic analogs of [D-Ala2 ,Leu']enkephalin were tested on the GPI stimulated transmurally at 0.05 Hz with pulse duration of lo-, s. Concentration of each analog re- quired to inhibit the maximum contraction by 50% (IC&) was estimated from the dose-response curve obtained at concentration range of 10-8-10-4~. The IC, values and the inhibitory potency of the analogs at a concentration of ~ O - , M were summarized in Table 1. The action of the analogs was reversible and was competitively inhibited by naloxone, e.g., inhibi- tion of the contraction by 5 at l O P 5 ~ was suppressed about 40% by naloxone at 1OP6w and 70% at lo-, M.

Receptor binding assay. The effect of the analogs on the binding of the opioid ligands ([3H]DALAMID, [,H]DADL, and [,HIDAGO) to the membrane frac- tions from rat brain was examined by the filtration method using Whatman GF/B glass filter as described by Maruyama (1 8). The concentration of each analog which produces 50% inhibition of the [3H]-labelled Iigands at 2 x l O P 9 ~ was shown in Table 1.

ACKNOWLEDGMENTS

We thank Dr. Kazuki Sato of Mitsubishi-Kasei Institute of Life Sciences for his advice on the synthesis of protected enkephalin analogs and Dr. Ukon Nagai of the same Institute for helpful discussions. The authors are grateful also to Instrument Center, Nagoya Institute of Technology for ' H NMR measurements, and to

MeTyr-DMeA1a-MeG1y-MePhe-MeLeu-oMe lo. As described above for the 'ynthesis Of 93 the 8 (77mg9 o.0g1 mmol) was treated with HC1/ dioxane and the product was chromatographed Over Dr. Mitsuo Hayashi of TOYO JOZO CO. Ltd. for I H NMR and carboxymethylcellulose (H+ form) using H, 0 as FAB-mass spectral measurements.

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Professor Masao Kawai Department of Applied Chemistry Nagoya Institute ofTechnology Gokiso-cho, Showa-ku Nagoya 466 Japan

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