Novel Dmt-Tic dipeptide analogues as selective delta-opioid receptor antagonists

Article abstract of DOI:10.1016/S0960-894X(99)00652-6

A series of Dmt-Tic analogues with substitution on the Tic aromatic ring has been synthesized and evaluated for opioid receptor affinity and activation. Incorporation of large hydrophobic groups at position 7 of Tic did not greatly alter the δ opioid receptor binding affinities of the dipeptides whereas substitution at position 6 substantially diminished their affinity. These modified Dmt-Tic peptides showed binding affinities as low as 2.5 nM with up to 500-fold selectivity for the δ versus μ opioid receptor and proved to be δ receptor antagonists.

Full text of DOI:10.1016/S0960-894X(99)00652-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 167±170
Novel Dmt-Tic Dipeptide Analogues as Selective Delta-Opioid
Receptor Antagonists
D. Page, a,* A. McClory, a T. Mischki, a R. Schmidt, a
J. Butterworth, b S. St-Onge, b M. Labarre, b K. Payza b and W. Brown a
a
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Department of Chemistry, AstraZeneca R&D Montreal, 7171 Frederick-Banting, Saint-Laurent, Quebec, Canada, H4S 1Z9
b
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Department of Pharmacology, AstraZeneca R&D Montreal, 7171 Frederick-Banting, Saint-Laurent, Quebec, Canada, H4S 1Z9
Received 8 October 1999; accepted 18 November 1999
AbstractÐA series of Dmt-Tic analogues with substitution on the Tic aromatic ring has been synthesized and evaluated for opioid
receptor anity and activation. Incorporation of large hydrophobic groups at position 7 of Tic did not greatly alter the d opioid
receptor binding anities of the dipeptides whereas substitution at position 6 substantially diminished their anity. These modi®ed
Dmt-Tic peptides showed binding anities as low as 2.5 nM with up to 500-fold selectivity for the d versus m opioid receptor and
proved to be d receptor antagonists. # 2000 Elsevier Science Ltd. All rights reserved.
The development of highly selective and potent opioid
agonists and antagonists has been slowed by the lack of
knowledge about the geometry of the ligand-binding
pocket. The direct evaluation of ligand receptor inter-
actions using X-ray di€raction analysis or NMR spec-
troscopy is complicated by the fact that the opioid
receptors, as members of the family of G-protein cou-
pled receptors, are embedded in the cell membrane.
Therefore, the e€orts to characterize the ligand binding
site have been focussed on extensive structure±activity
studies of endogenous opioid peptides such as enkepha-
lins, endorphins and dynorphins as well as the amphib-
ian opioid peptides deltorphin and dermorphin.
potency.5 In addition, the methylation increased the
hydrophobicity of the compound and added some con-
formational constraints on the aromatic ring. Further
N-terminal modi®cation added to the knowledge of the
spatial requirements for d-antagonism. N-mono and
N,N-dimethylation,6 reduction of the amide bond7 or
incorporation of b-methyl-20,60-Dmt8 resulted in analo-
gues with high d receptor anity and selectivity as well
as d antagonist potency, while alkylation with larger
groups such as diethyl, piperidine, pyrrolidine or pyr-
role decreased the potency as d antagonists and led to a
decrease in selectivity.9 In order to gain further insight
into the design of new opioid ligands we report herein a
novel class of Dmt-Tic ligands with substitution on the
Tic aromatic ring easily accessible by utilizing Suzuki
cross-coupling chemistry.
It was shown recently that opioid peptide analogues
containing a Tic (1,2,3,4-tetrahydroisoquinoline-3-car-
boxylic acid) residue in the 2-position are potent and
highly selective d antagonists.1 The resulting N-terminal
Tyr-Tic fragment has been introduced in a variety of
opioid peptides with di€erent receptor selectivity resulting
not only in a change of their biological properties but
also directing their anity towards the d receptor.2,3 In
fact, the smallest structure proven to be a d antagonist is
the Tyr-Tic dipeptide segment itself.4 Methylation of the
Tyr residue led to the Dmt-Tic dipeptide (Dmt=20,60-
dimethyl-tyrosine) which exhibited improved d opioid
anity and selectivity and enhanced d antagonist
The amino acid H-l-Tic(7±OH)±OH (1) was used as
starting material for all examples, the hydroxyl group
providing a handle for substitution at either positions 6,
7 or 8 (Scheme 1). Protection of the amine followed by
esteri®cation of the acid with trimethylsilyl diazo-
methane10 provided Boc-l-Tic(7±OH)±OMe (2). Forma-
tion of the tri¯ate (4) followed by Suzuki cross-coupling
with a series of boronic acids a€orded Boc-l-Tic(7±OR)±
OMe derivatives 5a±e. H-l-Tic(7±OH)±OH (1) could
also be speci®cally mono- or bis-brominated (6a±b)
which were in turn Boc-protected and esteri®ed to give
compounds 7a±b. Formation of the methyl ether fol-
lowed by Suzuki cross-coupling with boronic acids
a€orded compounds 9a±f.
*Corresponding author. Tel.: +1-514-832-3200; fax: +1-514-832-
3232; e-mail: daniel.page@astrazeneca.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00652-6

Â
D. Page et al. / Bioorg. Med. Chem. Lett. 10 (2000) 167±170
168
Scheme 1. (i) di-tert-butyldicarbonate, Na2CO3, H2O/dioxane, 16 h, quant.; (ii) TMSCHN2, MeOH/C6H6, 30 min, 99%; (iii) NaH, MeI, DMF,
30 min, 93%; (iv) tri¯ic anhydride, Et3N, CH2Cl2, 20 min, 85%; (v) Ar±B(OH)2, (Ph3P)4Pd, Na2CO3, toluene/EtOH, 70 ꢀC, 12 h, 75±95%; (vi) Br2,
AcOH, 1±12 h, 95%.
Scheme 2. (i) 1 M HCl/AcOH, 1 h, quant; (ii) Boc-d/l-Dmt-OH, EDC, HOBt, DIPEA, DMAP, DMF, 2±4 days, 15±55%; (iii) 1 M LiOH, EtOH/
H2O, Á, 3 h, quant.
After N-deprotection these amino acid derivatives were
used to prepare the di€erent Dmt-Tic dipeptides
(Scheme 2) by coupling with Boc-d/l-Dmt±OH11 yielding
compounds 10a±l. Peptide couplings were found to be
slow and gave low yields likely due to the bulky nature
of the amino acids. The best results were obtained using
a combination of EDC, HOBt and DMAP as coupling
reagents. Bis-substituted Tic derivatives 9a±c failed to
give any of the desired dipeptide products using di€erent
coupling strategies (HATU, EDC, HOBt, DPPA)
probably due to steric hindrance. Saponi®cation of the
methyl ester followed by removal of the N-protecting
group a€orded the di€erent dipeptides 11a±l.12 The
formation of small amounts of the respective diketo-
piperazines was observed but these were easily removed
by reverse-phase HPLC. The binding anities of dipep-
tides 11a±l were determined at the three opioid receptors
(d, m, k) (Table 1). The diastereomeric dipeptides were

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D. Page et al. / Bioorg. Med. Chem. Lett. 10 (2000) 167±170
169
Table 1. Binding anities of Dmt-Tic dipeptide analogues 11a±l on opioid receptors
R1
R2
R3
aa
d receptor
125I-Deltorphin II
IC50 (nM)
m receptor
125I-FK-33824
IC50 (nM)
k receptor
125I-DPDYN
IC50 (nM)
m/d
Ratio
Dmt-Tic
11a
11b
11b
11c
11c
11d
11d
11e
11e
11f
H
OH
OMe
OMe
OH
OH
OMe
OMe
H
H
H
H
Br
Br
Br
Br
H
H
H
H
H
H
H
H
Ph
H
H
H
H
Br
Br
Br
Br
H
H
H
H
H
H
H
H
H
H
H
l
d/l
d/l
l
1.6
236
894
2160
2280
2870
8130
1240
8240
1670
1940
635
n/d
587
2650
1140
142
37503
9290
9000
9440
n/db
>10000
n/d
>10000
>10000
5740
>10000
7560
>10000
>10000
2730
558
9.2
211
237
Ð
10.9
12.1
>10000
1080
571
d
l
1.1
d
14.4
505
15.9
48.1
Ð
155
546
211
10.5
546
Ð
l
2.5
122
Ph
Ph
d
l
13.2
81.7
3.8
4.9
5.8
13.5
52.7
2540
1160
1520
4-F-Ph
4-F-Ph
4-OMe-Ph
4-OMe-Ph
3-thiophene
2-benzo[b]furan
OMe
d
11f
l
11g
11g
11h
11i
11j
11k
11l
d/l
l
d/l
d/l
d/l
d/l
d/l
2440
>10000
7220
4640
>10000
>10000
>10000
>10000
OMe
OMe
4-F-Ph
4-OMe-Ph
6.2
3.1
aBased on chromatography retention time.
bn/d: Not tested.
separated by reverse phase HPLC13 and the compounds
with higher d receptor anities were assumed to be the
l-Dmt containing isomers based on the SAR of pub-
lished Dmt-Tic analogues.9
3-thiophene derivative (11h) showed good binding a-
nity at the d receptor albeit with poor selectivity. The
benzo[b]furan derivative (11i) displayed only moderate
anity for the d-receptor probably due to its larger size
creating some kind of steric hindrance. Introduction of
bulky substituents R2 (11j±l) at position 6 of Tic was
The presence of the hydroxyl group at R1 (compounds
11a and 11c) greatly reduces the binding anities of the
dipeptides towards the d opioid receptor, however,
when the hydroxyl group was methylated (11b and 11d)
the binding anities for the l isomer were restored.
This result indicates that an H-bond donating group is
detrimental to an ecient ligand-receptor interaction
whereas a sterically more demanding H-bond acceptor
is well tolerated. Furthermore, the fact that the 6,8-
dibromo compound 11d (l isomer) displayed an IC50 of
2.5 nM and a 500-fold selectivity for d over m receptor,
similar to the values observed for Dmt-Tic, indicates
that the hydrophobic binding pocket is `spacious'
enough to accommodate bulkier ligands.
Further substitutions at this 7-position (R1) of Tic with
bulkier hydrophobic groups seemed to con®rm the
hydrophobic pocket hypothesis. Introduction of a
phenyl group did not greatly alter the d receptor anity
since dipeptide 11e still showed low nM binding anity.
Addition of p-methoxybenzene (11g) produced one of
the most selective compounds for the d-receptor. The
anity of the p-¯uorobenzene analogue (11f) was
comparable to that of 11e but a loss of selectivity
was due to a 2-fold increased m-receptor anity. The
Figure 1. E€ect of 100 nM Dmt-Tic±OH analogues, on SNC80
GTP[g]35S binding dose response curve on human d receptor.

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D. Page et al. / Bioorg. Med. Chem. Lett. 10 (2000) 167±170
170
detrimental to d opioid receptor binding. Except for
compound 11d (l), which turned out to be a partial
agonist at 100 nM (EC50 28 nM), none of the dipeptides
showed d agonist activity up to 30 mM in the GTP[g]35S
functional assay. Analogues 11f (l) and 11g (d/l) were
shown to block the e€ect of the d selective opioid ago-
nist SNC-80 with Ke values of 11.5 and 38.6 nM,
respectively, compared to 5.3 nM for Dmt-Tic (Fig. 1).
9. Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Guerrini, R.;
Balboni, G.; Salvadori, S. DDT 1998, 3, 284.
10. Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm.
Bull. 1981, 29, 1475.
11. Dygos, J. H.; Yonan, E. E.; Scaros, M. G.; Goodmonson.
O. J.; Getman, D. P.; Periana, R. A.; Beck, G. R. Synthesis
1992, 741.
12. Selected analytical characterizations of the dipeptide ana-
1
logues: (11b, d/l isomers) H NMR (CD3OD) d 6.94 (d, 1H),
6.70 (m, 2H), 6.55, 6.44, 6.33, 6.28 (4s, 2H), 5.25 (m, 1H), 4.63
(m, 2H), 4.41 (m, 3H), 3.79 (m, 1H), 3.75, 3.72 (2s, 3H), 3.38
(d, 1H), 3.22 (m, 3H), 3.11 (m, 2H), 2.74 (m, 2H), 2.26, 2.18
(2s, 6H), 1.86 (dd, 1H); MS for C22H26N2O5 399.18 (MH+);
C22H26N2O5; calc: C 53.49%, H 5.26%, N 5.11%; found: C
Acknowledgements
The authors are thankful to Dr. K. Carpenter for NMR
analysis of the dipeptides. A sample of H-Dmt-Tic±OH
was kindly provided by Dr. Lawrence H. Lazarus
(NIEHS).
1
53.39%, H 5.19%, N 5.21%; (11d, l isomer) H NMR (CD3
OD) d 7.34, 7.27, 7.18 (3s, 1H), 6.47, 6.35, 6.31 (3s, 2H), 4.74
(d, 1H), 4.39 (m, 1H), 4.20 (m, 1H), 3.77 (m, 4H), 3.03 (m,
1H), 2.89 (m, 1H), 2.72 (dd, 1H), 2.27, 2.17 (2s, 6H), 1.71 (dd,
1H); MS for C22H24Br2N2O5 557.25 (MH+); C22H24Br2
N2O5; calc: C 41.79%, H 4.08%, N 4.09%; found: C 41.80%,
1
References and Notes
H 4.13%, N 4.10%; (11f, d/l isomers) H NMR (CD3OD) d
7.57 (m, 2H), 7.36 (m, 2H), 7.30±6.99 (m, 3H), 6.57, 6.44, 6.26
(3s, 2H), 5.36 (m, 1H), 4.81 (d, 1H), 4.63 (m, 1H), 4.51 (d, 2H),
4.39 (m, 1H), 3.82 (m, 1H), 3.50 (d, 1H), 3.23 (m, 3H), 3.11
(m, 2H), 2.84 (m, 2H), 2.27 (m, 6H), 1.98 (dd, 1H); MS for
C27H28N2O4 463.01 (MH+); C27H28N2O4; calc: C 58.55%, H
5.13%, N 4.61%; found: C 58.48%, H 5.10%, N 4.55%; (11g,
d/l isomers) 1H NMR (CD3OD) d 7.48 (m, 2H), 7.36 (m, 2H),
7.05 (dd, 1H), 6.97 (m, 2H), 6.57, 6.44, 6.29 (3s, 2H), 5.32 (m,
1H), 4.78 (d, 1H), 4.63 (m, 1H), 4.50 (d, 2H), 4.38 (m, 1H),
3.82 (m, 1H), 3.79 (m, 3H), 3.46 (d, 1H), 3.24 (m, 3H), 3.10
(m, 2H), 2.81 (m, 2H), 2.30 (m, 6H), 1.96 (dd, 1H); MS for
C28H30N2O5 475.02 (MH+); C28H30N2O5; calc: C 58.69%, H
5.25%, N 4.50%; found: C 58.78%, H 5.29%, N 4.43%; (11j,
d/l isomers) 1H NMR (CD3OD) d 7.48 (m, 1H), 7.39 (m, 2H),
7.15 (t, 1H), 7.06 (d, 1H), 6.92 (m, 2H), 6.43, 6.30 (2s, 2H),
5.15 (m, 1H), 4.37 (d, 1H), 4.25 (m, 1H), 4.13 (d, 1H), 3.96 (d,
1H), 3.90 (m, 1H), 3.61 (m, 2H), 3.15 (m, 3H), 3.00 (m, 2H),
2.75 (m, 2H), 2.10 (m, 6H), 2.04 (dd, 1H); MS for C28H30N2O5
475.15 (MH+); C28H30N2O5; calc: C 52.09%, H 4.67%, N
3.75%; found: C 52.14%, H 4.66%, N 3.84%.
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13. Preparative HPLC puri®cation was performed on a Gilson
system using a Luna C18 (250Â20.2 mm) column and one of
the two following gradients: Gradient IÐ20±50% B; Gradient
IIÐ30±80% B in 25 min (A 0.1% TFA in H2O, B 0.1% TFA
in CH3CN), ¯ow rate 40 mL/min at room temperature.