Low molecular weight non-peptide mimics of ω-conotoxin GVIA

Article abstract of DOI:10.1016/j.bmcl.2009.03.130

We report the synthesis and biological activity of a low molecular weight non-peptidic mimic of the analgesic peptide ω-conotoxin GVIA. The molecular weight of this compound presents a reduction by 193 g/mol compared to a previously reported lead. This co

Full text of DOI:10.1016/j.bmcl.2009.03.130

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2763–2765
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Low molecular weight non-peptide mimics of
x-conotoxin GVIA
b
c
b
Peter J. Duggan a, Richard J. Lewis b, Y. Phei Lok a, , Natalie G. Lumsden , Kellie L. Tuck , Aijun Yang
*
a CSIRO Molecular and Health Technologies, Clayton, VIC 3168, Australia
b Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld 4072, Australia
c School of Chemistry, Monash University, Clayton, VIC 3800, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and biological activity of a low molecular weight non-peptidic mimic of the anal-
gesic peptide -conotoxin GVIA. The molecular weight of this compound presents a reduction by 193 g/
mol compared to a previously reported lead. This compound exhibits an EC50 of 5.8 M and is accessible
in only six synthetic steps compared to the original lead (13 steps). We also report several improvements
to the original synthetic route.
Received 12 January 2009
Revised 21 March 2009
Accepted 25 March 2009
Available online 28 March 2009
x
l
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Keywords:
Conotoxin
GVIA
Peptidomimetic
Calcium channel blocker
Pain
The key aim of peptidomimetics is to discover small molecules
which mimic peptide function without the drawbacks associated
with large molecular weight peptides. In general, peptides cannot
be orally administered1 and have a higher tendency to elicit an
immunological response. Reduction in molecular weight is associ-
ated with better oral bioavailability,2 with smaller lead compounds
also being more amenable to further optimization. For compounds
targeting neuronal Cav2.2 channels, lower molecular weights are
especially important to address blood–brain barrier permeability.
With this aim in mind, we explored the structure–activity relation-
ship of a previously designed non-peptide mimetic and concur-
rently sought to reduce the molecular weight of our lead
compound.
and the subsequent requirement of blood–brain barrier permeabil-
ity, it is therefore of interest to develop a mimetic which is less
peptide-like and of molecular weight lower than 500 g/mol. Sev-
eral small molecule Ziconotide mimics are known.11–13 One recent
example of an orally active Cav2.2 blocker is NMED-160.5,9
Although development of this compound has been halted,14 there
is still strong interest in developing small molecule Cav2.2 block-
ers.6,15,16 Using the strongly binding
x-conotoxin GVIA as a start-
ing point, we have previously reported the design and syntheses
of two classes of mimetics.17–20 The benzothiazole class of mimet-
ics 1a–c are shown in Figure 1.
x
-Conotoxin GVIA is a calcium channel (Cav2.2) blocker iso-
lated from the marine snail Conus geographus.3,4
-Conotoxins that
target this voltage-gated channel exhibit analgesic properties. A re-
lated peptide,
-conotoxin MVIIA (Ziconotide or Prialt)5,6 has
x
x
recently been approved for the treatment of chronic pain. Of note,
the analgesic properties are effective in patients who are tolerant
to opioids and Ziconotide is amenable for long-term use without
causing dependence. Although Ziconotide is reported to be well-
accepted as an intrathecally administered drug,7,8 issues have
arisen with the poor pharmacokinetic profile associated with this
peptide.9,10 Since Cav2.2 channel blockers are primarily targeted
for the treatment of chronic pain, a drug which is administered or-
ally is ideal for this application. In order to facilitate oral delivery
* Corresponding author. Tel.: +61 3 9545 2573; fax: +61 3 9545 2446.
E-mail address: pheilok@yahoo.com (Y. P. Lok).
Figure 1. Non-peptide mimics of
low molecular weight, ‘truncated’ analogues 2a–b, 3a–b.
x-conotoxin GVIA 1a–c previously reported and
0960-894X/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.130

2764
P. J. Duggan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2763–2765
Compounds 1a, 1b and 1c (Fig. 1)17 inhibit rat neuronal Cav2.2
channels with EC50 of 3.18, 3.08 and 1.78 M, respectively.
pled with acid 6 to give the precursor 2c. The use of a phthalimide
l
protecting group in 6 gave a crystalline compound which was more
stable and easier to handle compared to the BOC-protected ana-
logue previously used.17
Although valid leads for further development, these compounds
possess high molecular weights in the range of 599–628 g/mol.
We report here the synthesis of low molecular weight, biologically
active analogues of 1b as well as several improvements to the ori-
ginal synthetic route which will facilitate the high-throughput par-
allel synthesis of other analogues.
A significant improvement to our earlier work is the preparation
of the key precursor phenol 5, prepared from commercially avail-
able 4-hydroxybenzaldehyde. Functionalization of this precursor
allows the attachment of various substituents that may not survive
the reductive amination step. For example, we previously prepared
the analogue of 3a where the amine was protected with a phthal-
imide group.17 Under non-anhydrous conditions, the phthalimide
ring was partially cleaved. This did not occur with the BOC protect-
ing group as shown in Scheme 2 for an alternative method of pre-
paring 3a.
Compound 1c was designed to mimic the Lys2, Tyr13 and Arg17
side chains of
x-Conotoxin GVIA. However, compound 1b showed
greaterselectivity forN-type (Cav2.2) versus P/Q-type(Cav2.1) chan-
nels while retaining reasonable activity17 and hence was chosen as a
lead. The omission of a hydroxyl group in 1b was advantageous from
a synthetic point of view. In this work, we designed and synthesized
four ‘truncated’ analogues of 1b, generating mimics of two amino
acid residues instead of three. Truncation of 1b gives compound 3b
which mimics the Tyr13 and Arg17 residues, and 2a which mimics
Compounds 3e and 3f were prepared by the methods outlined
in Scheme 2. These compounds are precursors that should enable
the generation of a large number of analogues.
the Tyr13 and Lys2 residues of
x
-conotoxin GVIA (Fig. 1).
In an effort to further simplify the structure of the mimic and
investigate the role of the benzothiazole ring system, we prepared
analogues of 3a–b where the benzothiazole group was replaced
with different aromatic ring systems as shown in Scheme 3. Alde-
hyde 7a, (Scheme 2) an alternative precursor to compound 3c, was
utilized as the precursor to compound series 8–10 (Scheme 3). The
use of the BOC group is significant for the preparation of com-
pounds 8–10 as the aromatic amines used caused ring-opening
of the phthalimide group. Compounds 8a and 8b are simplified
analogues of 3a and 3b, respectively, which do not contain the ben-
zothiazole ring system. Analogues 9b–c and 10b–c were designed
to investigate the effects of the diphenylmethylene group as this
moiety is prevalent in several calcium channel blockers, including
NMED-160. For this series of compounds we investigated the use of
guanidinium carboxamidine hydrochloride reagent to install the
guanidinium group for 8d and 10b. This reagent was used instead
of the bis-BOC protected guanidinium as this reagent avoids the
need for a final acidic deprotection step which may cleave the N-
benzyl bond. In general, we found that the deprotection of both
BOC groups to give 8b–10b required prolonged treatment with
4 M HCl in dioxane.
Previous molecular modeling studies on the benzothiazole core
of 1a–c revealed that rotation around the N-benzyl bond results in
two conformations.21 The Arg17-mimicking substituent can point
upwards or downwards relative to the plane of the benzothiazole
ring and hence may interact in the space intended for the Lys2
mimicking substituent. Therefore, we prepared the guanidine
substituted analogue 2b which represents one possible conforma-
tion. By similar reasoning, compound 3a was prepared. Compound
3a also represents an analogue of 1a where Arg17-mimicking sub-
stituent was replaced with
a propylamine chain (Fig. 1).
Compounds 2a–b contain an amide bond attached to the 2-amino-
benzothiazole and an ortho-substituted ring, whereas compounds
3a–b are para-substituted benzylamines. Comparisons between
2a–b and 3a–b will uncover any significant effects due to these
differences.
Compounds 2a–b, and 3a–c were prepared according to the
route shown in Scheme 1. The 2-aminobenzothiazole 417 was cou-
Compounds 2a–b, 3a–f, 8a–10a and 8b–10b were tested for N-
type calcium channel inhibition using radioligand-binding dis-
placement assay as previously reported.17 As shown in Table 1,
the truncated Tyr13- Lys2 mimic 2a and the corresponding gua-
nidylated analogue 2b were inactive. However, compound 3b gave
an EC50 of 5.8 lM which is only 2.7 lM lower than the preferred
lead compound 1b. This small reduction in activity is compensated
by a significant drop in molecular weight by 193 g/mol to 448 g/
mol compared to the original lead of 641 g/mol. In addition, the
time and cost of synthesis has also been reduced as compound
3b can be prepared in six steps, compared to 13 steps for com-
pound 1c. Interestingly, compound 3a, which contained an amino
Scheme 1. Synthesis of truncated analogues 2a–b and 3a–c. Reagents and
conditions: (a) (i) 4-hydroxybenzaldehyde, dry toluene, 4 Å molecular sieves, reflux
(ii) ethanol, NaBH4, reflux, 70%; (b) EDCI, HOBt, NMM, DMF, 6, 74 °C, 66%; (c)
N2H4.H2O, EtOH, THF, reflux, 56%; (d) N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-
carboxamidine, DCM, Et3N, 81%; (e) 2 M HCl/EtOAc, rt, 53%; (f) K2CO3, DMF, 50 °C,
54%.
Scheme 2. An alternative method for the preparation of 3c, and precursors 3e and
3f. Reagents and conditions: (a) K2CO3, DMF, 50 °C, 91%; (b) (i) dry toluene, 4 Å
molecular sieves, reflux; (ii) dry ethanol, NaBH4, reflux, 87%; (c) Bu4NF, (CH3)4SiN3,
THF, 50 °C, 88%.

P. J. Duggan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2763–2765
2765
prepared by addition of 1 molar equivalent of HCl in EtOAc did
not show any activity in the assay. The precursors 3e, 3f and 5
were also inactive.
The fact that none of the compounds with varied aromatic cores
8–10 were active suggests that the rigidity and/or hydrogen-bond-
ing interactions of the benzothiazole ring system are important for
activity. Additionally, the removal of the benzothiazole-fused sys-
tem shortens the span of the molecule towards any putative bind-
ing pockets. An increase in bulk due to the large diphenylmethyl
groups in 9a–b or 10a–b did not give any appreciable activity.
However, the presence of a guanidyl side chain in 9b and 10b gave
detectable micromolar activity compared to the amino analogues
9a and 10a. It could be argued that guanidylated substituent pos-
sesses higher basicity and a more delocalized charge that could en-
hance interaction at the binding site.
Scheme 3. Synthesis of compounds 8–10. Reagents and conditions: (a) (i) dry
toluene, 4 Å molecular sieves, reflux (ii) dry ethanol, NaBH4, reflux, 69–91%; (b) 2 M
HCl/EtOAc, 81–100%; (c) N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxami-
dine, DCM, Et3N, 58–62%; (d) 4 M HCl in dioxane, 0 °C, 81–98%; (e) guanidinium
carboxamidine hydrochloride, DMF, Hunig’s base, 47–85% (for 8a and 10b).
In summary, we have designed and synthesized compounds
2a–b and 3a–b which are ‘truncated’ analogues of a previously re-
ported lead compound.17 Compound 3b was found to retain signif-
icant activity despite the loss of nearly 200 g/mol in molecular
weight compared to the original lead compounds 1b–c. The discov-
ery of this compound opens a new avenue for our research where
future analogues of 3b are not only more drug-like, but these ana-
logues can be accessed in 5–6 steps instead of 12–13 steps (for 1a–
c). The reduction of molecular weight and reduction in number of
synthetic steps are important and necessary advancements for the
translation of this research into a pragmatic drug development set-
ting. The various improvements to the previously published17 ori-
ginal synthetic scheme that are reported here will also facilitate
the efficient preparation of analogues of 3b.
Table 1
N-type calcium channel binding results
a
Compound
EC50
(
lM)
1a
1b
1c
2a
2b
3a
3b
8a
8b
9a
9b
10a
10b
3.18
3.08
1.78
>100
33
76
5.8
34
21
>100
23
>100
12
Supplementary data
a
EC50 values were calculated from dose–response curves, with each data point
recorded in triplicate.
Supplementary data (details of experimental procedures and
spectroscopic data for all compounds) associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2009.03.130.
group instead of a guanidyl group did not show appreciable activ-
ity. This highlights the importance of the guanidyl group and also
may indicate that the higher basicity is required to retain activity.
The comparison between 2b and 3b may indicate that less rigidity
is required since 3b contains a more freely-rotating N-benzyl bond
instead of an amide bond. Furthermore, 3b which has the propyl-
guanidyl at the para-position of the ring gives a compound which
is more extended than the ortho-substituted 2b. The larger span
of 3b could be important as the compound is expected to mimic
a very large peptide. The dose–response curves for compounds
2b and 3a–b are shown in Figure 2.
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Figure 2. Dose–response curves for 2b, 3a and 3b. 95% Confidence intervals are
shown.