Stereospecific synthesis of (2S)-2-methyl-3-(2′,6′-dimethyl-4′-hydroxyphenyl)-propionic acid (Mdp) and its incorporation into an opioid peptide

Article abstract of DOI:10.1016/S0960-894X(00)00660-0

To examine the effect of replacing the N-terminal amino group in opioid peptides with a methyl group on biological activity, a stereospecific synthesis of the tyrosine analogue (2S)-2-methyl-3-(2′,6′-dimethyl-4′-hydroxyphenyl)-propionic acid (Mdp) was performed. The enkephalin analogue (2S)-Mdp-D-Ala-Gly-Phe-Leu-NH₂ turned out to be a quite potent δ opioid antagonist and a somewhat less potent μ antagonist, indicating that a positively charged N-terminal amino group is not a conditio sine qua non for the binding of opioid peptides to δ and μ receptors but may be required for signal transduction.

Full text of DOI:10.1016/S0960-894X(00)00660-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 323±325
Stereospeci®c Synthesis of (2S)-2-Methyl-3-(20,60-dimethyl-40-
hydroxyphenyl)-propionic Acid (Mdp) and its Incorporation Into
an Opioid Peptide
Yixin Lu, Grazyna Weltrowska, Carole Lemieux, Nga N. Chung and Peter W. Schiller*
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal,
Quebec, Canada H2W 1R7
Received 16 October 2000; accepted 13 November 2000
AbstractÐTo examine the e€ect of replacing the N-terminal amino group in opioid peptides with a methyl group on biological
activity, a stereospeci®c synthesis of the tyrosine analogue (2S)-2-methyl-3-(20,60-dimethyl-40-hydroxyphenyl)-propionic acid (Mdp)
was performed. The enkephalin analogue (2S)-Mdp-d-Ala-Gly-Phe-Leu-NH2 turned out to be a quite potent d opioid antagonist
and a somewhat less potent m antagonist, indicating that a positively charged N-terminal amino group is not a conditio sine qua non
for the binding of opioid peptides to d and m receptors but may be required for signal transduction. # 2001 Elsevier Science Ltd.
All rights reserved.
Until recently it has been assumed that the presence of a
positively charged nitrogen atom in opioid compounds
represents an absolute requirement for their interaction
with opioid receptors.1 The general assumption was that
the positive charge on the ligand would engage in an
electrostatic interaction with the negatively charged side
chain of an Asp residue located in the trans-membrane
domain III of opioid receptors. Alternatively, it was
suggested that a non-ionic interaction, such as chelation
of the quaternary ammonium group by multiple aro-
matic residues, may be important for the binding of
opioid compounds to the d opioid receptor.2 Recently,
somatostatin-derived cyclic hexapeptide analogues
lacking a positive charge were shown to be d opioid
antagonists with signi®cant d receptor binding anity
(Kdi =150±1070 nM).3 A neutral des-amino analogue of
a cyclic b-casomorphin analogue also turned out to be a
d antagonist with relatively modest d receptor anity
(Kdi =109 nM).3
peptide we chose the potent enkephalin analogue H-
Dmt-d-Ala-Gly-Phe-Leu-NH2 because substitution of
Dmt (20,60-dimethyltyrosine) for Tyr1 in opioid peptides
is known to generally increase d and m receptor binding
anity by at least one order of magnitude.4 The repla-
cement of the amino group in this analogue with a
methyl group required the development of a stereo-
speci®c synthesis of (2S)-2-methyl-3-(20,60-dimethyl-40-
hydroxyphenyl)-propionic acid (Mdp).
The stereospeci®c synthesis of (2S)-Mdp is outlined in
Scheme 1. Iodination of 3,5-dimethylphenol 1 using a
literature procedure5 a€orded 3,5-dimethyl-4-iodophe-
nol 2 (79% yield), which was then protected as the Boc
derivative (98% yield). Heck coupling of 3 with methyl
acrylate6 gave 4 in 61% yield. The trans con®guration of
4 was established by measurement of the coupling con-
stant (16.4 Hz) between the two alkene protons. Sub-
sequent catalytic hydrogenation, followed by basic
hydrolysis a€orded acid 6 in 88% yield. Incorporation
of Evans' chiral auxiliary (S)-( )-4-benzyl-2-oxazolidi-
none was performed in the standard manner7 to yield 7
(81% yield). Asymmetric methylation8 then furnished 8
as a single diastereomer9 in 71% yield after chromato-
graphic puri®cation. The chiral oxazolidinone auxiliary
and Boc group were then removed to give Mdp (10)10 in
96% yield.
To further investigate the role of the positive charge on
the N-terminal amino group of opioid peptides in the
interaction with their receptors, we decided to prepare
and pharmacologically characterize an enkephalin ana-
logue in which the amino group is replaced with the
neutral and almost isosteric methyl group. As parent
Peptides were prepared by manual solid-phase synth-
esis using a p-methylbenzhydrylamine resin and Boc
*Corresponding author. Tel.: +1-514-987-5576; fax: +1-514-987-
5513; e-mail: schillp@ircm.qc.ca
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00660-0

324
Y. Lu et al. / Bioorg. Med. Chem. Lett. 11 (2001) 323±325
protection according to a protocol published elswhere.11
After cleavage from the resin by HF/anisole treatment,
the crude peptides were puri®ed by preparative
reversed-phase HPLC. Their purity and structural
identity were established by analytical HPLC and
FAB-MS.
(MVD) according to published protocols.3 The GPI and
MVD assays are representative for m and d opioid
receptor interactions, respectively. In the rat brain
membrane binding assay, the enkephalin analogue (2S)-
Mdp-d-Ala-Gly-Phe-Leu-NH2 (11) showed quite high d
receptor binding anity (Kdi =11.7 nM) (Table 1). Its
anity for d receptors was only about 5 times lower
than that of [Leu5]enkephalin and 36 times lower than
that of the extremely potent parent peptide H-Dmt-d-
Ala-Gly-Phe-Leu-NH2 (12). On the other hand, 11 dis-
played only modest m receptor binding anity (Kmi =192
nM). Thus, replacement of the N-terminal amino group
in 12 with a methyl group lowered the binding anity to
a much larger extent at m receptors than at d receptors
and, consequently, peptide 11 turned out to be about 10
times more d-selective (Kmi =Kid=16.4) than 12
(Kmi =Kdi =1.53). In fact, peptide 11 is 4 times more d-
selective than [Leu5]enkephalin (Table 1). Both 11 and
12 showed no signi®cant binding anity for k receptors
(Kki >1 0 mM). Both in the GPI assay and in the MVD
assay, peptide 12 displayed subnanomolar agonist
potency with IC50s of 0.586 nM and 0.212 nM, respec-
tively (Table 2). Interestingly, the (2S)-Mdp1-analogue
(11) was found to be a moderately potent m opioid
antagonist in the GPI assay (Ke=154 nM) and an even
more potent d antagonist in the MVD assay (Ke=28.1
nM). Whereas a neutral des-amino analogue of a m
agonist peptide had previously been found to be a m
opioid antagonist,3 the data obtained with compound
11 demonstrated for the ®rst time that elimination of
the positive charge converted a d agonist peptide into a
d opioid antagonist as well.
Scheme 1. (i) KI, KIO3, MeOH, HCl; (ii) (Boc)2O, Et3N, DMAP,
H2O/THF; (iii) CH2ˆCHCOOMe, Pd(OAc)2, Et3N, (p-MePh)3P,
CH3CN, re¯ux; (iv) H2, Pd/C, 70 psi, 50 ꢀC, EtOAc; (v) 1 N aq
NaOH/THF; (vi) Et3N, PvCl, ether, 78 to 0 ꢀC, then treated with n-
BuLi, Xc, THF, 78 to 0 ꢀC; (vii) NaHMDS, THF, MeI, 78 to
25 ꢀC; (viii) LiOH, H2O2, THF/H2O; (ix) TFA, CH2Cl2.
Pharmacological Results and Discussion
Mu, d and k opioid receptor binding anities of pep-
tides 11 and 12 were determined in rat and guinea pig
brain membrane binding assays as described else-
where.11 For the determination of their in vitro opioid
activities, compounds were tested in bioassays based on
inhibition of electrically evoked contractions of the
guinea pig ileum (GPI) and the mouse vas deferens
These results indicate that elimination of the positive
charge through substitution of the N-terminal amino
group with a methyl group resulted in a neutral ligand
which was no longer capable of binding to and stabiliz-
ing an active conformation of the m or d receptor.
Obviously, the presence on the ligand of a positively
charged amino group capable of engaging in an elec-
trostatic or amino±aromatic interaction with an appro-
priate receptor moiety or domain is required for the
induction or stabilization of an active receptor con-
formation. There is good agreement between the recep-
tor binding anities of compounds 11 and 12 and their
Ke and IC50 values determined in the functional assays.
Peptide 11 is the most potent neutral d opioid antago-
nist reported to date. Its d receptor binding anity is
about 15 times higher than that of the well known d
opioid antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-
Aib-Phe-Leu-OH).12
Table 1. Binding anities of opioid peptide analogues at m and d
receptors
Compound
Kmi [nM]a
192Æ2
Kdi [nM]a
11.7Æ2.7
Kmi =Kdi
11 (2S)-Mdp-d-Ala-Gly-Phe-
Leu-NH2
16.4
12 H-Dmt-d-Ala-Gly-Phe-
Leu-NH2
[Leu5]enkephalin
0.492Æ0.075 0.322Æ0.059
9.43Æ2.07 2.53Æ0.35
1.53
3.73
aMean of 3 determinationsÆSEM.
Table 2. GPI and MVD assays of opioid peptide analogues
GPI
MVD
Compound
IC50 [nM]a
Ke [nM]a,b
154Æ25
IC50 [nM]a
Ke [nM]a,c
28.1Æ2.5
11 (2S)-Mdp-d-Ala-Gly-Phe-Leu-NH2
12 H-Dmt-d-Ala-Gly-Phe-Leu-NH2
[Leu5]enkephalin
0.586Æ0.085
246Æ39
0.212Æ0.51
11.4Æ1.1
aMean of 3±6 determinationsÆSEM.
bDetermined against the m agonist TAPP (H-Tyr-d-Ala-Phe-Phe-NH2).
cDetermined against the d agonist DPDPE (H-Tyr-c[d-Pen-Gly-Phe-d-Pen]OH).

Y. Lu et al. / Bioorg. Med. Chem. Lett. 11 (2001) 323±325
325
A non-stereospeci®c synthesis of racemic (R,S)-Mdp
and its substitution for Dmt in the dipeptide d opioid
antagonist H-Dmt-Tic-OH has recently been reported.13
H-(R,S)-Mdp-Tic-OH showed very low d and m recep-
tor binding anities and it was not characterized in the
functional GPI and MVD assays. The very low receptor
binding anities of this compound suggest that repla-
cement of the N-terminal amino group with a methyl
group has a more detrimental e€ect in the case of opioid
peptides containing the N-terminal H-Dmt-Tic-phar-
macophore than in the case of opioid peptides with a
non-cyclic d-amino acid in the 2-position of the peptide
sequence.
M. L.; Yao, W.; Liu, J.; Iwama, S.; Smith, A. B., III.; Hirsch-
mann, R. J. Med. Chem. 2000, 43, 551.
4. Hansen, D. W.; Stapelfeld, A.; Savage, M. A.; Reichmann,
M.; Hammond, D. L.; Haaseth, R. C.; Mosberg, H. I. J. Med.
Chem. 1992, 35, 684.
5. Hunig, S.; Schwarz, H. Liebigs Ann. Chem. 1956, 599, 131.
6. Dygos, J. H.; Yonan, E.; Scaros, M. G.; Goodmonson,
O. J.; Getman, D. P.; Periana, R. A.; Beck, G. R. Synthesis
1992, 741.
7. Evans, D. A.; Gage, J. R.; Leighton, J. L. J. Am. Chem.
Soc. 1992, 114, 9434.
8. Evans, D. A.; Ennis, M. D.; Mathre, D. J. J. Am. Chem.
Soc. 1982, 104, 1737.
9. A single diastereomer of 8 was con®rmed by HPLC analy-
sis. Analytical reversed-phase HPLC chromatography was
carried out on a Vydac 218-TP column (22Â250 mm) with a
linear gradient of 50±95% MeOH in aq 0.1% TFA at a ¯ow
rate of 1.0 mL/min (retention time=24.46 min).
10. Physical data for 10: [a]2D0 +45.9ꢀ (c 0.93, CH2Cl2); 1H
NMR (400 MHz, CD3COCD3) d 6.5 (s, 2H), 2.94±3.00 (m,
1H), 2.61±2.68 (m, 2H), 2.24 (d, 6H), 1.08 (d, 3H, J=6.8 Hz);
13C NMR (100.6 MHz, CD3COCD3) d 177.7, 155.9, 138.6,
128.1, 115.8, 40.2, 33.0, 20.5, 16.8; HRMS (FAB) m/e calcd for
C12H16O3 [M]+ 208.1099, found: 208.1095.
Acknowledgements
This work was supported by operating grants from the
Medical Research Council of Canada (MT-5655) and
the U.S. National Institute on Drug Abuse (DA-04443).
References and Notes
11. 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.
12. Corbett, A. D.; Gillan, M. G. C.; Kosterlitz, H. W.;
McKnight, A. T.; Paterson, S. J.; Robson, L. E. Br. J. Phar-
macol. 1984, 83, 271.
13. Salvadori, S.; Balboni, G.; Guerrini, R.; Tomatis, R.;
Bianchi, C.; Bryant, S. D.; Cooper, P. S.; Lazarus, L. H.
J. Med. Chem. 1997, 40, 3100.
1. Rees, D. C.; Hunter, J. C. In Comprehensive Medicinal Chem-
istry; Emmet, J. C., Ed.; Pergamon: New York, 1990; pp 805±
846.
2. Befort, K.; Tabbara, L.; Bausch, S.; Chavkin, C.; Evans,
C.; Kie€er, B. Mol. Pharmacol. 1996, 49, 216.
3. Schiller, P. W.; Berezowska, I.; Nguyen, T. M.-D.;
Schmidt, R.; Lemieux, C.; Chung, N. N.; Falcone-Hindley,