Design and biological evaluation of non-peptide analogues of omega-conotoxin MVIIA

Article abstract of DOI:10.1016/S0960-894X(99)00699-X

Omega-conotoxin MVIIA, a highly potent antagonist of the N-type voltage sensitive calcium channel, has shown utility in several models of pain and ischemia. We report a series of three alkylphenyl ether based analogues which mimic three key amino acids of the toxin. Two of the compounds have been found to exhibit IC₅₀ values of 2.7 and 3.3 μM at the human N-type voltage sensitive calcium channel. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(99)00699-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 345±347
Design and Biological Evaluation of Non-Peptide Analogues of
Omega-Conotoxin MVIIA
Stefan Menzler, a Jack A. Bikker, b Nirmala Suman-Chauhan a and David C. Horwell a,*
aParke-Davis Neuroscience Research Centre, Robinson Way, Cambridge CB2 2QB, UK
bParke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
Received 3 November 1999; accepted 13 December 1999
AbstractÐOmega-conotoxin MVIIA, a highly potent antagonist of the N-type voltage sensitive calcium channel, has shown utility
in several models of pain and ischemia. We report a series of three alkylphenyl ether based analogues which mimic three key amino
acids of the toxin. Two of the compounds have been found to exhibit IC50 values of 2.7 and 3.3 mM at the human N-type voltage
sensitive calcium channel. # 2000 Elsevier Science Ltd. All rights reserved.
o-Conotoxin MVIIA, a 25 amino acid peptide from the
venom of the marine cone snail Conus magus,1,2 is a
highly potent and selective blocker of the N-type vol-
tage sensitive calcium channel (VSCC).3 It has a binding
anity of <10 nM for the N-type VSCC, compared to
>1 mM for the L-type VSCC.4 N-type VSCCs are found
exclusively in neurons where they regulate intracellular
calcium concentration, which a€ects various important
neural functions such as neuronal excitability, neuro-
transmitter release and neurosecretory activity.5±8
Antagonists of N-type VSCCs would, therefore, o€er
great potential for therapeutic intervention.9 The syn-
thetic version of o-conotoxin MVIIA, Ziconotide1, has
demonstrated ecacy in animal models of traumatic
brain injury, focal cerebral ischemia and pain.10±13 For
severe neuropathic pain, it is more potent than mor-
phine and chronic administration does not elicit toler-
ance or lead to addiction.14 Ziconotide1 is currently in
clinical trials for the treatment of pain.
responsible for the binding of the toxin to the channel
pores have been determined to be Lys-2, Arg-10, Leu-
11, Tyr-13 and Arg-21.22 As the middle segment of
the peptide chain also plays a role in determining the
selectivity for the N-type VSCC,22,23 we focused our
attention on the three central amino acids Nos. 10, 11
and 13 (Fig. 1).
Following our `dendroid approach',24 side chain
mimetics of the three selected amino acids were attached
to a dendritic sca€old, which is composed of aromatic
branching units and adjustable linkers. Ether linkages
were preferred over ester or amide bonds since they are
expected to be more stable under physiological condi-
tions. These molecules possess a high degree of con-
formational ¯exibility. In contrast to rigid molecules,
the `dendroid motif' allows each pharmacophoric group
to ®nd the preferred conformation for receptor binding
independently. We argue that this facilitates the search
for a global energy minimum of the receptor±ligand
complex.
Encouraged by the clinical utility of o-conotoxin
MVIIA, we were interested in the possibility to produce
low molecular weight mimetics of this peptide. Lead
generation has been sought by a high volume screening
strategy.15±20 In this paper we disclose a ligand based
approach, because the three-dimensional solution
structure of the toxin is available from the Brookhaven
Protein Data Bank.21 Several of the key amino acids
Our starting point for this design strategy was the pre-
viously reported analogue 1 (Fig. 1).25 The activity of 1
at the N-type VSCC was evaluated in the IMR-32
assay.26 This parent template had the necessary spatial
requirements, but proved virtually inactive (19% inhi-
bition at 10 mM, triplicate determination). In an attempt
to reduce ¯exibility, we shortened the linkers between
the branching units and the pharmacophoric groups.
This modi®cation led to the closely related analogues 2
and 3,27 the synthesis of which is outlined in Scheme 1.
*Corresponding author. Fax: +44-1223-416712; e-mail: david.horwell
@wl.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00699-X

346
S. Menzler et al. / Bioorg. Med. Chem. Lett. 10 (2000) 345±347
Figure 1. Peptidomimetic design rationale.
Figure 2. Overlay of 2 (grey) with three key amino acids of o-con-
otoxin MVIIA (green).
Table 1. IC50 values for blocking N-type (IMR-32 assay) and L-type
(A7r5-assay) VSCCs
Compounds
IMR-32 IC50 (mM)
A7r5 IC50 (mM)
1
2
3
19% at 10 mM
3.3‹0.3 (n=3)
2.7‹0.9 (n=3)
69% at 10 mM
4.2 (n=1)
3.0 (n=1)
Scheme 1. Reagents and conditions: (a) 3,4-Dihydro-2H-pyran/p-
TsOH/EtOAc/0 ꢀC-rt/1 h, 68±78%; (b) 9-BBN/THF, then 2-iodo-
benzyl chloride/K3PO4/Pd(PPh3)4/dioxane/60 ꢀC/2 h, 46±70%; (c) 2-
iodopropane/NaH/H2O/DMF/0 ꢀC-rt/15 h, 43%; (d) NaOMe/MeOH/
rt/1 h, 60%; (e) NH2OH/MeOH/rt/24 h, 57%; (f) Ac2O/120 ꢀC/4 h,
82%; (g) NaOMe/MeOH/rt/30 min, 94%; (h) PPh3/DIAD/THF/0 ꢀC-
rt/2 h, 86%; (i) NaOMe/MeOH/rt/1 h, 85%; (k) NaH/DMF/0 ꢀC-rt/2
h, 88±89%; (l) Dowex 50WX2-400/MeOH-PhMe 5:1/1 h, 88±93%; m)
Ra-Ni/4 bar H2/EtOH/rt/5 h, then EtOH:AcOH-H2O 20:1:1 12 h, 40±
46%.
In the IMR-32 assay, 2 and 3 were found to be approxi-
mately equipotent (IC50 3.3 and 2.7 mM, respectively,
see Table 1). Obviously, the position of the phenolic
hydroxyl group is not crucial. Ongoing SAR studies will
reveal if this site is amenable to further modi®cation or
even truncation.
The design of 2 is supported by molecular modelling.
Figure 2 shows an overlay of 2 and the three key amino
acid side chains of o-conotoxin MVIIA. The overlay
was generated with GASP 2.05 on a Silicon Graphics
Octane workstation running IRIX 6.5.3.28 Structure 2
was built and energetically optimised using the standard
SYBYL 6.5 force ®eld.29 The middle segment of the
toxin was used as a rigid template. As depicted in Figure
2, the pharmacophoric groups of 2 and the amino acid
side chains of the toxin overlap with high stringency.
A7r5 rat vascular smooth muscle cells (Table 1).30,31
Analogue 1 gave 69% inhibition at 10 mM; 2 and 3 were
able to antagonise the L-channel with IC50 values of 4.2
and 3.0 mM, respectively.
In summary, a series of three small-molecule mimetics
of o-conotoxin MVIIA has been synthesised. Two
compounds possess micromolar activity in blocking N-
type VSCCs in the IMR-32 assay and L-type VSCCs in
the A7r5-assay. They are interesting leads for the
development of novel antinociceptive agents. Further
evaluation of the pharmacophoric groups and the den-
droid core architecture is in progress.
To assess the selectivity for the N-type VSCC, 1±3 were
also tested for functional activity at the l-type VSCC in

S. Menzler et al. / Bioorg. Med. Chem. Lett. 10 (2000) 345±347
347
Acknowledgements
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23. Ramachandran, J.; Nadasdi, L.; Gohil, K.; Kristipati, R.;
Tarczy-Hornoch, K.; Gaur, S.; Singh, T.; Bell, J.; Miljanich,
G. In Perspectives in Medicinal Chemistry; Testa, B., Ed.;
VCH: Weinheim, 1993; pp 374-388.
The authors wish to thank Dr. Michael Ra€erty and
Dr. Thomas Malone for their co-operation, and Joanne
Schmidt for testing our compounds in the IMR-32
assay. We also thank Cerep (France) for conducting the
A7r5-assay, Dr. Giles Ratcli€e for NMR spectra and
Jane McGu€og for mass spectra.
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(d, J=6.1 Hz, 6 H, CH(CH3)2), 1.98 (s, 3H, CH3COO ), 2.88
(m, 2H, Ar-CH2-CH2-C6H4OH), 2.96 (m, 2 H, Ar-CH2-CH2-
C6H4OH), 3.23 (t, J=6.1 Hz, 2 H, Ar-O-CH2-CH2-Ar), 4.26
(t, J=6.1 Hz, 2H, Ar-O-CH2-CH2-Ar), 4.58 (m, 1 H,
CH(CH3)2), 4.95 (s, 2 H, Ar-CH2-O-Ar), 6.13±6.21 (m, 3H,
arom.), 6.70±7.83 (m, 12H, arom.). 13C NMR (100 MHz,
CD3OD) d 180 (CH3COO ), 168 (C(NH2)2), 162 (2 C), 161,
156 (C-OH), 147, 142, 136, 134, 131 (4 C), 130 (3 C), 129 (2 C),
127, 116 (3 C), 97 (2 C), 95, 71, 70, 69, 38, 36 (2 C), 24
(CH3COO ), 22 (2 C, CH(CH3)2). HRMS (FAB) calc. for
[M+H]+ 525.2753, found 525.2737. Analysis calcd for
C35H40O6N2: C 71.90; H 6.90; N 4.79. Found: C 71.47; H
1
6.86; N 4.74. 3xAcOH: H NMR (400 Mhz, CD3OD) d 1.25
(d, J=6.1 Hz, 6 H, CH(CH3)2), 1.90 (s, 3 H, CH3COO ), 2.83
(m, 2 H, Ar-CH2-CH2-C6H4OH), 2.93 (m, 2 H, Ar-CH2-CH2-
C6H4OH), 3.15 (t, J=6.1 Hz, 2 H, Ar-O-CH2-CH2-Ar), 4.18 (t,
J=6.1 Hz, 2 H, Ar-O-CH2-CH2-Ar), 4.48 (m, 1 H, CH(CH3)2),
4.87 (m, 2 H, Ar-CH2-O-Ar), 6.04±6.12 (m, 3 H, arom), 6.56±7.74
(m, 12 H, arom). 13C NMR (100 MHz, CD3OD) d 180
(CH3COO ), 168 (C(NH2)2), 162 (2 C), 161, 158 (C-OH), 148,
144, 142, 136, 131 (3 C), 130 (3 C), 129 (2 C), 128,127, 121,
116, 114, 97 (2 C), 95, 71, 70, 69, 38, 36 (2 C), 24 (CH3COO ),
22 (2 C, CH(CH3)2). HRMS (ES) calcd for [M+H]+ 525.2753,
.
found 525.2751. Analysis calcd for C35H40O6N2 H2O: C 69.75;
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