Synthesis and Cav2.2 binding data for non-peptide mimetics of ω-conotoxin GVIA based on a 5-amino-anthranilamide core

Article abstract of DOI:10.1071/CH07327

A simple and efficient method has been developed for the synthesis of two anthranilamide-based non-peptide mimetics of ?-conotoxin GVIA. These anthranilamide derivatives aim to mimic the K2, R17, and Y13 residues of the peptide. The synthetic route described enables the rapid synthesis of anthranilamide analogues with identical alkyl chain lengths. The target compounds show affinity to rat N-type voltage gated calcium channels (Ca _v2.2) with EC₅₀ values of 42 and 75 ?M. CSIRO 2008.

Full text of DOI:10.1071/CH07327

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2008, 61, 11–15
Synthesis and Cav2.2 Binding Data for Non-Peptide
Mimetics of ω-Conotoxin GVIA based on
a 5-Amino-Anthranilamide Core
Peter J. Duggan,A Jonathan M. Faber,B Janease E. Graham,A,B
Richard J. Lewis,C Natalie G. Lumsden,C and Kellie L. TuckB,D
ACSIRO, Molecular and Health Technologies, Bag 10, Clayton South VIC 3169, Australia.
BSchool of Chemisty, Monash University, Clayton VIC 3800, Australia.
CInstitute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia.
DCorresponding author. Email: kellie.tuck@sci.monash.edu.au
A simple and efficient method has been developed for the synthesis of two anthranilamide-based non-peptide mimetics
of ω-conotoxin GVIA. These anthranilamide derivatives aim to mimic the K2, R17, and Y13 residues of the peptide.
The synthetic route described enables the rapid synthesis of anthranilamide analogues with identical alkyl chain lengths.
The target compounds show affinity to rat N-type voltage gated calcium channels (Cav2.2) with EC50 values of 42
and 75 µM.
Manuscript received: 13 September 2007.
Final version: 29 November 2007.
Introduction
N-type VGCC receptors.[8] However,
a related peptide
ω-conotoxin MVIIA (Prialt, Zinconotide), isolated from Conus
magus, was recently approved for clinical use for chronic
pain where morphine-based therapeutics are not efficacious.[19]
Another ω-conotoxin, CVID, isolated from Conus catus, reached
Phase II clinical trials and was reported to have a more favourable
therapeutic index than that of ω-conotoxin MVIIA.[20] Although
successful analgesics, both peptides require intrathecal admin-
istration, i.e. injection near the spinal chord, to mediate
beneficial effects, owing to a poor pharmacokinetic profile
typical of peptide-based therapeutics.[19,21] NeuroMed’s orally
available small molecule N-type VGCC blocker, NMED-160,
which reached Phase II clinical trials,[11,22] has recently been
abandoned.[23] This highlights the need for development of more
small molecule N-typeVGCC blockers.An alternative approach,
undertaken by our group, is the rational design of small molecule
non-peptide mimetics of ω-conotoxin GVIA that act as N-type
VGCCblockers.[24–26] Asthebindingepitopesanddefinedstruc-
ture of ω-conotoxin GVIA have been extensively studied, this
peptide is an ideal candidate for mimicry.[14,27,28] The solution
structure of the native peptide was solved and published by Pal-
largyandNortonin1999,[28] andalaninescanningundertakenby
Lew[27] has identifiedY13 and K2 as the most important amino
acids for receptor binding. These amino acids, along with R17,
also identified as important for receptor affinity, are part of the
suggested receptor binding site on ω-conotoxin GVIA.[14,27,29]
Recently our group reported the synthesis and biological
activity of anthranilamide (1) and benzothiazole (2) derivatives
as non-peptide mimetics (see Fig. 2)[24,25] of ω-conotoxin GVIA
that were designed to project the amino acids K2, Y13, and
R17 with similar αβ bond vectors to those present in the native
peptide. Binding affinities for four of the non-peptide mimetics
are summarized inTable 1. For benzothiazole derivative binders,
The complex venom produced by marine snails of the genus
Conus has provided medicinal chemists with the inspiration for
the development of novel therapeutics.[1] Bioactive components
of this venom include both low molecular weight compounds,[2]
such as 5-hydroxytryptamine and serotonin, as well as pep-
tides including conantokin,[3–5] conanlukin,[6] conopressin,[4]
contryphan,[7] and the ω-conotoxins.[8] The attention of our
researchgrouphasbeendrawntotheω-conotoxinsowingtotheir
highly potent inhibition[9,10] of N-type voltage gated calcium
channels (VGCCs, Cav2.2), involved in the pain transmission
pathway.[11,12] These ω-conotoxins are a disulfide-rich class of
polypeptides that are conformationally defined by the inhibitor
‘cysteine knot’ motif, a cysteine four loop structure formed by
three intramolecular disulfide bonds.[13]
ω-Conotoxin GVIA (Fig. 1)[14] isolated from Conus geogra-
phus shows the most potent N-type VGCC binding of all
conotoxins and shows attenuation of response to acute pain
(ED50 0.12 µg kg−1) at 40 times greater potency than that
of morphine (ED50 4.36 µg kg−1).[15,16] An additional advan-
tage of ω-conotoxin GVIA over opioid-based analgesics is that
there is no development of tolerance or addiction on repeated
ω-conotoxin administration.[17,18]
Despite these promising discoveries, the native peptide has
limited clinical potential, owing to very strong binding to the
1
10
20
CKSOGSSCSOTSYNCCRSCNOYTKRCY - NH2
Fig. 1. The amino acid sequence of ω-conotoxin GVIA showing cysteine
connectivity, an amidated C terminus and hydroxylated prolines represented
by O.[14]
© CSIRO 2008
10.1071/CH07327
0004-9425/08/010011

12
P. J. Duggan et al.
H2N
O
K2
O
N
Y13
O
S
N
O
O
NH2
K2
O
HN
O
Rꢁ
Y13
HO
N
H
O
N
1a R ꢀ NꢀC(NH2)2
1b R ꢀ NH2
R17
R
2a Rꢁ ꢀ OH
2b Rꢁ ꢀ H
NH2
R17
H2N
Fig. 2. ω-Conotoxin GVIA mimetics base on anthranilamide and benzothiazole cores.
Table 1. Binding potencies (µM) for ω-conotoxin GVIA and mimetics
1a, 1b, 2a, and 2b to N-type and P/Q-type voltage gated calcium
channels
resulted when the hydroxyl and guanidinium functional groups
were omitted in previous scaffolds.[24,25]
Mimetics with identical projection chains were synthesized
in five steps in accordance with the synthetic route shown in
Scheme 1. The chain lengths of n = 4 and 5 were chosen for
projection of R17 and K2 mimics.
95% confidence intervals shown in parentheses[24,25]
Compound
EC50 N-type [µM]
EC50 P/Q-type [µM]
ω-conotoxin GVIA
3.0 × 10−5
12 (1.4–90)
111 (70–180)
176 (140–220)
42 (34–53)
1a
1b
2a
2b
3.5 (2.5–4.8)
13.1 (9.5–18.1)
1.9 (1.6–2.3)
3.8 (2.9–4.9)
Results and Discussion
Commercially available 2-amino-5-nitrobenzoic acid was
coupled with 4-phenoxyaniline using N,Nꢀ-dicyclohexyl-
carbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP).
Owing to the low nucleophilicity of the para-nitroaniline moiety,
its protection was not required for this step. The nitrobenzene 4
was then reduced by palladium-catalyzed hydrogenation to give
the dianiline derivative 5, which, owing to its susceptibility to
oxidation was immediately treated with two equivalents of the
acyl chloride, formed in situ from the corresponding acid, to
give the resulting dibromide intermediates 6a or 6b. Treatment
of the dibromides 6a or 6b with sodium azide and catalytic
amounts of 18-crown-6 in DMF gave the consequent diazides 7a
or 7b. Reduction of the diazides 7a or 7b to the target diamines
3a or 3b was achieved by palladium-catalyzed hydrogenation
at atmospheric pressure. Reverse phase chromatography and
recrystallization were used to purify both diamine targets 3a
and 3b before biological testing.
The diamines 3a or 3b were tested for rat brain N-type
VGCC binding affinity using a previously described radio-
ligand displacement assay, which employs 125I-GVIA as the
radioligand.[30] Micromolar range N-type VGCC binding was
observed for both mimetics 3a and 3b with EC50 values of 75 and
42 µM, respectively.A Target diamines 3a and 3b showed com-
parablebutweakerbindingstrengthwiththatobservedforearlier
mimetics (1a, 1b, 2a, and 2b) presumably owing to the inclusion
of simplified K2 and Y13 mimicking functionalities. Unfortu-
nately the relative weakness of receptor binding shown by 3a
and 3b made the narrowing of confidence intervals difficult.
26 (20–32)
O
O
NH2
K2
HN
HN
O
Y13
n
N
H
n ꢀ 4 or 5
NH2
R17
n
O
3
Fig. 3. The general structure of the non-peptide ω-conotoxin GVIA
mimetics prepared and tested in the present study.
the omission of a hydroxyl group in theY13 mimic resulted in lit-
tle loss of binding ability. Similarly, in the anthranilamide series,
an R17 mimic functionalized as an amine (1b) showed similar
binding to that of the inhibitor with a guanidinium functionalized
R17 mimic (1a) (Table 1).[24,25]
In the present publication, we report the synthesis and binding
studies for amino-anthranilamide derivatives 3 (Fig. 3). The key
modification in this class of derivatives is the incorporation of
an amide linkage for the R17 mimic, replacing the ether linkage
in 1. This change enabled the rapid synthesis of inhibitors with
the same K2 and R17 projection chain lengths. Other changes
in design are the omission of the hydroxyl functionality of the
Y13 mimic and replacement of the guanidinium group with
an amine. These changes greatly simplified the synthetic path
and it was anticipated that no great reduction in N-type VGCC
affinity would occur, given that only a small loss in potency
Conclusion
Two N-type VGCC binders have been synthesized and show
binding in rat brain radioligand binding assays in the micromolar
A95% confidence intervals for diamines 3a and 3b are 11–520 and 8–220 µM, respectively.

Synthesis and Cav2.2 Binding Data for Non-Peptide Mimetics of ω-Conotoxin GVIA
13
O
NH2
NO2
O
O
NH2
NO2
(a)
HO
N
H
4
(b)
O
O
Br
O
O
HN
O
NH2
NH2
n
(c)
N
N
H
H
6a n ꢀ 4
6b n ꢀ 5
5
HN
Br
n
O
O
(d)
O
O
NH2
O
N3
O
HN
HN
O
HN
HN
n
n
N
H
N
H
(e)
7a n ꢀ 4
7b n ꢀ 5
3a n ꢀ 4
3b n ꢀ 5
NH2
N3
n
n
O
O
Scheme 1. Reagents and conditions: (a) DCC, DMAP, 4-phenoxyaniline, THF, 48 h, 20%; (b) H2, Pd/C, THF;
(c) ClOC(CH2)nBr,TEA, DCM, 0◦C → rt, 6a 40%, 6b 52% over two steps; (d) NaN3, 18-crown-6, DMF, 7a 98%,
7b 98%; (e) H2, Pd/C, MeOH, 3a 24%, 3b 57%.
range.B Thus this synthetic route provides a fast and efficient
synthesis of N-type VGCC binders with varying K2 and R17
projection length with comparable binding affinity to the more
synthetically demanding mimetics previously described.
sodium benzophenone ketal. Solutions were dried over anhy-
drous magnesium sulfate (MgSO4) or sodium sulfate (Na2SO4).
2-Amino-5-nitro-N-(4-phenoxyphenyl)benzamide 4
To a vigorously stirring solution of 4-phenoxyaniline (2.45 g,
13.2 mmol), DMAP (0.33 g, 2.7 mmol) and DCC (2.82 g,
13.7 mmol) in THF (50 mL) was added 2-amino-5-nitrobenzoic
acid (2.03 g, 11.2 mmol) in THF (20 mL) dropwise under nitro-
gen. When TLC analysis showed no remaining starting material
(typically 48 h), the insoluble urea (N-Nꢀ-dicyclohexylurea) was
removedbyfiltrationandtheresiduerinsedwithTHF.Thefiltrate
was then concentrated to one quarter of its volume and the prod-
uct precipitated by addition of hexane. The solid was collected
and washed with dichloromethane (DCM) to yield the aniline
4 (0.68 g, 20%) as a yellow crystalline solid, mp 210–212◦C.
νmax/cm−1 3466, 3346, 3292, 1641, 1620, 1591, 1510, 1487,
1410, 1310, 1250, 1132, 837. δH (400 MHz, [D6]acetone) 9.90
(bs, 1H, NH), 8.62 (d, 1H, J 2.5, Ar–H), 8.07 (dd, 1H, J 2.6 and
9.2, Ar–H), 7.80–7.40 (m, 2H, Ar–H), 7.41 (bs, 2H, NH2), 7.35–
7.37 (m, 2H, Ar–H), 7.13–7.09 (m, 1H, Ar–H), 7.03–7.00 (m,
4H,Ar–H), 6.93 (d, 1H, J 9.2,Ar–H). δC (100 MHz, [D6]acetone)
167.1, 158.6, 156.4, 154.3, 137.1, 135.4, 130.7 (2C), 128.4,
126.4, 123.9, 123.4 (2C), 120.0 (2C), 119.2 (2C), 117.5,
117.1. Found m/z 350.1144 [M + H]+. C19H16N3O4 requires
350.1141.
Experimental
General Procedures
Melting points were recorded on a Stuart melting point apparatus
and are uncorrected. Proton (1H) NMR spectra were recorded, at
300 or 400 MHz and carbon (13C) NMR spectra were acquired
at 75 or 100 MHz on a Bruker AM 300 spectrometer or a
BrukerAvance 400 DRX spectrometer. NMR spectra were refer-
enced to residual solvent peaks (chloroform (δH 7.26, δC 77.0),
acetone (δH 2.05, δC 29.8, 205.9), methanol (δH 4.87, 3.30,
δC 49.86)). To aid the assignment of NMR spectra, correla-
tion spectroscopy (COSY) and heteronuclear multiple quantum
coherence (HMQC) spectroscopy were used. Infrared spectra
wererecordedonaPerkin–Elmer1600infraredspectrometerand
refer to nujol mulls or solutions in chloroform applied as a thin
film on NaCl plates. Low resolution mass spectra were recorded
on a Micromass Platform spectrometer. Accurate mass determi-
nations were carried out at high resolution on anAgilent G1969A
LC-TOF system with reference and mass correction at 4000V
capillary voltage for electrospray ionization (ESI). Reverse
phase silica gel used was octadecylsilane C18 Chromatorex
DM1020T and was obtained from Fuji Silysia. Starting mate-
rials and reagents were purchased from Sigma-Aldrich and were
used without purification.THF was distilled under nitrogen from
2,5-Diamino-N-(4-phenoxyphenyl)benzamide 5
To a solution of the aniline derivative 4 (200 mg, 0.57 mmol)
in THF (5 mL) was added 10% palladium on charcoal (60 mg,
BIt has not yet been proved that the K2-Y13-R17 mimetics bind to the N-type voltage gated calcium channels in the same manner as ω-conotoxin GVIA.[24,26]

14
P. J. Duggan et al.
0.06 mmol). After degassing and flushing with hydrogen, the
mixture was stirred under an atmosphere of hydrogen at 22◦C
for 4 h, then filtered through Celite to afford a green solu-
tion of the title compound (9), which was used for subsequent
transformations without further purification. m/z (ESI, 70 eV)
320.1 [M + H]+.
28.4, 28.3, 25.2, 24.2. m/z (ESI, 70 eV) 670.1 [M − H]−. Found
[M + H]+ 672.1082, required for C31H36Br2N3O4 672.1073.
2,5-Bis-(5-azidopentanamido)-N-(4-phenoxyphenyl)
benzamide 7a
A solution of the dihalide 6a (270 mg, 0.42 mmol), sodium azide
(135 mg, 2.08 mmol) and 18-crown-6 (1 mg, 0.004 mmol) in
dry DMF (3 mL) was stirred at RT overnight. Diethyl ether
(20 mL)wasaddedandthemixturewashedwithsat. NaHCO3(aq)
(3 × 10 mL). The organic layer was dried, filtered and the sol-
vent removed under vacuum to afford title compound 7a as a
solid (234 mg, 98%). νmax/cm−1 3282, 2097, 1652, 1592, 1506,
1488, 1405, 1232. δH (400 MHz, [D6]acetone) 10.42 (bs, 1H,
NH), 9.83 (bs, 1H, NH), 9.21 (bs, 1H, NH), 8.41 (d, 1H, J 8.89,
Ar–H), 8.21 (d, 1H, J 2.3, Ar–H), 7.79 (d, 2H, J 9.0, Ar–H), 7.57
(dd, 1H, J 2.4 and 9.0, Ar–H), 7.41–7.36 (m, 2H, J 7.4, Ar–H),
7.15–7.06 (m, 1H, Ar–H), 7.05–6.97 (m, 4H, Ar–H), 3.40–3.35
(m, 4H, CH2–N3), 2.46–2.41 (m, 4H, –CO–CH2–), 1.80–1.72
(m, 4H, CH2–CH2–N3), 1.71–1.62 (m, 4H, –CO–CH2–CH2–).
δC (100 MHz, [D6]acetone) 171.8, 171.5, 168.1, 158.6, 154.5,
135.5, 135.3, 130.7 (2C), 130.6, 124.0, 123.7, 123.4 (2C), 122.3,
122.7, 120.1 (2C), 119.7, 119.2 (2C), 51.8, 51.7, 37.6, 36.9,
29.1, 29.0, 23.4, 23.3. m/z (ESI, 70 eV) 568.2 [M − H]−. Found
[M − H]− 568.2418, required for C29H30N9O4 568.2421.
N,Nꢀ-(2-(4-Phenoxyphenylcarbamoyl)-1,4-
phenylene)bis(5-bromopentanamide) 6a
A vigorously stirred solution of 5-bromopentanoic acid (163 mg,
0.90 mmol) in DCM (3 mL) and DMF (5 µL) was cooled to
−5◦C before dropwise addition of a solution of oxalyl chlo-
ride (85 µL, 0.98 mmol) in DCM (1 mL). The mixture was
then allowed to warm to room temperature (RT) and after stir-
ring for 1 h, the volatiles were evaporated under a stream of
nitrogen. Following this, the residue was redissolved in DCM
(10 mL), cooled to 0◦C, and triethylamine (246 µL, 1.77 mmol)
was added dropwise with stirring. A freshly prepared solution
of 2,5-diamino-N-(4-phenoxyphenyl)benzamide 5 (0.30 mmol)
in THF (5 mL) was then added dropwise over 20 min. The
mixture was allowed to warm to RT and stirred for 15 h. The
reaction was quenched with water (5 mL), the organic layer sep-
arated, and the aqueous phase washed with DCM (2 × 5 mL).
The organic extracts were combined and washed with 1 M
NaOH(aq) (3 × 10 mL), 1 M HCl(aq) (3 × 10 mL), and brine
(2 × 15 mL). The solution was dried, filtered, and the solvent
removed under vacuum to afford title compound 6a (231 mg,
40%) as a solid, mp 125–132◦C. νmax/cm−1 3284, 1715, 1652,
1592, 1538, 1506, 1488, 1410, 1246. δH (400 MHz, [D6]DMSO)
10.37 (bs, 1H, NH), 10.01 (bs, 1H, NH), 9.90 (bs, 1H, NH),
7.88 (d, 1H, J 2.03, Ar–H), 7.83 (d, 1H, J 9.08, Ar–H), 7.70
(d, 2H, J 8.96, Ar–H), 7.64 (dd, 1H, J 1.80 and 8.56, Ar–H),
7.35–7.31 (m, 2H, Ar–H), 7.10–7.07 (m, 1H, J 7.4, Ar–H),
7.01–6.94 (m, 4H, Ar–H), 3.56–3.45 (m, 4H, –CH2–Br),
2.35–2.16 (m, 4H, –COCH2–), 1.84–1.82 (m, 4H, –CH2–
CH2–Br), 1.70–1.33 (m, 4H, –COCH2–CH2–). δC (100 MHz,
[D6]acetone) 171.8, 171.6, 168.3, 157.3, 152.1, 135.0, 134.8,
131.8, 129.9 (2C), 123.4, 123.3, 121.9 (2C), 121.6, 120.1, 119.3
(2C), 119.0, 117.8 (2C), 35.3, 35.2, 34.7, 34.6, 31.7, 31.5, 23.7,
23.6. m/z (ESI, 70 eV) 642 [M − H. Found [M + Na]+ 666.0574,
required for C29H31Br2N3NaO4 666.0579.[16]
2,5-Bis-(6-azidohexanamido)-N-(4-phenoxyphenyl)
benzamide 7b
Title compound 7b was prepared from a solution of the dihalide
6b (192 mg, 0.29 mmol), sodium azide (92 mg, 1.42 mmol) and
18-crown-6 (1 mg, 0.004 mmol) in dry DMF (3 mL) following
the same method used to produce diazide 7a. The diazide 7b
was isolated as light yellow solid (176 mg, 98%). νmax/cm−1
3284, 2096, 1712, 1652, 1592, 1538, 1506, 1487, 1411, 1245.
δH (400 MHz, [D6]acetone) 10.41 (bs, 1H, NH), 9.83 (bs, 1H,
NH), 9.20 (bs, 1H, NH), 8.40 (d, 1H, J 9.0, H3), 8.21 (d, 1H, J
2.3, H6), 7.79 (d, 2H, J 9.0, H3ꢀ), 7.56 (dd, 1H, J 2.4 and 9.0,
H4), 7.39–7.35 (m, 2H, H8ꢀ), 7.14–7.09 (m, 1H, J 7.4, H9ꢀ),
7.03–7.00 (m, 4H, H4ꢀ and H7ꢀ), 3.36–3.29 (m, 4H, CH2–N3),
2.41–2.37 (m, 4H, –CO–CH2–), 1.73–1.61 (m, 4H, CH2–CH2–
N3), 1.46–1.29 (m, 4H, –CO–CH2–CH2–), 0.94–0.87 (m, 4H,
CH2–CH2–CH2–N3). δC (100 MHz, [D6]acetone) 172.0, 171.3,
168.1, 158.9, 153.9, 136.1, 135.3, 130.7 (2C), 130.6, 123.8,
123.6 (2C), 123.3, 121.8, 120.1, 119.9 (2C), 119.7, 118.9 (2C),
52.0, 51.9, 38.4, 37.3, 27.1, 27.0, 24.5, 24.4, 20.5, 20.4. m/z (ESI,
70 eV) 596 [M − H]−. Found [M − H]− 596.2734, required for
C31H34N9O4 596.2739.
N,Nꢀ-(2-(4-Phenoxyphenylcarbamoyl)-1,4-phenylene)
bis(6-bromohexanamide) 6b
Following the same method used to prepare 6a, the title
compound 6b was prepared from 6-bromohexanoic acid
(143 mg, 0.73 mmol), oxalyl chloride (68 µL, 0.78 mmol), DCM
(3 mL), DMF (5 µL), triethylamine (196 µL, 1.41 mmol), 2,5-
diamino-N-(4-phenoxyphenyl)benzamide 5 (0.28 mmol) and
DCM (10 mL). The title compound 5a was isolated as a solid
(99 mg, 52%), mp 120–127◦C. νmax/cm−1 3280, 1714, 1652,
1592, 1538, 1506, 1486, 1411, 1244. δH (400 MHz, [D6]acetone)
10.40 (bs, 1H, NH), 9.82 (bs, 1H, NH), 9.20 (bs, 1H, NH), 8.40
(d, 1H, J 9.0,Ar–H), 8.21 (d, 1H, J 2.3,Ar–H), 7.81 (d, 2H, J 9.0,
Ar–H), 7.59 (dd, 1H, J 2.4 and 9.0, Ar–H), 7.39–7.35 (m, 2H, J
7.4, Ar–H), 7.13–7.09 (m, 1H, Ar–H), 7.04–7.00 (m, 4H, Ar–H),
3.49–3.46 (m, 4H, –CH2–Br), 2.40 (m, 4H, –COCH2–), 1.89–
1.86 (m, 4H, –CH2–CH2–Br), 1.73–1.68 (m, 4H, –COCH2–
CH2–), 1.53–1.48 (m, 4H, –CH2–CH2–CH2–Br). δC (100 MHz,
[D6]acetone) 171.8, 171.5, 168.0, 158.5, 154.3, 135.4, 135.3,
130.7, 130.6 (2C), 123.8, 123.5, 123.4 (2C), 123.2, 122.6, 120.0
(2C), 119.7, 119.1 (2C), 38.3, 38.0, 34.5, 34.4, 33.2, 32.9,
2,5-Bis-(5-aminopentanamido)-N-(4-phenoxyphenyl)
benzamide 3a
A degassed mixture of diazide 7a (234 mg, 0.41 mmol) and Pd/C
(43 mg, 0.04 mmol) in methanol (5 mL) was stirred for 24 h
under an atmosphere of hydrogen at 25◦C. The reaction mix
was then filtered through Celite and the solvent removed under
vacuum to afford the target diamine 3a as a light yellow oil
(207 mg, 98%), which was purified by reverse phase chromatog-
raphy (1:1 MeOH/H2O) and recrystallized from methanol/water
(51 mg, 24%). νmax/cm−1 3287, 2938, 1659, 1590, 1506, 1489,
1404. δH (400 MHz, CD3OD) 8.05–8.03 (m, 2H, Ar–H), 7.64
(d, J 8.04, 2H, Ar–H), 7.53 (dd, J 1.50 and 8.53, 1H, Ar–H),
7.36–7.32 (m, 2H, Ar–H), 7.10–7.08 (m, 2H, Ar–H), 7.00–6.97
(m, 4H, Ar–H), 2.69–2.61 (m, 4H, CH2–NH2), 2.46–2.37 (m,

Synthesis and Cav2.2 Binding Data for Non-Peptide Mimetics of ω-Conotoxin GVIA
15
4H, –CO–CH2–), 1.76–1.63 (m, 4H, CH2–CH2–NH2), 1.58–
1.49(m, 4H, –CO–CH2–CH2–). δC (100 MHz, CD3COD)174.5,
174.2, 168.9, 159.0, 155.5, 136.1, 135.2, 134.5, 130.9 (2C),
128.6, 127.2, 124.5, 124.3, 123.9 (2C), 121.1, 120.3 (2C), 119.6
(2C), 42.1, 42.0, 38.1, 37.5, 33.0, 32.8, 24.1, 24.0. m/z (ESI,
70 eV) 518 (M + H)+. Found (M + H)+ 518.2761, required for
C29H36N5O4 518.2767.
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2,5-Bis-(6-aminohexanamido)-N-(4-phenoxyphenyl)
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The title compound 3b was prepared from diazide 7b (176 mg,
0.29 mmol) and Pd/C (31 mg, 0.03 mmol) in methanol (5 mL)
following the same method used to prepare diamine 3a. Diamine
3b was isolated as a light yellow oil (137 mg, 85%), which was
purified by reverse phase chromatography (1:1 MeOH/H2O) and
recrystallized from methanol/water (90 mg, 57%). νmax/cm−1
3302, 2935, 1654, 1590, 1505, 1489, 1404. δH (400 MHz,
CD3OD) 8.06–8.02 (m, 2H, Ar–H), 7.65 (d, J 8.36, 2H, Ar–
H), 7.53 (dd, J 1.69 and 8.72, 2H, Ar–H), 7.37–7.32 (m, 2H,
Ar–H), 7.12–7.07 (m, 1H, Ar–H), 7.01–6.98 (m, 4H, Ar–H),
2.63–2.55 (m, 4H, CH2–NH2), 2.42–2.35 (m, 4H, –CO–CH2–),
1.74–1.63 (m, 4H, CH2–CH2–NH2), 1.58–1.31 (m, 8H, –CO–
CH2–CH2–CH2–). δC (100 MHz, [D6]acetone) 172.3, 172.2,
168.1, 158.7, 154.4, 135.4, 135.3, 130.7 (2C), 124.0 (2C), 123.9,
123.3, 122.5, 120.1(2C), 120.0(2C), 119.4, 119.14, 119.12, 51.8
(2C), 38.1, 37.3, 33.0, 32.9, 26.97, 26.94, 25.64, 25.63. m/z (ESI,
70 eV)546.3[M + H]+. Found[M + H]+ 546.3078, requiredfor
C31H40N5O4 546.3080.
Biological Methods
Radioligand binding assays were run in triplicate in 96-well
plates at room temperature as previously described.[30] Each
assay contained test compound, radiolabelled peptide (125I-
GVIA for N-type channels or 125I-MVIIA for P/Q-type chan-
nels) and 8 µg of crude rat brain membrane added last. All
dilutions were made in assay buffer (20 mM HEPES, 75 mM
NaCl, 0.2 mM EDTA, 0.2 mM EGTA, 2 µM Leupeptin, 2 µL
apoprotinin (to 30 mL assay buffer) and 0.1% BSA, pH 7.4).
Final volume in each well was 150 µL. After shaking for 1 h,
membrane was filtered (Wallac, Finland, glass fibre filters pre-
soaked in 0.6% polyethyleneimine) and washed with 20 mM
HEPES, 125 mMNaCl, pH7.4onaTomtecharvester.Afteraddi-
tion of scintillant, radioactivity bound to the filter was counted
using a 1450 MicroBeta (Wallac, Finland). The data was ana-
lyzed using GraphPad Prism 2.0 (GraphPad Software, Inc., San
Diego, USA).
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C. I. Schroeder, Bioorg. Med. Chem. 2004, 12, 4025. doi:10.1016/
J.BMC.2004.05.040
[25] J. B. Baell, P. J. Duggan, S. A. Forsyth, R. J. Lewis, Y. P. Lok,
C. I. Schroeder, N. E. Shepherd, Tetrahedron 2006, 62, 7284.
doi:10.1016/J.TET.2006.05.041
Acknowledgements
The authors wish to acknowledge the Monash–CSIRO Collaborative
Research Support Scheme for the funding for the present project as well
as the top-up scholarship received by J.E.G. Monash University is also to be
acknowledged for the MGS received by J.E.G.
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