Enkephalin analogues with 2′,6′-dimethylphenylalanine replacing phenylalanine in position 4

Article abstract of DOI:10.1016/S0960-894X(00)00665-X

Four Leu-enkephalin (Enk) analogues containing 2′,6′-dimethyphenylalanine (Dmp) in position 4 were prepared and tested for their receptor binding and biological activities. Among the analogues prepared, [2′, 6′-dimethyltyrosine¹, D-Dmp⁴]Enk was found to be an antagonist toward μ and δ opioid receptors with pA₂ values of 6.90 and 5.57, respectively.

Full text of DOI:10.1016/S0960-894X(00)00665-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 327±329
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Enkephalin Analogues with 2 ,6 -Dimethylphenylalanine Replacing
Phenylalanine in Position 4
Yusuke Sasaki,* Mariko Hirabuki, Akihiro Ambo, Hidekazu Ouchi
and Yutaka Yamamoto
Tohoku Pharmaceutical University, 4-1 Komatsushima 4-chome, Aoba-ku, Sendai 981-8558, Japan
Received 28 July 2000; accepted 14 November 2000
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AbstractÐFour Leu-enkephalin (Enk) analogues containing 2 ,6 -dimethyphenylalanine (Dmp) in position 4 were prepared and
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0
1
4
tested for their receptor binding and biological activities. Among the analogues prepared, [2 , 6 -dimethyltyrosine , d-Dmp ]Enk
was found to be an antagonist toward m and d opioid receptors with pA values of 6.90 and 5.57, respectively. # 2001 Elsevier
2
Science Ltd. All rights reserved.
Introduction
only a few into other biologically active peptides.17,18
We present here novel Leu-enkephalin (Enk) analogues
containing 2 ,6 -dimethylphenylalanine (Dmp) in posi-
tion 4, and their opioid receptor binding and biological
activities in comparison with the parent molecules.
1
3
4
0
0
Two aromatic amino acids, Tyr and either Phe or Phe
are important structural elements in opioid peptides
1
that interact with the opioid receptors. Recent struc-
ture±activity studies of opioid peptides have demon-
0
Dmt) in place of Tyr produces compounds with vastly
0
strated that the introduction of 2 ,6 -dimethyltyrosine
1
(
improved opioid receptor binding anities.
Chemistry
2
�16
Via the
combination of Dmt with 1,2,3,4-tetrahydro-isoquino-
Scheme 1 illustrates the synthetic route to Dmp. For the
synthesis of a key intermediate, 2-iodo-m-xylene (7),
commercially available 5 was reacted with sodium tri-
line-3-carboxylic acid (Tic), some d opioid receptor
1
3,14
0
12
selective antagonists, including Dmt-Tic-OH,
1
N,N -
5
10
19
diMe-Dmt-Tic-OH,
DIPP-NH2
and DIPP[c],
methylstannane according to Yamamoto et al., fol-
have been developed. Conformational studies of these
peptides have revealed that the topology of the two
aromatic rings induced by the two unusual amino acid
residues and increased hydrophobicity imparted by the
Dmt residue are critical to the high activity and d
lowed by reaction with iodine according to the method
of Ohno et al.20 The reaction of 7 with methyl 2-acet-
amidoacrylate, Dygos's method,21 led to 8, followed by
saponi®cation to yield 9. The catalytic hydrogenation of
9 then yielded racemic Ac-Dmp (10). For the optical
resolution, 10 was converted to its dipeptide derivatives,
Ac-dl-Dmp-Arg-OMe (11), which were easily separated
9
,11,14
receptor selectivity.
Very recently, we also demon-
1
strated that [Dmt ]Leu-enkephalin is an exceedingly
potent opioid agonist and that the modi®cation of the
16
22
into each diastereoisomer by preparative HPLC. Acid
1
0
0
Tyr aromatic nucleus by 2 ,6 -dimethylation imparts a
1
hydrolysis and neutralization of each isomer yielded the
l- (12) and d-Dmp (13), which were converted to their
N -Fmoc derivatives (14 and 15). The synthesis of all
6
high enzymatic stability to the peptides. From these
ndings, modi®cation of a Phe aromatic nucleus at
a
23
®
0
0
24
position 3 or 4 of an opioid peptide by 2 ,6 -dimethyla-
tion can be considered to be another interesting
approach in the design of opioid mimetics with unique
biological activity. Nevertheless, there has been no con-
clusive demonstration that the phenyl ring-methylated
phenylalanine is incorporated into opioid peptides but
Enk analogues (1±4 in Table 1) was performed via a
solid-phase method using Fmoc chemistry, as pre-
viously described.1
6,25
Biological Results and Discussion
Table 1 shows the results of receptor binding anity of
the analogues, which were determined using rat brain
*
2
Corresponding author. Tel.: +81-22-234-4181; fax: +81-22-275-
013; e-mail: ysasaki@tohoku-pharm.ac.jp
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00665-X

3
28
Y. Sasaki et al. / Bioorg. Med. Chem. Lett. 11 (2001) 327±329
2
5
l-Dmp and Dmt1 replacements produced 3 with
markedly improved anities for both receptors. This
analogue exhibited 40-fold and 110-fold higher anities
than 1 for the m and d receptors, respectively, but a
4
synaptosomes, as previously reported. Replacement of
4
Phe in Enk by l-Dmp led to formation of 1 with a m
anity comparable to Enk but with an approximately
2-fold reduced d anity, suggesting that this process of
displacement is detrimental to d receptor anity, but
not to m receptor anity. The d-Dmp replacement of
Enk (2) induced a drastic reduction in binding anity to
both receptors, a ®nding in accordance with some
reports that l-con®guration at the fourth residue in Enk
is an important factor for high receptor anity and
1
1
16
5-fold lower anity than [Dmt ]Enk for both recep-
tors, possibly due to slight changes of an active con-
formation by the simultaneous dimethylations of two
aromatic nucleuses. It should be noted that the other
combination of d-Dmp and Dmt replacements pro-
duced 4, which retained a m anity nearly equivalent to
that of Enk and with a modest d anity, showing again
4
4
1
2
6
opioid activity. On the other hand, a combination of
3 2
Scheme 1. Synthetic route to Fmoc-l-Dmp (14) and Fmoc-d-Dmp (15): (a) Me SnNa, DME, ice-salt bath, 2 h; (b) I /THF, rt, 3 h; (c) methyl 2-
2
ꢀ
acetamidoacrylate/Pd(OAc) /Et
2
N/MeCN, (2-MeC H ) P, re¯ux, 24 h; (d) 1 M NaOH/dioxane, rt, 2 h; (e) H (4 kgf/cm )/10% Pd-C/AcOH, 70 C,
3 6 4 3 2
ꢀ
.
48 h; (f) HCl Arg-OMe/Et
Na CO /aq MeCN.
3
N/DCC/HOBt/DMF, 0 C to rt, 5 h; (g) preparative HPLC; (h) concd HCl, re¯ux, 8 h; (i) pH 4±6/H O; (j) Fmoc-OSu/
2
2
3
Table 1. Opioid receptor binding anity of Dmp replacing Enk analogues
Binding anity, K
i
ÆS.E. (nM)
Selectivity
a
b
Peptide
m
d
m/d
Tyr-Gly-Gly-Phe-Leu (Enk)
2.42 Æ 0.93
0.0068 Æ 0.0030
1.25 Æ 0.29
1.43 Æ 0.71
1.69
0.22
0.07
0.28
0.19
0.14
1
c
[
[
[
[
[
Dmt ]Enk
4
0.031 Æ 0.011
17.7 Æ 4.2
l-Dmp ]Enk (1)
4
d-Dmp ]Enk (2)
2505 Æ 169
8924 Æ 4098
0.158 Æ 0.034
40.9 Æ 11.5
1
4
Dmt , l-Dmp ]Enk (3)
0.030 Æ 0.011
5.61 Æ 0.59
1
4
Dmt , d-Dmp ]Enk (4)
a
3
Versus [ H]DAMGO.
3
b
Versus [ H]deltorphin II.
Data cited from ref 16.
c
Table 2. In vitro biological activity of Dmp replacing Enk analogues
GPI (m)
MVD (d)
Peptide
IC50 (nM)a
pA
2
IC50 (nM)a
pA
2
GPI/MVD
Tyr-Gly-Gly-Phe-Leu (Enk)
103 Æ 30
0.55 Æ 0.17
808 Æ 101
>10,000
Ð
Ð
Ð
Ð
Ð
6.90c
22.2 Æ 4.3
0.17 Æ 0.02
624 Æ 103
>10,000
Ð
Ð
Ð
Ð
Ð
5.59
4.64
3.24
1.29
Ð
1.38
Ð
1
b
[
[
[
[
[
Dmt ]Enk
4
l-Dmp ]Enk (1)
4
d-Dmp ]Enk (2)
1
4
Dmt , l-Dmp ]Enk (3)
2.00 Æ 0.51
1.45 Æ 0.26
1
4
d
Dmt , d-Dmp ]Enk (4)
>10,000
>10,000
a
Values are the mean of 4±8 experiments Æ S.E.
b
c
Data cited from ref 16.
Against endomorphin 2 as an agonist.
Against deltorphin II as an agonist.
d

Y. Sasaki et al. / Bioorg. Med. Chem. Lett. 11 (2001) 327±329
329
1
the e€ectiveness of Dmt replacement for maintaining
high receptor anity.
8. Guerrini, R.; Capasso, A.; Sorrentino, L.; Anacardio, R.;
Bryant, S. D.; Lazarus, L. H.; Attila, M.; Salvadori, S. Eur. J.
Pharmacol. 1996, 302, 37.
9
. Salvadori, S.; Balboni, G.; Guerrini, R.; Tomatis, R.;
In vitro biological activity was evaluated using isolated
guinea pig ileum (GPI) and mouse vas deferens (MVD)
tissue samples, as previously reported.27 The GPI tissue
contains predominantly m receptors, while MVD
includes d receptors. Analogue 1 showed reduced activ-
ity, and was 8- and 30-fold less potent than Enk in the
GPI and MVD assays, respectively. Analogue 2 was
devoid of activity in both assays as expected from the
binding data. Consistent with the binding data, 3 had a
high potency in both assays. Notably, analogue 4 was
also devoid of activity in both assays despite the fact
that this analogue showed potent m anity and modest
d anity. This analogue turned out to be a potent m
Bianchi, C.; Bryant, S. D.; Cooper, P. S.; Lazarus, L. H. J.
Med. Chem. 1997, 40, 3100.
10. Schiller, P. W.; Schmidt, R.; Weltrowska, G.; Berezowska,
I.; Nguyen, T. M.-G.; Dupuis, S.; Chung, N. N.; Lemieux, C.;
Wilkes, B. C.; Carpenter, K. A. Lett. Pep. Sci. 1998, 5, 209.
11. Wang, C.; McFadyen, I. J.; Traynor, J. R.; Mosberg, H. I.
Bioorg. Med. Chem. Lett. 1998, 8, 2681.
12. Schiller, P. W.; Fundytus, M. E.; Merovitz, L.; Wel-
trowska, G.; Nguyen, T. M.-D.; Lemieux, C.; Chung, N. N.;
Coderre, T. J. J. Med. Chem. 1999, 42, 3520.
1
3. Capasso, A.; Guerrini, R.; Balboni, G.; Sorrentino, L.;
Temussi, P.; Lazarus, L. H.; Bryant, S. D.; Salvadori, S. Life
Sci. 1996, 59, PL93.
1
4. Bryant, S. D.; Salvadori, S.; Cooper, P. S.; Lazarus, L. H.
Trends Pharmacol. Sci. 1999, 19, 42.
5. Salvadori, S.; Guerrini, R.; Balboni, G.; Bianchi, C.; Bry-
antagonist and weak d antagonist. The pA values of 4
2
were 6.90 against endomorphin 2, as a m agonist in the
GPI assay, and 5.57 against deltorphin II, as a d ago-
nist, in the MVD assay. No signi®cant antagonist
activity was observed with 2 in either assay. The results
of 4 are of interest in light of recent observations that
1
ant, S. D.; Cooper, P. S.; Lazarus, L. H. J. Med. Chem. 1999,
42, 5101.
16. Sasaki, Y.; Suto, S.; Ambo, A.; Ouchi, H.; Yamamoto, Y.
Chem. Pharm. Bull. 1999, 47, 1506.
1
7. Majer, P.; Slaninova, J.; Lebil, M. J. Peptide Protein Res.
1994, 43, 62.
8. Sagan, S.; Josien, H.; Karoyan, P.; Brunissen, A.; Chas-
saing, G.; Lavielle, S. Bioorg. Med. Chem. 1996, 4, 2167.
9. Yamamoto, Y.; Ouchi, H.; Tanaka, T.; Morita, Y. Het-
Dmt-d-Phe-NH and its C-terminally extended analo-
2
28
gue are m receptor antagonists. However, it is possible
that the antagonist action of 4 is attributable to the
presence of the two dimethylated aromatic amino acids
at positions 1 and 4 because the two newly synthesized
peptides, Dmt-d-Dmp-NH2 and Dmt-Gly-Gly-d-Phe-
Leu, have no antagonist activity, but did show a weak
agonist activity in the GPI assay (our unpublished
results). In conclusion, the present study demonstrated
that Dmp is useful as a Phe surrogate in the design of
opioid mimetics with unique biological activity. Analo-
gue 4 may lead to more potent and selective novel m
receptor antagonists. Further studies are now in progress.
1
1
erocycles 1995, 41, 1275.
20. Ohno, A.; Tsutsumi, A.; Yamazaki, N.; Okamura, M.;
Mikata, Y.; Fujii, M. Bull. Chem. Soc. Jpn. 1996, 69, 1679.
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O. J.; Getman, D. P.; Periana, R. A.; Beck, G. R. Synthesis
1992, 741.
2
2. Medium-pressure HPLC was performed on a Develosil
LOP ODS column (3Â30 cm, Nomura Kagaku) using a
CH
CN� 0.06% TFA solvent system.
3. An aqueous solution of the HC1 hydrolysate was adjusted
to pH 4±6 with Na CO yielding l- or d-Dmp as a colorless
3
2
2
3
precipitate. Determination of the l and d con®gurations was
carried out using l-amino acid oxydase according to a pre-
viously described method (Toth, G.; Lebl, M.; Hruby, V. J
Chromatogr. 1990, 504, 450). The result showed that Dmp
derived from the dipeptide analogue, which eluted later in the
preparative HPLC, turned out to have an l-con®guration, and
References and Notes
1
. Hruby, V. J.; Gehrig, C. A. Med. Res. Rev. 1989, 9, 343.
. Hansen, D. W., Jr.; Mazur, R. H.; Clare, M. In Peptides:
the other analogue, which eluted more quickly, was a d-anti-
ꢀ
2
Structure and Function; Deber, C., Hruby, V. J., Kopple, K.
pode. l-Dmp (12): [a]
D
+75.1 (c=0.73, 0.1 N HCl), R
f
0.56
on a chiral TLC plate (Macherey-Nagel, Germany, 5Â20 cm,
ꢀ
D., Eds.; Pierce Chemical Co.: Illinois, 1985; pp 491±494.
3
CH CN:H O:MeOH=4:1:1). d-Dmp (13): [a]
� 69.0
3
2
D
. Chandrakumar, N. S.; Yonan, P. K.; Stapelfeld, A.;
Savage, M.; Rorbacher, E.; Contreras, P. C.; Hammond, D. J.
Med. Chem. 1992, 35, 223.
f
(c=0.73, 0.1 N HCl), R 0.43. Both l- and d-Dmps were con-
verted to Fmoc derivatives using Fmoc-OSu, following a
ꢀ
standard method. Fmoc-l-Dmp (14): mp 196±198 C, [a]
D
ꢀ
ꢀ
4
. Hansen, D. W., Jr.; Stapelfeld, A.; Savage, M. A.; Reich-
man, M.; Hammond, D. L.; Haaseth, R. C.; Mosberg, H. I. J.
Med. Chem. 1992, 35, 684.
� 26.8 (c=0.5, MeOH). Fmoc-d-Dmp (15): mp 195±197 C,
ꢀ
[a]
24. All analogues reported here gave satisfactory FAB MS
D
+34.8 (c=0.5, MeOH).
5
. Pitzele, B. S.; Hamilton, R. W.; Kudla, K. D.; Tsymbalov,
S.; Stapelfeld, A.; Savege, M. A.; Clare, M.; Hammond, D. L.;
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7
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S. D.; Crescenzi, O.; Guerrini, R.; Picone, D.; Tancredi, T.;
28. Capasso, A.; Amodeo, P.; Balboni, G.; Guerrini, R.;
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